TH F 9‘8 NV! 688 ITY LIBRARIES IHIIHHI'HllllllllHllHllllHHlMIIll WI 3 1293 0089 This is to certify that the dissertation entitled EXTRACHROMOSOMALLY MAINTAINED TRANSFORMATION VECTORS OF THE LIGNIN DEGRADING FILAMENTOUS FUNGUS PHANEROCHAETE CHRYSOSPORIUM presented by THOMAS ALLAN RANDALL has been accepted towards fulfillment of the requirements for p1 .D degree in M 7’ M I i /M( 6 Major pilfessor Date ”A” MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 (W W \ LIBRARY Michigan State University \ ,4 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE ‘_ 53‘ I; it! MSU Is An Affirmative Action/Equal Opportunity Institution cumming-9:1. 7“ EXTRACHROMOSOMALLY MAINTAINED TRANSFORMATION VECTORS OF THE LIGNIN DEGRADING FILAMENTOUS FUNGUS PHANEROCHAETE CHRYSOSPORIUM BY THOMAS ALLAN RANDALL A DISSERTATION Submitted to . Michigan State University . 1n partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1990 ew- "' a 1’ (2 ABSTRACT EXTRACHROMOSOMALLY MAINTAINED TRANSFORMATION VECTORS OF THE LIGNIN DEGRADING FILAMENTOUS PHANEROCHAETE CHRYSOSPORIUM BY THOMAS ALLAN RANDALL Ehaneroggaete Chrysgspggium is a lignin degrading white rot basidiomycete fungus. There is considerable interest in this organism due to its applications in biopulping, xenobiotic degradation and in bioconversion of lignocellulosic materials to feeds, fuels, and chemicals. Hence, the physiology, genetics, and molecular biology of this organism is being studied extensively. To further the molecular genetic analysis of this fungus, we constructed vector pRRlz that transforms 2. Chrysospogium to G418 resistance. Vector pGlz-l, a derivative pRRlz, was isolated from a pRRlz transformant and was also shown to transform 2. ghrysosporium. This vector appeared to result from a rearrangement between pRRlz and an endogenous plasmid(pME). Vector pGlZ-l was shown to be maintained in B. ghrysgspgrigm in a low copy, circular, extrachromosomal state and was recovered intact from fungal transformants by E. 99;; transformation. The vector was stably transmitted to basidiospore progeny of the transformants. A similar mode of transformation was found to occur with two other vectors, YIpS-lggp,r and pAN7-1, which transform 2. QQIyggspgxigm to G418 and hygromycin resistance, respectively. Plasmid p12-6, a common derivative of pGlZ-l and YIpS- nr, was also shown to transform 2. ghzxgggpgzigm. Southern blot analysis of p12-6 transformants shows directly that the vector is maintained extrachromosomally, although not autonomously. Two classes of plasmids(p12-6 and p511), were found to be recovered by g. 99;; transformation using total DNA of p12-6 transformants. The lag; gene inserted into p12-6, was shown to be passed intact through E. ghrysgspgrium suggesting that p12-6 has a potential as a shuttle vector. When the Bear determinant was inserted into the coding region of a genomic clone containing the lignin peroxidase _ HZ, the resulting plasmid was shown to transform 2. Chrysospozium to G418 resistance, was maintained in.a high copy number autonomous form in the transformants, and was recoverable by E. 991i transformation. ACKNOWLEDGEMENTS I would like to thank my parents. Allen, Russ, Dudley and the boys. Don Primerano, Amy Johnson, Moriko Ito, Rich Shwartz, Andy Black, Bob and Jan and all in his lab for being fun to be around. Shree Dhawale and Satya Kakar for teaching me fungal genetics. I would like to thank my committee, Dr. Sue Conrad, Dr. Martha Mulks, Dr. Ron Patterson, and Dr. Lee McIntosh for advice on the project. I would like to thank Dr. C.A. Reddy for allowing me to work on something interesting. iv. TABLE_QE_QQNIENI§ page List of Tables ...................................... viii List Of FigureSOOOOOOOOOOOOO ..... O 0000000000000 0.0... x IntrOductiODOOOOOO......OOOOOOOO OOOOOOOOOOOOOOOOOOOOO 1 Literature Review....... ................... . ......... 5 I. Lignin Degradation in 2. Chrysosporium ..... ... 5 1. The structure of lignin..................... 5 2. Microbiology of lignin ...................... 6 3. The Physiology of lignin degradation in 2. ghrysosporium.. ..... . ............ ........ 8 4. Enzymes involved in lignin degradation...... 11 A. Liggin perOXidaseS(LIP).....OOOOOOOOOOOOOO 11 B. Mn dependent peroxidases(MNP) ...... ..... 13 C. H202 producing enzymes.................... 14 II. Genetics and Molecular Biology of 2. czlgisosporiEEOOOO0.0.0.000...00.00.... ....... O. 15 1. IntrOductionoo.........OOOOOOOOOOOOOOOO ..... O 15 2. Genetic analysis of 2. Chrysosporium......... 16 A. Life cycle................................ 16 B. Genetics.................................. 17 C. Ligninolytic and cellulolytic mutants..... 19 3. Molecular Biology of 2. chrysospo;ium........ 22 III. Fungal Transformation Systems................. 27 1. Transformation of Saccharomyces cerevisiae.... 27 A. Integration Vectors.. ..... . ..... ............ 29 B. Autonomous Vectors.......................... 34 C. Centromere Vectors.......................... 38 2. Transformation of Filamentous Fungi........... 39 3. Neurosporg czgs a............................. 41 A. Integrative Transformation.................. 44 B. Studies on the Development of a Shuttle Vector System.................. 50 4. Aspergillus nigglag_.......................... 57 A. Integrative Transformation... ..... .......... 58 B. Studies on the Development of a Shuttle Vector System... ..... .......... 65 C. Transformation of other Aspergillus species......................... 72 i. Aspergillus niger...... ........... ...... 73 ii. Aspergillgs oryzae...................... 76 iii. Other Agpgrgillug species....... ....... . 77 V. 5. Other Filamentous Fungi....................... A. Egdospora anserina.......................... B. Ehycomyces blakesleeanu .................... C. Schizophyllum commune....................... D. Coprinus Qinereus........................... E. Ascobolus immersus.......................... 6. Fungi of Industrial Importance................ A. Penicillium chrysogenum..................... B. erhalosporium acremgnium................... C. llucor CirCineIIOideSOOOOOO......OOOOOOOO0.0. D. TriChOdema reesei.......OOOOOOOOOOOOO...... E. Ehanerochaete chrysosporium................. 7. Fungal Plant Pathogens........................ A. Ustilago maydis............................. B. Egsarium species............................ 8. Other Fungal Transformation Systems........... 9. Conclusions................................... REFERENCESOOOO.........OOOOOOOOOOOOOOOOO......OOOOOOO CHAPTER 1. A NOVEL EXTRACHROMOSOMALLY MAINTAINED TRANSFORMATION VECTOR FOR THE LIGNIN DEGRADING BASIDIOMYCETE PHANEROCHAETE QflBX§Q§2QBlflM.......................... Abstract....................................... Introduction................................... Materials and Methods.......................... Results........................................ Discussion..................................... Acknowledgements............................... Literature cited............................... CHAPTER 2. THE NATURE OF EXTRACHROMOSOMAL MAINTENANCE OF TRANSFORMING VECTORS IN THE FILAMENTOUS BASIDIOMYCETE EHANEBQQHAEIE QHRXOSEQBQM. Summary........................................ Introduction................................... Materials and Methods.......................... Results........................................ Discussion..................................... Acknowledgements............................... References..................................... vi. 78 79 84 86 87 89 92 92 95 97 98 100 101 101 105 108 121 126 159 160 161 162 165 182 186 187 192 193 193 195 196 211 213 214 CHAPTER 3. REARRANGEMENT OF TRANSFORMATION VECTORS IN EEAHEBQQEAEIE QEBISQSEQBIQM VIA RECOMBINATION WITH AN ENDOGENOUS PLASMID.. Summary........................................ Introduction................................... Materials and Methods.......................... Results and Discussion......................... Acknowledgements............................... References..................................... CHAPTER 4. AN IMPROVED TRANSFORMATION VECTOR FOR THE LIGNIN-DEGRADING WHITE ROT BASIDIOMYCETE BEANEBQQHAEIE QHBZSQSPORIUM............... Summary........................................ Introduction.......... ..... ......... ......... .. Materials and Methods.......................... Results and Discussion............. ...... ...... Acknowledgements............................... References....... .............................. APPENDIX A. USE OF A SHUTTLE VECTOR FOR THE TRANSFORMATION OF THE WHITE ROT BASIDIOMYCETE, PHANEROCHAETE c 080 CO.........OOOOOOOOOOOOOOOOO Abstract........................ ...... ......... Introduction................................... Materials and Methods.......................... Results and Discussion......................... Acknowledgements............................... References..................................... vii. 216 217 217 219 220 229 230 231 232 233 235 236 252 253 257 258 258 259 260 262 263 I OF TABLE Page LITERATURE REVIEW 1 .Nucleotide and amino acid homology among 2. ghryggspgrigm lignin peroxidase genes........... 24 2 Selection markers and transformation vectors used forWWOOOOOOOOOOOOOOOOOOO00...... 43 3 Selection markers and transformation vectors used for Aspergillus nidulans................... 59 4 Selection markers and transformation vectors used for other Aspergillus species.............. 74 5 Transformation markers used in recently described fungal transformation systems......... 109 6 Vectors and selection markers used across genus/ species lines for selection of transformants.... 113 7 Transformation vectors for expressing reporter genes from Eghezignig cgli...................... 118 8 Heterologous expression of genes of scientific, commercial and medical interest in filamentous fungi..................... ...... .... 119 CHAPTER 1 1 Transformation of E. 9911 with 2231 and M991 digested DNA from G418-resistant pG12-1 transformants of 2. ghgyggspgrig_............... 175 2 Transformation of E. lei with exonuclease III- Sl nuclease treated genomic DNA of G418-resistant pGlZ-l transformants of 2. gnryggspgrium........ 177 3 Stability of pG12-1 through vegetative growth under non-selective conditions.................. 180 viii. CHAPTER 2 Genotypes of E. coll strains used in this study. 205 Recovery of plasmids from total_DNA of p12-6 transformants in {egg and recA E.flustrainSOOOOOOO......OOOOOO.....OOOOOOOO. 206 Recovery of plasmids from total DNA of pSV7 transformants of P. Chrysosporium in E.2911Dasaeoeooeeoeoeeeeoeeeeeeeee 000000000000 210 APPENDIX A Transformation of 2. ghrysosporium with vectorpRRIZOCIOOOOOOOOOOO0.0.0.0000............ 261 ix. FIGURE LITERATURE REVIEW Page 1 2 The structure of lignin.......... ........ . ...... 7 Types of integrative transformation events observed in fungi............ ............ 32 Common transformation vectors used across species lines ............................ 112 CHAPTER 1 A schematic illustration of plasmid vectors pRRlz and pGlz-100000000000000000......OOOOOOOOO 167 Southern hybridization of 2. Chrysosporium total genomic DNA to the ME-l fragment of pGlz-IOOOOOOO......OOOOOOOOOOOO0.00.00.000.00... 169 Southern blot analysis of polymerase chain reaction(PCR)-amplified and unamplified DNA from G418 resistant basidiospore progeny of pG12-1 transformants................ ..... ....... 171 Exonuclease-Sl digestion of total DNA from two 2. ghxysgspgrigm transformants.................. 176 Southern blot analysis of PCR amplified DNA G418-resistant pG12-1 transformants of E. cnrysgspgrium after 10 subcultures on non-selective medium.................... ........ 181 CHAPTER 2 Restriction map of plasmids pG12-1,p12-6, and p511..........OOOOOOOOOOOOOOOOO..0... ....... 198 Southern blot analysis of total DNA of pG12- -1 and p12-6 transformants of 2. gnrygggpgzigm..... 202 Construction of plasmid pSV7.................... 209 CHAPTER 3 PCR analysis of YIpS-Lanr and pAN7—1 transformants of B. gnrysosporium ..... .......... 221 X. Restriction map of YIpS-ggnr and its derivatives rescued n E. 9911 from the total DNA of YIpS-kgn transformants.................. ....... Restriction map of pAN7-1 and its derivatives rescued in E. 9911 from the total DNA of pAN7-1 transformants............................ CHAPTER 4 Restriction map of pUGLGl:kan................... Southern analysis of genomic DNA from pUGLGlzkan traDSfomants.......OOOOOOCOOOIOOOOOO0.0.0.0.... Restriction and Southern blot analysis of transformant DNA................................ Confirmation of circular and fungal origin of the transforming plasmid in a G418-resistant 2. 999y999999199 transformant................... Analysis of individual basidiospore progeny of a pUGLGlzkan transformant of 2. c9;ysospo;ium..... APPENDIX A Construction of vector pRR12.................... Recovery of pRRlZ from B. 9ggysos90rium transtomantSOOOOOOO00.0.0.0...0.00.00.00.00...O xi. 224 226 238 240 242 245 248 259 262 1 INTRODUCTION Lignin is a highly complex, heterogeneous, amorphus, three dimensional aromatic polymer that is a major structural component of all woody plants(l). Lignin is second only to cellulose in being the most abundant structural component of terrestrial plants. Its role in these plants is to provide structural rigidity and because of its recalcitrance to biodegradation, to provide the first line of defense against plant pathogens(2). Lignin, because of its relative resistance to microbial degradation, is an important limiting factor in the earth's carbon cycling and in the efficient conversion of plant biomass to feeds, fuels, and chemicals(1). Lignin degradation in nature is predominantly carried out by the white rot fungi. There has been much interest in the lignin degrading system (LDS) of the basidiomycete fungus 2999999999999 9hzy99999919m because it is able to rapidly 'and completely degrade lignin(3): grows well at a relatively high temperature (39-40°C); and conidiates profusely(4-6). For those reasons and others, 2. 9912999999199 has become a model organism for the study of lignin degradation(2,3). The interest in the lignin degrading capacity of this fungus is primarily due to the potential industrial applications of lignin degradation(2,3,7). B. ghzygoségrium has also attracted world wide attention recently because the LDS of this fungus has also been implicated in the detoxification of a broad range of recalcitrant man-made environmental pollutants(8,9). The LDS of 2. 9912999999199 is expressed strictly during secondary metabolism in response to nutritional starvation(4). Recent investigations have focused on the biochemistry, genetics, and the molecular biology of the lignin degrading system of 2. chgysosporium in order to better understand the process of lignin degradation in nature(8,9). Two major families of extracellular enzymes, designated lignin peroxidases(LIP) and manganese dependent peroxidases(MNP), which are involved in lignin degradation have been identified(8,9). Genes encoding several LIP and MNP isozymes have been cloned and sequenced. Both LIP and MNP gene families consist of multiple genes with very high sequence homology. Many other enzymes, such as glucose oxidase(gox), glyoxal oxidase, p-etherases, laccases, ring cleavage enzymes, and demethylases, that have been implicated in the lignin degradation pathway, have been described(8,9). Various secondary metabolic mutants altering the pattern of expression of LIPs, MNPs, glucose oxidase have been isolated in order to better define the regulatory pathway(s) controlling the expression of these genes(10-14). The transformation systems for the well studied yeasts 3 and filamentous fungi have led to advances in two broad areas. First, the varied types of vectors used in both the yeast and fungal transformation systems has been utilized in concert with the classical genetic approaches to yield considerable advances in the understanding of the genetics and physiology of these lower eukaryotes. Secondly, these transformation systems have been the models for the development of similar systems for many of the less well characterized fungi, many of which are important in agriculture and medicine and for which gene transfer systems are either not available or are primitive. To facilitate more effective genetic manipulation of these fungi, of which £.i9n:ysospo;ium is a prime example, recombinant DNA technology can in many cases be used to develop these fungi to their full potential. The first step in exploiting the biotechnological potential of these organisms is to develop a DNA transformation system for these organisms and to clone and characterize specific genes of interest. Comparisons between the transformation systems developed for single celled yeasts and the filamentous fungi have also illustrated marked differences in the mechanism by which DNA transformation is achieved in these organisms. This has resulted in detailed knowledge concerning the genetic processes operating in these organisms. Transformation has also been used to understand the regulation of transcription of genes of interest and to 4 understand the nature of important elements of the chromosome. Attempts to extend this knowledge to the filamentous fungi has lead to a better understanding of the transformation process in filamentous fungi and towards the development of more sophisticated and useful vectors. These transformation systems are now being utilized in the genetic analysis of various filamentous fungi. This literature review will be concerned with two areas of interest in relation to this thesis: 1) the recent advances in lignin degradation by 2. 9h9ysosgorium; and 2) the phenomenon of DNA transformation in fungi. LITERATURE REVIEW 1. Lignin Degradation by g. chgysosporium 1. The Structure of Lignin Lignin exists in nature as a major cell wall component of all vascular plants, accounting for 25% of the dry weight of plants(l). The other major components of plants, cellulose and hemicellulose, account for approximately 50 and 25%, respectively.1 Lignin in plants has two major roles: 1) it provides much of the structural support needed for trees to stand; 2) it provides a barrier to the attack of plants by bacterial and fungal pathogens. The lignin polymer is a heterogenous aromatic polymer formed through the peroxidase mediated dehydrogenative polymerization of three cinnamyl alcohol derivatives: p- coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol(2; Fig. 1). These three compounds are present in variable amounts in different plant species and within the tissue of a single plant(3,4). The lignin monomers are linked primarily by C-C and C-O-C linkages which are relatively resistant to microbial degradations. The amounts of the individual lignin monomers as well as the relative proportions of intermonomer linkages are also highly variable between different species of plants and between different tissues of the same plant. Furthermore, the 6 subunits which comprise lignin are organized in a very irregular fashion with no repeating 1inkages(Fig. 1). This is in contrast to the other major components of wood, cellulose and hemicellulose, which are have an ordered arrangement of defined linkages. Thus, the heterogeneous, amorphous, and three dimensional structure of lignin ideally serves a protective role in nature against plant pathogens 2. Microbiology of Lignin Relatively few groups of bacteria and fungi have been reported to degrade lignin, and none have been convincingly shown to use lignin as a sole energy source for growth in pure culture(4,5). White rot fungi are known to degrade all the three major components of wood: cellulose, lignin, and hemicellulose(3,4,7). Among the white rot fungi, 2. 9hzy99999zigm has been shown to completely mineralize lignin to CO more rapidly than most others(6). 2 ‘2. 9hry99999rigm has become the model system for the study of lignin degradation for many reasons. It is one of the fastest growing of the white rot fungi: has a rapid rate of lignin degradation; the physiological and cultural parameters for maximizing growth and lignin degradation have been defined: the enzymes associated with lignin degradation and the genes encoding several of them have been extensively documented(8,9). In addition, genetic studies on B. 991199999919m, while primitive compared to o ”gaze-Emmett @ou '0‘ “N3 I "if. .. W3" 1 I. "a“? 3>‘:'°‘2°“ «303‘ ’ #3:.“ . neon "l'i/ Ki" $8. . '— IP“*”::°ji fig NJ::12 £2 at a. . '2' w poet-newt 0|:th n'.ocu3,Iz-Nveedtevyl ole“ . N.'lz'°cus'm m Fig. 1. The structure of lignin. The structure shown illustrates the major linkages of a representative angiosperm lignin. At bottom left are the structures of the three precursor alcohols which predominate in the lignin structure. Figure is taken from ref. 9. 8 other fungi, have been extensive, as compared to other white rot fungi, and many auxotrophic(1S-18) and secondary metabolic(10-14) mutants have been characterized. Soft rot and brown rot fungi also attack wood but have not been studied in detail. The soft rot fungi cause softening of the wood tissue, primarily by attacking the polysaccharide components, although low levels of lignin degradation have been observed(19). The brown rot fungi mainly degrade the polysaccharides but also cause a limited degradation of lignin. This limited degradation is thought to be due to the inability of the brown rot fungi to efficiently attack the aromatic rings of the cinnamyl alcohol derivatives(20). Several genera of eubacteria, including 9999999my999, 1122:1113. We. and images—W have been reported to degrade different forms of natural or modified lignins(2,21): however the degradation is relatively slow and there is no convincing evidence for the complete mineralization of lignin to CO by bacteria in pure 2 culture. 3. The Physiology of Lignin Degradation in g. 9991999999199 Lignin degradation in 2. 9911999999199 is a tightly regulated event and occurs only in a limited range of physiological conditions. Lignin degradation is strictly a secondary metabolic event(22-24). The appearance of the lignin degrading system(LDS: measured as the degradation of 9 4C-labelled synthetic lignin[DHP] to C02) is triggered in 1 response to nitrogen, carbon, or sulfur limitation(22,25,26). Lignin degradation in nitrogen limited cultures is strongly repressed by the addition of exogenous nitrogen sources such as N114+ or L-glutamate(26, 27) and the latter has been shown to play an important role in the regulation of secondary metabolism in g. 9h91999999199(27). The onset of secondary metabolism in 2. 9991999999199 is preceded by a ten fold increase in the intracellular levels of cyclic AMP(cAMP) suggesting that cAMP plays a role in the onset of lignin degradation(28,29), but the exact role of cAMP is not known. Since lignin degradation is an oxidative process, oxygen is an important requirement. A comparison of the rates of lignin degradation under different oxygen partial pressures showed that the amount of lignin degraded at 5% O is only 10% of that degraded at 21% 02, and that 2 cultures grown under 100% 0 showed the highest level of 2 lignin degradation (26). Agitation of cultures has been found to completely inhibit lignin degradation in wild type strains in certain culture media(30). This inhibition can be relieved by the addition of low levels of detergents such as Tween-20 or Tween-80 to shaking cultures, although the rate of agitation still remains critical for optimal ligninolytic activity in these cultures(31,32). Hydrogen peroxide plays an important role in lignin degradation by wood-rotting fungi(33-36). Several wood. 10 rotting fungi have been shown to produce substantial levels of H202 from various substrates(37,38). An increase in the specific activity for H 02 production coincides with the 2 onset of ligninolytic activity in low nitrogen cultures(39). The critical involvement of 3202 was further established with the isolation of lignin peroxidases(see below) which are strictly dependent on H202 for activity(40,41). Evidence has been published which indicates that glucose oxidase, and perhaps glyoxal oxidase and other enzymes, play an important role in lignin degradation by 2. chrxseseeriun(42.43)- The appearance of the enzymes of the LDS, as measured 14 14C lignin to C0 by 2, is not dependent on the presence of lignin in the medium, although higher levels of these enzymes are produced when lignin is present(44). As lignin itself cannot cross the cell membrane, it was thought that this effect may be due to a by-product of lignin degradation. Lignin monomers, dimers, and several secondary metabolites produced during lignin degradation were then tested and it was found that lignin dimers as well as veratryl alcohol, a typical secondary metabolite produced by 2. 9999999999199, increased the level of lignin degradation in nitrogen limited cultures(45,46). . ll 4. Enzymes involved in Lignin Degradation A. Lignin Peroxidasea(LIP) Lignin peroxidases are a family of H202 dependent, heme containing, glycosylated extracellular enzymes(Mr of 39-43 kD), which catalyze the Ca-Cb cleavage of lignin model compounds and other phenolic and aromatic compounds(40,41,47-49). Lignin peroxidases are relatively non-specific and contain one protoheme IX(prosthetic group) per molecule. Various spectral analyses(50,51) showed that the native enzyme contains a high-spin iron and the reduced enzyme contains a high-spin, pentacoordinate ferrous iron. The number of lignin peroxidase isozymes produced by different strains of B. 9999999999199 varies from 3 to 15 (30,49,52). All of these isozymes catalyze oxidation of veratryl alcohol to veratrylaldehyde in the prescence of H202(30), the typical assay for lignin peroxidase activity. The levels of different LIP isozymes vary under different culture conditions(30,53) indicating a very complex . regulation of the production of these enzymes. It has been found that the highest lignin peroxidase enzymatic activity is seen when the cultures are grown under air during primary metabolism and then shifted to 100% 0 during 2 secondary metabolism(45). Addition of veratryl alcohol to secondary metabolic cultures has been shown to result in up to a four fold increase in the level of lignin peroxidases produced(54). It was subsequently found that veratryl 12 alcohol increased the levels of some of the lignin peroxidase isozymes while having no effect on others suggesting that it could induce the production of a higher level of a specific subset of the lignin peroxidases(30). The addition to cultures of certain trace minerals was also shown to enhance lignin degradation(30) and levels of lignin peroxidase activity. The.lignin peroxidases of B. 999ysos9orium strain BKM-F have been more completely characterized than those of other strains. The FPLC profile of the extracellular fluid from BKM-F cultures grown for 5 days in low nitrogen medium showed multiple heme protein peaks. Six of which(designated H1,H2,H6,H7,H8, and H10) have veratryl alcohol oxidizing activity(26) while the four others(H3, H4, H5, and H9) were shown to have manganese peroxidase activity(see below). The lignin peroxidases H2 and H8 are the major proteins produced in dimethylsuccinate buffered cultures, and H2 and H6 show the highest specific activity. Polyclonal antibody raised against H8 has been found to crossreact to all lignin peroxidases(30) suggesting their structural relatedness. V8 protease digestion analyses indicate that H1 and H2 produce almost identical peptide patterns: H8 was quite similar to these except for two missing peptides and H6 and H8 produced patterns different from H1, H2, and H10(30). These results suggested that multiple genes encode lignin peroxidases, although in some cases the multiplicity of these enzymes due to post- transcriptional differences could not be ruled out. These 13 enzymes have several potential biotechnological applications. LIP enzymes, either purified or in culture with other enzymes of the LDS could be utilized in the production of a range of small molecular weight chemical feedstocks from the lignin polymer. In commercial pulping procedures, which produce high levels of toxic halogenated aromatic byproducts, a biopulping alternative using LIPs has been shown to dehalogenate these chemicals, thus LIP enzymes have potential in processes to detoxify these toxins. B. Mn++ Dependent peroxidases(MNP) A second group of extracellular glycosylated heme proteins which require both H202 and Mn++ to oxidize a variety of lignin model compounds and model dyes such as poly R-481 and poly B-411(55,56) are designated manganese dependent lignin peroxidases. These are different from lignin peroxidases in that they do not exhibit veratryl alcohol oxidation activity(57). The Mn of the MNPs is II to MnIII. The MnIII oxidized from Mn then transfers a single electron to the lignin model compound resulting in an unstable radical which, as in the case of lignin peroxidases, is then resolved to several different breakdown products. The exact contributions of MNPs to the lignin degradation process is still unclear, although studies with a L195 mutant containing only MNP activity can 14 still degrade 14C lignin to C02, although at a lower 14 level(13). Ligninolytic cultures reportedly produce as many as six Mn++ peroxidases, which, based on their similarity in peptide digestion patterns and amino acid sequences, are probably encoded by distinct but highly related genes(52). The availability of these two classes of peroxidases should allow for a more selective production of chemical feedstocks and treatment of waste byproducts once more is known concerning the specific substrate differences between the LIPs and MNPs, and the possible differences in activities between the isozymes of each family. These aspects are currently under investigation. C. H202 Producing Enzymes There is a temporal correlation between the appearance of H202 production and lignin degradation activity. The fact that the key lignin degrading enzymes(LIPs and MNPs) require H O for their catalysis clearly showed that H20 2 2 plays an integral role in lignin degradation but the 2 physiological source of H 02 in lignin degrading cultures 2 was not known. Kelley et.al.(33,42) found that the primary enzyme involved in the production of H202 during secondary metabolism in B. 9999999999199 was glucose oxidase. This catalyzes the oxidation of D-glucose to H202 and D- glucolactone in the prescence of molecular oxygen. Glucose oxidase activity, like LIP and MNP activity, was found to be a secondary metabolic event triggered by either nitrogen or carbohydrate starvation and was repressed by adding 15 exogenous nitrogen(NH4+ or glutamate) to lignin-degrading cultures. Since the identification of glucose oxidase as the primary source of H202 during lignin degradation, several other H202 producing enzymes have been identified. The Mn++ dependent peroxidase described above has also been shown to produce H202 by the oxidation of NADH or NADPH(58), although this enzyme is present in the extracellular fluid in very low levels and it appears unlikely that the cell would have an abundant supply of NADPH as a source of H202. Two others enzymes produced by B. 9991999999199 have been shown to produce H202: these are fatty acyl CoA oxidase(59) and glyoxal oxidase(43), both of which are present at low levels and their actual significance in producing H202 in lignin degrading cultures is not clear at present. II. Genetics and Molecular Biology of 2. Writes 1. Introduction The enzyme system involved in degrading lignin to C02 is necessarily very complex and is believed to consist of a large number of enzymes. The genes encoding the lignin degrading enzyme complex have not been well characterized to date. Several lignin peroxidase cDNAs have been cloned l6 and sequenced(60-62) and six different structural genes for lignin peroxidases alone, each encoding a different isozyme, have recently been described by Zhang and Reddy(63). Several other groups have reported the cloning, sequencing, and structural analysis of the lignin peroxidase gene encoding H8 protein(64-70). Two manganese peroxidase cDNAs have recently been cloned and sequenced(71,72). A detailed molecular genetic analysis of the lignin peroxidase and manganese peroxidase genes, including the regulation of expression of these genes and an understanding of the genetic regulation of secondary metabolism in B. 9991999999199 and the ability to manipulate it, would not only lead to the development of strains with increased lignin degrading capacity, but would provide information concerning how secondary metabolism is controlled in fungi. 2. Genetic analysis of_2. 9991999999199~ A. Life Cycle. 2. 9991999999199 is a filamentous basidiomycete fungus(6). As in other filamentous fungi, the structure of the mycelium leads to an undefined intracellular environment with respect to the traditional concept of cellular growth. It grows vegetatively as a mycelium, and lacks clamp connections. Cell walls are septate, but not regularly spaced, and the fungus is coenocytic. Vegetative 17 growth occurs by apical extension at the leading edge of the mycelium. B. 9991999999199 can different into several major spore types. Conidia, which are products of mitotis, are produced under most culture conditions starting at approximately the third day of growth and a majority contain one to two nuclei per conidium(15). Basidiospores, which are haploid meiotic products containing two genetically identical(homokaryotic) nuclei, are produced in a specialized fruiting structure termed the basidium. The diploid stage of growth of 2. ch91sos9orium is transient(restricted to karyogamy in the basidium). The production of basidiospores can be induced only in certain environmental conditions, for example by growing the fungus on cellulose, at 30°C and under continuous light(73). B. Genetics. Much of our knowledge concerning the genetic system of 2. 9991999999199 and the methods for its' genetic analysis have been developed by Gold, Broda, and coworkers. Whether 2. 9991999999199 is homothallic or heterothallic has not been determined unequivocally(74,75). A genetic system has been developed, based on either mycelial fusion(anastomosis), or forced fusion of protoplasts to investigate genetic phenomena such as complementation(15- 17,76). Media to induce colonial growth have been developed. These generally contain sodium deoxycholate(0.01%) and sorbose(l-10%), which have been 18 shown to induce colonial growth in many fungi(77). A replica plating technique has also been developed(78). 2. 9991999999199 is haploid, and cells are multinucleate, with up to 15 nuclei per hyphal cell(15). The nuclear content of conidia varies; approximately 60 % of conidia are mononucleate with the rest being bi-or multinucleate(15). The multinucleate conidia are often heterokaryotic, containing nuclei of differing genotypes, and result in the development of stable heterokaryotic progeny from a single conidium. Some of the strains such as ME446(ATCC 34571), which has been extensively studied by a number of laboratories have been shown to be naturally heterokaryotic(79). Since the basidiospores have been shown to be binucleate and homokaryotic(74), isolation of basidiospores from heterokaryotic strains can be used to produce homokaryotic isolates of a desired genotype. Isolation of auxotrophs has been done by standard mutagenic techniques(15-17). Complementation studies between auxotrophs have resulted in classification of a collection of leucine auxotrophs into three complementation . groups(18). Complementation between auxotrophs can be maintained simply by having populations of two or more mutant nuclei within a mycelium, due to the cross feeding of nuclear products within a common cytoplasm. The parental genotypes can be re-isolated from such a heterokaryon by inducing basidiospore formation. No recombination can be achieved between two haploid nuclei of different genotypes unless meiosis is induced and 19 basidiospores are produced. No linkage analysis between auxotrophs has been performed and the number of chromosomes is still undetermined. To bypass the traditional accumulation of linkage data based on the isolation of mutants, Broda and co-workers have begun work on the determination of a linkage map of the E. chr1soS9o9ium chromosomes using restriction fragment length polymorphisms(RFLPs; 79,80). These studies showed that many of the LIP genes are closely linked in 2. 9991999999199 ME446(81): evidence to date suggests the presence of at least seven linkage groups(80). c. Ligninolytic and cellulolytic mutants In recent years, a number of investigators have focused their attention on isolating a variety of metabolic mutants which would serve as important tools in understanding the biochemistry and molecular biology of lignin degradation by this fungus. Two classes of pleiotrophic mutants designated phenol oxidase(99x; 10,11) and glucose oxidase(g9x; 12,35) have been isolated. The 999 mutants lack LIPs, MNPs, glucose oxidase and other secondary metabolic activities. Revertants of these mutants have resulted in the restoration of all the lost activities, suggesting that the 999 mutants are lacking a global regulatory element involved in expression of secondary 20 metabolic activities, including the LDS. Another class of regulatory mutants which specifically lack the ability to produce LIP enzymes but retain MNP enzyme activity has been isolated(13). The decoupling of LIP and MNP activity in these mutants suggests a further level of regulation controlling the exression of these seperate classes of enzymes. Lignin degradation is limited to secondary metabolism and is induced by nitrogen depletion while high levels of nitrogen are strongly inhibitory to the lignin degrading enzyme system. A mutant has recently been isolated which is able to express the lignin degrading complex in high nitrogen cultures and is designated a nitrogen deregulated mutant(999; 14). This mutation appears to result from the constitutive expression of a regulatory protein controlling the LDS enzymes and genetic studies to confirm this are in progress. This 999 mutant may be the first step toward the development of a strain which can constitutively degrade lignin. A different approach toward strain improvement has also been applied. Growth of 2. 9991999999199 in a medium containing a lignin model compound covalently bound to glycine found that the cleavage of glycine from this compound was dependent on the appearance of the LDS. This may allow for a selection of a strain able to use this glycine adduct as a sole nitrogen source, in which presumably the LDS would be deregulated(82). Major potential industrial uses of E. 9991999999199 include applications in the biopulping of paper and 21 degradation of recalcitrant xenobiotics such as dioxins(83). To produce paper, lignin has to be selectively stripped from wood, leaving the cellulose and hemicellulose. Present chemical treatments required to accomplish this produce many toxic aromatic by-products, which E. 9991999999199 has been shown to detoxify(84-86). Several cellulase deficient mutants of 2. ch91soS9or1um have been described which retain high levels of lignin degrading activity, yet do not degrade or have marginal ability to degrade cellulose(87,88). Such a seperation of cellulase degradation from lignin degradation would be absolutely necessary in an industrially useful strain since the wild type would degrade the cellulose and hemicellulose along with the lignin.- While wild type 2. 9991s039o91u9 strains are among the fastest and most efficient lignin degraders in nature, this degradation is relatively slow. The development of a genetically altered strain combining high levels of LDS with a minimum of industrially counterproductive enzyme activities(cellulases and hemicellulases) may allow the development of a process for biopulping of wood which would, from an environmental standpoint, be a major improvement over present industrial processes and save the industry billions of dollars in energy and chemical costs. 22 3. Molecular Biology of g. s s c To complement the above described attempts at studying the genetics of lignin degradation in 2. 9991999999199, procedures for the isolation and study of the genes encoding various enzymes involved in lignin degradation are needed. In addition, a means of reintroducing these genes back into E. 9h91soS99riu9 to analyze the expression and regulation of these genes and of secondary metabolism in general is required. The genome size of B. 9991999999199 is approximately 4 x107 bp, twice that of 9. 9999119199(89). Recently protocols have been developed for the isolation of DNA and RNA from 2. 9991999999199(90-92). The initial isolation of cDNAs of lignin peroxidase genes used oligonucleotide probes. These were synthesized based on the amino acid sequence of tryptic digests of the major lignin peroxidase, BS. The isolation of cDNAs encoding two structurally different lignin peroxidases has been accomplished from a library of clones enriched for a population of mRNAs expressed specifically during secondary metabolic growth in ‘2. 9991999999199(60,91).. Two classes of cDNAs, CLG4 and CLGS, were isolated which code for two distinct lignin peroxidase genes, H2 and 310, respectively, based on comparison of the N terminal amino acid sequence of these proteins to the respective cDNA sequence(53). These cDNAs have 71.5% DNA homology, and 68.5% protein homology to each other(61). Another cDNA(ML-1) with significant homology to 23 the CLGS cDNA, and which appears to encode lignin peroxidase H8, has also been isolated(62). Recently, Zhang, et.al. have cloned six genomic clones corresponding to lignin peroxidases from E. c991SOS909199 BKM-F(63, Zhang, Y.z., C.A. Reddy, A. Rasooly, submitted). This was done by screening a genomic library of strain BKM- F in YRplZ with both CLGS and CLG4 cDNA clones. The genes isolated were found to form two gene familes based on DNA hybridization. Five of the genes are highly homologous to one another and the CLGS cDNA, while the other showed hybridization only to the CLG4 cDNA clone. All genes have structural differences, based on major differences in their restriction maps. Two of these genes, GLGl(corresponding to CLG4 and encoding H2) and GLGZ(corresponding to CLGS and encoding H10), have been sequenced. GLGZ was shown to have eight introns, ranging in size from 50-62 bp(63, Zhang, Y.z., C.A. Reddy, A. Rasooly, submitted), quite similar in size and structure to introns in other filamentous fungal genes(93). The GLGI gene has nine introns ranging is size from 50-63 bp(Naidu, P., Y.z. Zhang, and C.A. Reddy, manuscript in prep.). 'Several laboratories have cloned and sequenced the lignin peroxidase gene encoding the H8 protein or its close relatives(64-70; Table 1). A summary of these results indicates that these genes are regulated at the RNA level(59,63) with 1.5 kb mRNA transcripts homologous to the above genes being present only in nitrogen limited cultures in secondary metabolism but not in nitrogen limited cultures prior to the onset of secondary metabolism or in 24 .auo>auoouaou .nsacuua cvvu: Sawhomnonxucu .m can 553 Sawhomuonxhau .m 60.3 0:00 mama on» no euccaua; on. v and 0 .voauaucopa coon no: on: mg: 52 venous» caououm and oauaooma one .o«= van o: .m: uoaavaxouom casuau ovooco aao>auocmncu cocoa oMMq pcc muud .NMHJ .aqu no noocosumn vaoc ocaac vocuaac o3» casuaz evaoc ocaEc vosouua no oocuccouemn .AOsma .anInvvamea .aOan .ao: .9. guess: use cascavooz no vozuoe on» no coauuuauavoe a .Euuooham oxmuzno an vocaeuoump on 0:93 5:33 nocauooaos: 92.0qu «o oucusouuomn so mm mm so on as so on .ms as uaoaa m» as am mm am no as as so as vcaa vonuaanaac: , .suvoa can sauu>ueuum no no so on an on as «a as mono awn-aanamc: .suoom can snua>¢evam as as no on so sm on am as cacao cm as as on as no an as no as was em as as so am as so on as as ova: «macasOnaopz sm_ no so on mm so no ss so ss ososo vouuaansu a ..a. um mean» so as so am as am am on as omaa no .oo .so as as mm mm. as as as so as mnaa van-aansdca .sunoa can snuu>ueu¢a as as as us as as as no as «man aoan aoaa mean «can ma: was some oaaa mmaa «sag ooceuOuom nuaauc ozaz< eccco eecvaxouon cacoaa scwuomwoachu .m ocoac *00aoaos can: ocaec vs: coauocaosz .n canua 25 nitrogen sufficient cultures. 31 analysis has been used to map the 5' ends of these transcripts and indicated that single(64) or multiple(68) transcription initiation sites can be present. These genes contained typical eukaryotic . sequence motifs thought to be essential for transcription(TATA boxes 78-89 bp upstream of the first AUG codon: and CAAT boxes 105-168 bp upstream of the first AUG codon), and, where data are available, a potential polyadenylation signal in the 3' untranslated portion of these genes. Lignin peroxidase genes appear to constitute a multigene family. In E. 9991999999199 BKM-F, 5 of the 6 LIP genes which have been cloned are highly homologous to each other based on Southern analysis and sequence data, and in general have 80-90 % homology at the sequence level(Naidu and Reddy, unpublished data), while the sixth gene has approximately 70 % sequence homology to the above family. Similar levels of homology are seen at the protein level. Features similar to other cloned peroxidase genes are seen in these LIP genes, in particular a potential active site containing two histidine residues. The multiplicity of LIP structural genes should, for the most part, account for the abundance of LIP isozymes present in B. cnrxsgangrium: attempts to make a one to one correlation of gene to protein are being made, although it is likely that not all of the genes encoding LIP isozymes have yet been cloned. Recent evidence has indicated that other white rot 26 basidiomycetes also have genes that have significant homology to the LIP genes of g. 9991999999199. A lignin peroxidase cDNA from 2919919 9991999 with 60% homology to the 2. 9991999999199 lignin peroxidases has been described(94). Based on cross hybridization with the genes from 2. 9991999999199, several genes from I99me99s 1999199199 have recently been cloned. This was done using the CLGS cDNA as an heterologous probe to screen a lambda EMBLB genomic library and indicated that this fungus appeared to contain a LIP gene family. More interesting, where some genetic linkage of LIP genes in 2. 9h91soS9o919m ME446 based on RFLP data has been demonstrated(81), six of the 1. 1999199199 genes appear to be very tightly linked in three subgroups. Three different subclones of the EMBL3 library each have two genes with LIP homology seperated by 1 to 2 kb. Sequence data from one of these genes shows that it is most related to the CLG4 cDNA of B. 9991999999199 and appears in all aspects to be similar to the LIP genes discussed above(Black and Reddy, manuscript in preparation). Two cDNAs encoding seperate manganese peroxidase genes have recently been sequenced(71,72); Southern blotting data indicate that these enzymes also form a family of related genes(72). A potential peroxidase active site containing two histidine residues has been found in each, and the. cDNAs appear to encode proteins with an Mr of 37 kD. Similar to the LIP isozymes, these genes appear only during secondary metabolism and appear to be transcriptionally 27 regulated(72). The reason for the multiplicity of lignin peroxidases and manganese peroxidases is not clear at this time, but several laboratories are studying this issue both at the biochemical and genetic level. III. Fungal Transformation Systems 1- Transformation of fiasshsrgmzsgs egrsziaigg fiasshargmxsss ssrgxisiag. a budding Yeast with well defined haploid and diploid stages, has long been a focus of genetic analysis due to its well studied genetic system. This has allowed detailed genetic analyses due to the fact that it can be technically manipulated with relative ease. 5- 9999119199 is the model for the development of transformation systems for lower eukaryotes and this subject has been reviewed in depth(95,96). An extensive variety of transformation vectors have been developed for 5. 9999119199. Transformation protocols and strategies for the development of vectors for this organism have been the basis for later protocols developed for other yeasts and filamentous fungi. For these reasons, a general outline of yeast transformation systems will be given as an introduction to a discussion of the transformation systems of filamentous fungi. Cell walls of yeasts are almost exclusively composed of mannose(mannoproteins) and glucose polymers (glucans:97). The most commonly used transformation procedure 28 involves the degradation of the yeast cell wall using cellulolytic and other cell wall digesting enzymes. This creates spheroplasts which are then incubated with polyethylene glycol in the prescence of CaCl2 to make the cell membrane permeable to the passage of free DNA into the cell. The various enzymes used to digest the cell wall in transformation protocols include cellulase, chitinase, p-glucoronidase, glucanase, and pectinase. These enzymes are used either individually(in relatively pure preparations such as glusulase[fi-glucoronidase]) or in combinations in various less well defined commercial preparations such as novozym 234(Novo Laboratories, Inc. Danbury, CT) or "Lysing Enzymes"(sigma Chemical Co, St. Louis M0.). The practice of rendering bacterial cells competent for DNA transformation by treatment with various cations(98) led to attempts to reproduce this phenomenon with whole cells of yeast. It was found that Li+, and to a lesser extent other monovalent alkali cations, could induce compentence of yeast cells(99) and transformation protocols similar in concept to the standard 5. 9911 transformation were developed. These protocols bypassed the need for regeneration of spheroplasts which is often low in spheroplast transformations. However, the transformation frequency obtained in lithium acetate transformations is usually lower than that obtained for the corresponding spheroplast transformation(99). Several other transformation procedures have been 29 developed for 9. 999ev151ae. They include fusion of spheroplasts from different genotypes(100). This procedure would be most useful for gene exchange between yeasts which cannot mate. Fusion of yeast spheroplasts with E. 9911 minicell spheroplasts(101) has also been induced in the presence of polyethylene glycol. It has been reported that the transformation frequency of an autonomous vector(pGY14, a 2pm plasmid based vector) using this method was found to be several fold higher than the standard spheroplast transformation system. More recently methods to direct transformation to mitochrondria or nuclei by shooting microprojectiles have been developed(102,103). In this procedure, small tungsten particles coated with the plasmid of interest are used to bombard whole yeast cells. A plasmid containing the mitochrondrial gene for cytochrome oxidase III(pQoni3) was used to transform a respiration deficient strain of 9. ce99v1s1a9(9913-) to a respiration competent state and this was shown to be due to integration of the transforming sequences into the mitochrondrial genome(102). A. Integration Vectors A number of initial transformation protocols employed vectors which transformed by integrating into the yeast chromosome. These are commonly designated YIp vectors. Hinnen et al.(104) used a plasmid designated pYe19910. This vector contained bacterial sequences from ColEl, and 30 the yeast L992 gene(encoding fi-isopropylmalate dehydrogenase), isolated by complementation of a 1999 strain of E. 9911. This vector was used to transform a 1992 auxotroph of 9. 999evisiae to prototrophy. Southern blotting analyses identified three types of integration . events(Fig. 2; after Hinnen et al.; 104). The largest class(Type I; linked insertion) of transformants had a single crossover event in a region of homology between the vector and host chromosome resulting in two adjacent copies of this gene (one functional and one nonfunctional) and the integration of the intact ColEl. The second class(Type II; unlinked insertion) of transformants were due to recombination of the vector into non-homologous regions of the genome. The third class(Type III; gene replacement) were transformants in which the wild type transforming yeast sequence precisely replaced the mutant sequence in the chromosome and all bacterial sequences were lost. This was similar to a gene conversion event or a double crossover. One of the most commonly used integration vectors has been YIp5(105). This contains a chromosomal fragment of yeast containing the 9399 gene and the E. 9911 plasmid pBR322, which contains an 991 and the tetracycline and ampicillin resistance markers. Its usefulness in the construction of more sophisticated yeast vectors will be discussed below. Homology to yeast DNA was found to be important in the integrative transformation event in 9. 9999119199 as the type II transformants which integrated by an apparent non- 31 Figure 2. Types of integrative transformation events observed in fungi. The thin linear lines_repre§ent chromosomal DNA. The thick line labeled m or m indicates an arbitrary non-functional and functional fungal selection marker, respectively which are highly homologous at the DNA sequence level. The thin line in the circular vector indicates DNA with no homology to the fungal chromosome represented. A) Type I transformation is strictly by non- homologous recombination into the chromosome resulting in two generally unlinked copies of the selection marker. B) Type II transformation is by a single crossover between regions of homology, resulting in two tightly linked copies of the selection marker. C) Type III transformation is by a double crossover, resulting in complete excision of the non- functional chromosomal copy of "m", leaving one copy of the selection marker in the chromosome. The most common variant of type A and B which are observed in fungi occurs when tandem copies of the vector integrate resulting in multiple linked copies of the selection marker. 33 homologous event were found to be exceedingly rare. Creating a linear vector by a single restriction cut within the yeast sequence was found to increase the transformation frequency of these vectors, up to a thousand fold compared to the circular integration vector(106). Linearization also directed the integration of the vector to the site at which it was linearized. Both events indicated that the recognition of a homologous stretch of DNA was very important in the integration process in yeast. Integration events can also be defined by traditional genetic analysis. A haploid transformant(prototroph with L992 complementing gene) can be crossed to a Leu- haploid of the opposite mating type and the resulting diploid then sporulated to form a four spored ascus. The four meiotic progeny of the cross then segregate in a standard Mendelian segregation pattern of 2 L£Q2+:2 1992-. The stability of the phenotype was also seen through mitosis. When the L£n2+ transformant was grown under nonselective conditions for several generations(usually at least ten) and then screened for the precence of the vector, virtually 100% of the progeny exhibited the complemented phenotype(107). Vectors integrated into the chromosome of 9. 9999119199 were generally not recoverable by using total genomic DNA from individual transformants to transform 9. 9911. Recovery was, however, achieved by digesting transformant DNA with a suitable restriction enzyme which leaves the antibiotic resistance marker and the plasmid 991 34 functional, ligating the resulting linear chromosomal fragments, and using the ligation mixture to transform 9. 9911. Using the appropriate restriction enzyme was also shown to result in the recovery of the original unaltered vector when two or more were tandemly integrated. B. Autonomous Vectors Two strategies have been used to develop vectors which can be maintained within the yeast nucleus in an intact, extrachromosomal form. Such vectors serve as shuttles for moving genes into and out of the yeast. The first approach is based on using portions of the endogenous 2p plasmid of 9. 9e9911s19e(108) to drive autonomous replication of the vector. The 2p plasmid is maintained in 9. 9999119199 isolates at 30-50 copies per cell, yet has no apparent selective advantage to the organism(109). The advantages of an extrachromosomal YEp(yeast episomal plasmid) vectors are several. These vectors can be easily recovered in E. 9911. Libraries constructed in these vectors can be routinely used to isolate genes complementing 9. 9999119199 mutations. Genes present on such a vector can also be maintained at a much higher copy number than if in the chromosome. Beggs(110) developed the first 2p based yeast-E. 9911 shuttle vectors, pJDBZ48 and pJDBZlQ. These vectors contained, besides 2p sequences, pMB9, a derivative of ColEl of 9. 9911 for replication and selection in bacteria, and the 9992 gene of 35 §- 9219215199- The second major class of yeast autonomous vectors are called yeast replicating vectors(YRp). These vectors are based on putative chromosomal replication origins termed "9:9": for autonomous replication sequence(105,107). Davis and coworkers(105,111) inserted a fragment of yeast chromosomal DNA containing the 1321 gene(encoding N-(5'- phosphoribosyl anthranilate isomerase) complementing a 9991 auxotroph into pBR332. It was found that the resulting vector, YRp7, was maintained as an autonomous vector and could transform at a significantly higher frequency(500- 5000 Trp+ yeast transformants per pg DNA for YRp7) than an integrating vector and concluded that the chromosomal fragment complementing the 9991 mutation also contained an 999 element, 9991, allowing for this high frequency of transformation. These YRp vectors have similar advantages to those described for YEp vectors. Further studies found that similar sequences allowing YIp5(described above) to be maintained as an autonomous plasmid could be isolated from a variety of eukaryotes(107). All 999' identified in yeast which have been sequenced contain an 11 bp core consensus sequence( 5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and these elements are postulated to be chromosomal origins of replication(112). Further studies indicated that 999' generally function as replication origins only in the yeast from which they have been isolated and that 999’ are not generalized broad host range replicons(113). These replicative vectors have several features which 36 distinguish them from integrative vectors. Southern hybridization analysis of total genomic DNA from transformants containing an autonomously replicating plasmid show that these plasmids are maintained as low molecular weight elements, distinct from the chromosomal DNA(110). These vectors are extremely unstable through both mitosis and meiosis. Meiotic analysis showed a high percentage of abnorma1.segregation patterns(4+:0-,3+:1-, 1+:3-, 0+:4-) indicating a marker which is behaving in a non Mendelian manner that is characteristic of extrachromosomal elements. Mitotic analysis showed that such plasmids are lost very quickly during nonselective growth. Plating of a transformant which has undergone 10 generations of nonselective growth on selective media shows very low frequency of prototrophs containing the transforming vector(1-5%; 114). However, variations in relative stabilities of different autonomous plasmids has been reported and this is discussed below. Vectors based on the 2p plasmid in general are significantly more stable than those based on 999 elements due to plasmid maintenance and partitioning functions(115) resident on the 2p sequences. An 999 vector, YpR141, was examined along with a variety of 2p vectors and found that the 2p vectors had a higher stability. A further study comparing an 999 vector(p8293) to a 2p vector(YEp13) showed that the 999 vector had a much higher segregation bias toward the mother cell than the 2p vector. This resulted in pSZ93 being maintained in a much smaller fraction of the 37 cells of a population. Furthermore, this difference in segregation bias between the two types of vectors was found to be eliminated in a strain lacking an endogenous 2p plasmid. This indicated that 2p specific genes influence the mitotic segregation of 2p vectors(114). There are several other means by which the stability of replicative vectors can be increased. pJDBZlQ was used to transform 9. 9999119199 to leucine prototrophy and an analysis of the types of plasmids present in individual transformants was made(116,117). Some transformants were found to have lost both pJD3219 and the endogenous 2p plasmid; in this case a new plasmid, pYX, was found which contained only 2p plasmid sequences and the 9992 gene with no bacterial DNA sequences. The vector lacking bacterial sequences was found to be maintained non-selectively much better than the parental pJD3219. 2p vectors, have also been shown on rare occasions were shown to integrate into a resident 2p plasmid, thus offering a means of replicating along with the stable endogenous plasmid. Similar types of 999 based plasmids which contain only yeast sequences have been tested for stability. Vector TRP R1(that contains only the yeast chromosomal fragment complementing 9991' auxotrophs and a closely linked 999 element; 118) is a model for these. This vector is small(~1 kb) and quite stable mitotically, but the lack of bacterial sequences restricts their usefulness as shuttle vectors. However, these vectors were found to be useful for the analysis of the maintenance of plasmids in yeast. Studies showed that 38 TRP R1 was be maintained stably in yeast during nonselective growth for up to 100 generations at an extremely high copy number(100 per cell). c. Centromere Vectors The in depth development of the molecular genetic system of 5. 99:9219i99 has also allowed the construction of other more specialized classes of vectors which are rather unique to the yeast system to date. The first class, centromere vectors, contain a chromosomal centromere on a YRp plasmid and combine the advantages of the integrative and autonomous vectors into one stable, autonomous vector(119). The initial identification of a centromere was that of CENIII from chromosome III(120). CENIII was identified by analyzing a series of plasmids containing overlapping chromosomal DNA fragments from the LEQZ-QENIII-Qnglg region of chromosome III inserted in the shuttle vector pLC544. One of these plasmids, pYe(99919)1 was found to behave mitotically and meiotically similar to a YIp vector, although it was maintained autonomously as a mini- chromosome. The plasmid pYe(gpglg)1 segregated through meiosis in a 2+:2- fashion for all markers tested and showed a mitotic stability of 95-97% compared with less than 1% for pLC544. The presence of a centromere also reduces the copy number of a plasmid to approximately one per cell. Further development of these vectors, primarily by 39 linearization and the addition of telomeres has created a class of vectors termed yeast artificial chromosomes(121) which can be shown to behave similarly to actual yeast chromosomes. These are particularly useful in constructing gene libraries of higher eukaryotes(principally mammals) where inserts of 100 kb to 1 Mb are possible, thus reducing screening problems inherent in large bacterial libraries and offering a more accurate analysis of higher mammalian systems(122). 2. Transformation of Filamentous Fungi In the last ten years, extensive progress has ben made on the DNA transformation systems for many filamentous fungi. These transformation systems, including the methodology, vectors, and selection markers used were first modeled after those developed for 9. 99:9visige. There are considerable differences in the nature of maintenance of the transforming vectors in filamentous fungi in comparison to that in single cell yeasts. Studies on transformation of the traditional fungal model systems, such as H999999999 and 5999rg11199, as well as more recent results obtained with other genera of fungi of industrial or agricultural importance are reviewed here. Several recent reviews have also extensively covered the progress in this rapidly expanding field(123-127). Transformation in filamentous fungi was first 40 demonstrated using modifications of yeast spheroplast transformation systems. Cell walls of filamentous fungi have a diverse range of components including cellulose, chitin, and other various polysaccharides usually complexed with proteins(128), and mannose and glucose polymers. The composition of the cell wall can vary depending on the morphogenic state of the fungus. Thus, with spheroplast based transformation systems, the choice of spheroplasting enzyme is usually dependent on the organism being studied and its morphogenic state. The generation of spheroplasts has been achieved using various p-glucoronidase preparations(glusulase, novozym 234, p-glucoronidase alone). The most common osmotic stabilizers used for suspension of the spheroplasts during and after cell wall degradation are sorbitol and M9804. The types of cell wall degrading enzyme and the osmotic stabilizer used were shown to have a marked influence on the regeneration frequency of spheroplasts, which is often quite low and can be the limiting factor in fungal spheroplast transformations(129). Additional modifications, including coating the DNA to be transformed with heparin to limit exposure to nucleases, and the addition of dimethyl sulfoxide to help permeablize the shperoplast membrane, have been used in some protocols(130). Sorbose is often employed in the selective media to restrict mycelial growth and induce colony formation(131). The most complete analyses of the effectiveness of various spheroplasting techniques and osmotic stabilizers has been done using E. 999999 and 41 299999999 99999199 as models and should be referred to (129,130). Mycelial transformation procedures, rather than conidial transformation systems, have also been developed for several fungi(130,132,133) and these appear to be effective alternatives. In addition to spheroplast transformation systems, both lithium acetate(134), liposome mediated(135) and biolistic(103) transformation systems have been developed for N. 999999. In the lithium acetate method, conidia were treated with lithium acetate followed by polyethylene glycol incubation and a heat shock. When the vector pSD10, containing bacterial sequences, the N. 999999 9999+ gene, and sequences from a mitochondrial plasmid of N. 999999 was used for transformation by the lithium acetate method, the transformation frequency was found to be comparable to the spheroplast transformation system(9 transformants per pg vs. 1-5 per pg for early spheroplast systems; 130). In the liposome protocol, n. 999999 total chromosomal DNA without a vector was encapsulated in phosphatidylserine and was then used in the standard spheroplast transformation protocol. Transformation frequencies for DNA coated with liposomes were generally found to be 20-40 fold higher than that for naked DNA molecules. 3- 8132212223 213113 The genetics and cytology of the filamentous ascomycete H999999999 999999 has been extensively studied 42 since the early part of the century and many advances in the understanding of basic genetic concepts have been accomplished with this fungus(136). Attempts at DNA mediated transformation of N. 999999 auxotrophs began in 1961 when the complementation of a pyridoxin mutant was attempted by incubating conidia from this strain with total genomic DNA from the wild type(137,138). Improvements were made in this technique over the next 15 years(135,139- 41) but no great advances were made until recombinant DNA technology became available. Complementation of auxotrophs has been the predominant selection marker in N999999999 transformations(Table 2), although the dominant selection markers benomy1(142) and 6418(143) have also been successfully used. Benomyl(methyl-1-[butylcarbamoyl]42- benzimidazole carbamate) specifically binds to fi-tubulin, and causes a lethal depolymerization of microtubules. A tubulin gene containing a point mutation in the tubulin protein that is resistant to benomyl-mediated depolymerization and can be used as a selection marker. A series of N. 999999 vectors based on benomyl resistance have now been constructed(144) and these have found use in a wide variety of other filamentous fungi(Tab1e 5). G418 is an aminoglycoside antibiotic to which many eukaryotic cells are sensitive. Kanamycin resistance determinants of E. 9911 encode an aminoglycoside phospotransferase which inactivates both kanamycin as well as G418 and has also been used as selection marker in other filamentous fungi(Table 5). However, sensitivity of N. 999999 to other 43 rant: 2 Selection markers and transformation vectors used for Neurospora crassa. ‘Selection Marker Vector Reference 9992f (catabolic pVK88 145 dehydrogenase) ' pALs-l 170 ' pALs-l, pALs-z 171 ' pVKS? 130 ' p803, pJP102 154 ' pSD3, pvxsv, pVKBB 152 ' pMXZ 173 ' pJP12 149 ' pDV1001 150 am (glutamate C10 150 dehydrogenaee) ' 010 146 ' pBClO, pEBlO 143 999r (benomyl resistance) Tllb, pBT3 142 ' psvso 161 ' pJD21,71,81,82, pRMlll 144 in; (myo-inositol-I- 3333030 173 synthase) 999:1 (33, reps, PRAI‘) pNCZ 152 G418 (geneticin) pBClO, pEBlO 143 Bialaphos pJA4 336 aanthranilate synthaee (AS),indoleglycerolphosphate synthase (IGPS), phospo- ribosylanthranilate isomerase (PRAI). 44 common antibiotics such as hygromycin B, which has also been used widely in fungal transformation protocols(Table 5), has not been reported to the best of our knowledge. A. Integrative Transformation The first recombinant vector used in E. 999539 transformation, pVK88, contained the homologous 9992+ gene(catabolic dehydroquinase) inserted into pBR322(145) to enable complementation of an N. 999999 99:2- auxotroph. Similar to the yeast integration vectors, these vectors transformed H- 999ss9 by integration into the chromosome. Genetic analyses of individual transformants showed that the complementing genetic marker generally followed a Mendelian segregation pattern and that the transforming phenotype is stable under nonselective mitotic growth, suggesting integration of the vector in a stable manner into the chromosome. In addition to these stable transformants, a number of abortive transformants, which can be seen on the initial selection plates but cannot be maintained by further transfers onto selective media, were also observed in these experiments. It is thought that in these abortive transformants the phenotype is expressed only transiently and that the marker is not able to stably integrate into the chromosome. The frequency of transformation in N. 999999, using primarily the 99:2+ and 99+(g1utamate dehydrogenase) genes as markers varied from one to several 45 thousand transformants per pg DNA based on the selection marker and the reporting 1aboratory(143,145,146). Further improvements in methodology have raised the transformation frequency in n. 999999 up to 50,000 transformants per pg DNA(147). These high frequencies have been found to be dependent on the batch of the cell wall digesting enzyme used and the investigator indicating a considerable variability in both the preparations of enzymes and the protocols used. The three types of integrative transformation events previously observed in 9. 999evisiae were also described with N. 999999. Transformants showing unlinked insertions(type 115 Fig. 2) were the most common irrespective of the selective markers used(145,146,148-51). The complementing gene, with or without accompanying bacterial sequences, integrated at a site in the chromosome unlinked to the defective chromosomal allele of the selection marker. Gene replacements(type III) and linked insertions(type I) were also be observed, but much less frequently. Attempts to direct integration at the homologous locus by restriction digestion within the complementing fungal DNA portion of the transforming vector has given mixed results. Marzluf and co- workers(149,151,152) have most completely analyzed this question in N. 999999 with several different selection markers and vectors. With the 9992+ gene as the marker inserted separately into three different pBR322 based vectors, pSD3, pVK57, and pVK88, that differed only in the 46 size of the n. 999999 DNA insert, the type II unlinked transformants were predominant. When pSD3 was linearized at any one of several restriction sites within the fungal marker gene of the vector, no increase in the frequency of replacement(type III) or linked insertion(type I) was seen as compared to the circular form of pSD3(151). In a seperate study, vector pJP12, in which the 99+ gene of the vector was disrupted with the 99:2+ gene, the feasibility of gene disruption in N. 9raSS9 was tested(149). Ten of 117 transformants studied showed a precise gene disruption, indicating that this process occurred, although at a low level. In contrast, when the vector pNC2, containing the 999-1+ gene (a trifunctional polypeptide encoding three enzyme activities in the biosynthesis of tryptophan) was used for transformation, the type of integration event was found to be strictly dependent on the strain of N. 999ssa used(152). n. 999999 999:1, 191 gave a high frequency of type I events, whether the vector was circular or linearized within the fungal marker gene of the vector, while linearization in the pBR322 part of the vector I reduced the linked events. In contrast, 9. C99ss9 999:1, 99:2, 999:2, 191 gave predominantly unlinked transformants irrespective of whether the vector was circular or linear. A different report found that varying the size of the homologous region in the vector(the 99 gene in N. 999999 inserts ranging from 2.7 kb to 9.1 kb) influenced the frequency of homologous integration. This frequency increased with the size of the insert(153). 47 In summary, it appears that the type of integration events can be influenced by a number of factors, structure of the transforming plasmid, selection marker and strain used for transformation and the size of the insert. While in both yeast and 3. 999999 all three types of integrative transformation events have been observed, there are extreme differences in the types of events which predominate. In 9. 9999119199, linked insertions and directed gene disruptions are the rule, due to the high level of homologous recombination between incoming vectors and chromosomal sequences(106). In contrast, no broad generalizations can be made concerning the type of integration events that are permissible in H- C99S9a with respect to sequence requirements. The mechanism of integration appears to be different considering the high frequency of unlinked transformants seen in B- C9a559 transformation experiments and the types of events which do occur need to be determined empirically. Attempts to recover vectors integrated into the chromosome of n. 999999 transformants, by E. 9911 transformation, have generally been unsuccessful(150,154) barring a few exceptions(see below). In one case in which the recovery of a vector by E. 9911 transformation was reported was later found to be due to contamination of the transformant DNA with an E. 9911 stock of the transforming vector(155,156). To achieve complementation of an appropriate 3. 999999 auxotroph transformed with a genomic library a more cumbersome procedure termed sib selection 48 was developed(147). In this procedure H- 999999 genomic library in a plasmid vector is divided into smaller pools and each pool of plasmids is then used to transform the appropriate auxotroph to the desired prototrophy. Whichever pool gives transformants is divided into smaller pools and the procedure is repeated until the pools contain individual plasmids; at this point the plasmid containing the gene complementing the mutation can be identified. This procedure avoids the requirement to recover the actual clone from the individual transformant, which can often be accomplished only by constructing a lambda library of a positive transformant and screening for foreign vector DNA inserts, which can be more time consuming than sib selection. Both structural and regulatory genes have been isolated using this sib selection procedure(157-159). To make the process of sib selection easier, a cosmid has been constructed to reduce the rounds of selection and transformation required to identify a specific gene. The cosmid pJBB, containing pBR322 and a 999 site(160), was modified to include the benomyl resistant fl tubulin gene of E. 999999(see above) to result in vector pSV50(161; Fig. 3). This cosmid has been used to clone many genes by complementation of structural mutants(161) and has also been used to isolate the A and a mating type idiomorphs from N. 999999(162). ‘It has also been found that cotransformation can be very efficient(163). In this technique, an unselected plasmid along with a seperate selected marker on a different plasmid are both transformed 49 into the fungus, and in many cases(up to 43% of the transformants) both plasmids can be introduced into the same transformant. This should be useful in conjunction with sib selection when the library of interest does not have an appropriate selectable marker for the H999999999 strain being transformed. The only generally applicable method used for the recovery of vectors from integrated transformants has been digestion of the total DNA of the fungal transformant DNA by an appropriate restriction enzyme, followed by ligation and transformation of E. 9911. As vectors often are inserted randomly into the fungal genome, this always results in the recovery of adjacent fungal sequences also and does not constitute a generalized shuttle vector - system(164). Even this direct recovery of transforming vectors by the restriction digestion-religation method was difficult with u. C99ss9 until recently, because transforming DNA was shown to be methylated at cytosine positions to a high degree in H- 999999(165). Consequently, when the transformant fungal DNA was reintroduced into commonly used 9. 9911 cloning strains such as H8101 and LE392, which contain the methylcytosine restriction system, it is immediately degraded. This problem has been overcome using 9. 9911 strains which are lacking methylcytosine restriction systems, defined by the 999A and 9998 genes encoding endonucleases which cleave only when the C residue in the recognition site is methylated(166). 50 3. Studies on the development of a shuttle vector sytem. Shuttle vectors developed for DNA transformation of 9. 9999919199 were used as the model for developing shuttle vectors for filamentous fungi. It was assumed that modifications of the procedures used to create efficient yeast shuttle vectors could result in similar success with other fungi. The primary focus at the time was on isolation of replicons suitable for the fungus either by isolating 999 elements or by the use of endogenous plasmid sequences as the basis for a replicative vector. A99 elements, as discussed above, are those genomic sequences that allow autonomous replication of yeast integrating vectors, such as YIpS in 9. 9999919199. Putative 999 elements from n. C99ss9 have been isolated in 9. 9999919199 using this approach. However, these heterologous 999 elements were found to be ineffective in promoting autonomous replication of vectors when transformed into n. 999999(145). This led to attempts to isolate 999 elements by transforming H- 999999 with integrating vectors containing libraries of 3. 999999 genomic DNA in an attempt to duplicate the results obtained with yeast. Paietta and Marzluf(152) developed several 9. 999999 999 vectors containing n. 999999 genomic DNA fragments cloned into pSD3(described above). These 999 insertions increased the transformation frequency 51 approximately 10 fold in comparison to vectors without the putative 999. However, these vectors were also found to integrate into the chromosomal DNA of the transformants, suggesting that the putative autonomous replicating vectors might be present only in a very small population of the transformants. Hence, the same investigators then used an alternate approach in which they tried to rescue autonomous vectors by using DNA isolated from pooled colonies of the transformants on a plate to transform E. 9911 followed by isolation of the plasmid from ampicillin resistant E. 9911 transformants. Using this approach, H- 999999 was transformed with pSD3 and grown in liquid culture for 1,2,3, or 4 days, after which DNA was prepared from the total mycelial mass isolated from each of the four cultures. It was found that the transforming vector could be isolated in a time dependent manner from these cultures, with the isolation being most difficult on the 4th day. This transient isolation of transforming vector was thought by the investigators to represent a stage in the process of transformation when the original vector was still circular, prior to the integration of the vector into the chromosome. Alternatively, it was considered that the transient isolation of the transforming vector was due to the possibility that these vectors recovered in E. 9911 were from abortive fungal transformants in the pooled population in which the plasmid vector was being diluted out. It was not determined in these experiments whether or not the vector recovered had actually replicated in the fungus.. In 52 a further attempt to isolate 9999999999 999 elements, E- 999999 mycelial spheroplasts were transformed with a genomic library of H- 999999 sequences in the transforming vector pFBl4, containing the 99:2+ gene in pBR322(130). This resulted in the isolation of genomic fragments by sib selection(see above) which could transform 9. 999999 at a higher frequency, yet were still found to be integrated into the chromosome(130). Some wild type strains of n. c99ssa and Ne99oS9o99 1999999919 have distinct types of relatively small(3-5 kb) endogenous circular mitochondrial plasmids(167) that encode monomer length RNA transcripts(168). These plasmids were frequently lost during cultivation in the laboratory with no apparent effect on growth of the fungus, and thus appear to have no essential function, except that some of these plasmids are implicated in the senescence of strains which contain them(167). These plasmids have been shown to have no homology to mitochondrial DNA. Further analyses of one of these plasmids showed that this plasmid closely resembles Group I mitochondrial introns, which are capable of self splicing and often are able to insert and be deleted from mitochondrial genes at precise locations. The mitochondrial plasmids of E. 999999 are structurally similar to these introns and may represent either a progenitor to these introns or spliced introns which have aquired the ability to replicate autonomously(168). One of the small mitochondrial plasmids, the Labelle plasmid of n. 1999999919, has been used as the basis for a 53 E. 999999 shuttle vector designated pALS-1(169). This vector, when transformed into E. 999999, was shown to exist as a circular extrachromosomal element and was visible in Southern blots of total undigested DNA from transformants as an extrachromosomally maintained element. The vector was recovered intact from the DNA of some of the transformants, although a deleted version, pALS—z, which lost all fungal plasmid sequences, was also occasionally recovered from some of the transformants. Subsequent analysis of pALS-2 indicated that it contained rearranged pBR325 sequences and only the 99:;+ fungal selection marker(170). Another 9. 99a539 vector, lambda C-10 was used to transform 9. 999999 to 99+(146). The results showed that the maintenance of the vector sequences in the transformants was meiotically unstable, which was consistent with autonomous maintenance. However, Southern blot analysis indicated that the sequences were maintained in a high molecular weight form, suggesting chromosomal integration. A similar result using a different 99 vector, pJR2, was found by the same investigators(171). These contradictory results may be explained by the RIP effect(see below). Transformation of H- 999999 with vector pMK2, a 15.2 kb plasmid containing the E. 999999 LaBelle mitochondrial plasmid, pBR322, and the 99:2+ gene for selection, resulted in transformants in which plasmids could consistently be recovered by E. 9911 transformation(172). In an in depth 54 analysis of one of these transformants, two types of altered plasmids, pMK2-3(8.4 kb) and pMK2-5(10.5 kb) were recovered; neither could retransform 99:2- 9. 999999 to prototrophy. pMK2-3 was shown to have acquired sequences of the E. 999999 chromosome. Plasmid pMK2-5 was found to be a dimer consisting mainly of pBR322 sequences. No homology to the 99:2+ selection marker or to the LaBelle plasmid was found although homology to the mitochondrial genome was demonstrated in pMKZ-S. Both these vector were shown to have replicated in 9. 9ra959. This was demonstrated by a 99m methylation assay based on the following rationale: If the vector had replicated in E- 999999 then it will have lost the characteristic methylation by the endogenous 999 methylase at the N6 position of adenine at the sequence GATC found in 999* strains of E. 9911 in which the transforming plasmid was propagated. Therefore, vector DNA that had replicated in H- 999999 would be susceptible to digestion by M991, which is active on DNA only in the absence of adenine methylation. Southern blots of 9991 digests of pMKZ transformants containing pMK2-3 and pMK2-5 indicated that vector sequences were digested, thus showing that they had been replicated in E. 999999. Southern blot analysis of total transformant DNA from which pMK2-3 and pMK2-5 were recovered indicated only chromosomal integration(172). These results, along with the fact that pMK2-3 contained chromosomal 9. 999999 sequences not originally present on pMK2, indicated that the recovery of these plasmids was most likely due to 55 alternate excision products from the chromosome, and not to the presence of automonous plasmids. Genetic analysis of pMK2-3 and pMKZ-S transformants also indicated meiotic instability, which can be attributed to RIP activity and not to true autonomous maintenance of vectors. A similar type of recovery of altered plasmids was observed in a seperate study(173) in which a cosmid library of 9. 999999 was used to complement a 9. 99aSS9 191- mutant. Two transformants which complemented this deficiency were obtained and plasmids of various sizes were recovered from both these transformants. Interestingly, none of the recovered plasmids contained the desired 191i gene. Genetic analyses indicated that these transformants showed some meiotic instability, complete mitotic stability, and were linked to the 91:9 locus, which is closely linked to the wild type 191 allele. The latter two pieces of data indicate integrative transformation, while the meiotic data could be explained by the RIP effect. Southern analyses indicated that the transforming cosmid was maintained as an extrachromosomal element, although these data were not shown. The structure of the recovered plasmids was not analyzed and it was not ruled out that these were due to excision from the chromosome. Detailed analysis of other fungal transformation systems(see sections below) with either integrative vectors or putative autonomous vectors have often shown either mitotic or meiotic instability of the vectors which, based on the yeast model, would suggest autonomous maintenance of ‘ 56 vectors. Two explanations for this instability have been made. First, head to tail integration of two or more copies of the transformation vector into the chromosome can generally be accompanied by genetic instability and occasional recovery of the intact vector from transformants. Southern analysis in most of these cases showed only chromosomal integration. Thus, these transformation events are best explained as recombination between adjacent integrated vectors resulting in the excision of intact vectors or of altered vectors containing junction fragments of adjacent chromosomal DNA. Secondly, it has been found that there is an endogenous 99 9999 ‘ methylation activity in 9. 999999 termed RIP(rearrangement induced premeiotically, or repeat induced point mutation; 174-177). In 9. ssa, there are few duplicated sequences aside from DNA encoding ribosomal RNA. The transformation of 9. 999999 generally results in the introduction of a second allele of a single copy gene in a haploid chromosome(174). It was found that duplicated sequences in one haploid were not stable through meiosis. This phenomenon was termed RIP and this activity is specifically active prior to karyogamy(175) in the perithecium(meiotic structure). The RIP activity identifies duplicated sequences, which are then subject to heavy cytosine methylation. Subsequent deamination of methylcytosines results in a high level of G-C to A-T mutations in the targeted duplicated sequences(176). This heavy mutation frequency often results in non-functional copies of genes 57 which have been duplicated or triplicated, and this non- functionality of these genes mimics instability of the marker through meiosis(177). The exact natural function of the RIP phenomenon in the cell is unknown. However, RIP can be used in to create mutations by transformation in E- 999999. In this approach, the gene to be inactivated would be transformed into 9. 999999 which results in a transformant with duplicate copies of the gene in question. When meitoic spores are isolated from such transformants, some will have both the transformed gene and endogenous gene irreversibly inactivated due to RIP activity, creating a specific mutation(174). It also appears that methylation of duplicated transformed sequences occurs in other fungi, resulting in similar meiotic instability of some integrated sequences(see below). Thus true evidence for autonomous replication of vectors, or rearranged derivatives thereof appears scarce to date, although some cases of true autonomous replication in fungi have been reported very recently(see below). More detailed specifics of such cases are discussed in the following sections. 4. Lgngrgillng nisglgng Many Species of Aansrgillns. such as A. nigsr. A. 9999991, and 9. 999999, are of importance due to their ability to produce large quantities of industrially important extracellular enzymes such as amylases and various secondary metabolites such as aflatoxins(178). A. 58 91991999 is the most widely studied from both the genetic and molecular standpoint and has offered considerable advances in our understanding of the regulation of gene expression(179) and of developmental processes in filamentous fungi(180). A variety of vectors with multiple applications have now been developed for 99999911199 species, as these organisms are well suited, as an alternative to 9. v's'ae, as hosts for the high level of expression of foreign eukaryotic proteins(181- 185). Also, transformation systems developed for Aspergillus species, in combination with those described above for 9. ssa, give a comprehensive picture of the types of molecular genetic manipulations and analyses that can be expected to be made in other filamentous fungi which are important in agricultural and industrial applications. A. Integrative Transformation. As with E- 999999, the initial transformation protocols developed for A. 91du1ans were either conidial or mycelial spheroplast transformations using vectors that contained 9. 91991999 genes which complemented appropriate auxotrophic mutations(see Table 3 for a survey of selection markers used). Ballance et al. used the vector pFBG, which contained the 9999 gene of n. 999999 to complement a 9x99 mutant of A. 91991999(186). Additional 9. 999999 regulatory(187) and structural(188) genes have 59 um: Selection markers and transformation vectors used for Aspergillus nidulans‘. Selection Marker Vector Reference 9991 (orotidine-5'-phosphate pFBG 186 decarboxylase ' ' pF86, pDJBl, pDJBZ 132 ' ' pDJBl, pRKBl, pRK82 217 ' ' pAIpGH1,4 196 ' ' pDJBl, pDJ812.1 194 9999 (acetamidase) p3SRZ 188 ' p3SRZ, p38R2rr, pBSRZmo 187 9999 (ornithine transcarbamylase) pSa143 337 ' ' pHAZ, pMAZ-C 218 ' ' pMTZOl 214 " ' pm, le 215 ' ' leSS 133 9999 (tryptophan biosynthesis complex) pnrzoi 1:0 ' ' ' pKBYZ 235 911r (ATP synthese, subunit 9) puwz, pMWIl 228 ' ' pr3deltaE 215 999: (catabolic dehyroguinase) p831 338 999 (hygromycin phosphotransferasec) pAN7-1 210 ' ' pDHZS 227 999 (pro1ine catabolism gene cluster) pAN222 339 ' (riboflavin') 131.01 222 993‘ pAIpG)“ 19s 9999 (isocitrate lyase) pAcu22 223 95-91" ' pHIONA-l 324 9192 (nitrate reductase) pAN301 325 9199' pS'I‘A14 237 951 (pyruvate kinase) pGW402,403 340 99 (adenosine triphosphate sulfurylase) pFB7S 341 aSe1ection marker is from homologous organism unless noted. from N. crassa cfrom Escherichia coli fused to A. nidulans transcription signals from Coprinus cinereus c ispcrgillus orysae 60 been found to complement corresponding 9. 9idu1ans mutants. In one case the acetyl-coenzyme A synthetase gene from each fungus was cloned and found to respond correctly to regulatory signals in the heterologous fungus(188). Tilburn et al.(189) used the plasmid p38R2(190), which contains the 9999 gene encoding acetamidase, which allows a dominant selection for growth using acetamide as the sole nitrogen source. Vector pBSRz contains a genomic fragment of A. 9i991ans with the 9999 gene cloned into pBR322 and has become important as a dominant selection marker in a variety of fungal transformation systems(Table 6, Fig. 3) for which no auxotrophic markers are available. The initial reports of A. 9idu1a9s transformation showed integration into the chromosome being the norm. Generally, transformation frequencies varied from several to several hundred transformants per pg DNA(132,191). The types of integration events seen were linked insertion and unlinked insertion, with no gene replacements being observed. As seen with 9. ss , both stable and abortive transformants can be observed, with up to 96% abortive in some cases(189). Vectors with different selection markers yielded different proportions of the three classes of transformants. For example, selection for the 9999 gene resulted in predominantly direct replacement events, leading to the excision of both the non-functional gene and pBR322 sequences(192,193) while the above reports with p38R2 gave transformants which were unlinked to the 9999 gene. 61 Homology of the transforming sequences to the chromosome does not appear to be a general requirement for a successful integrative transformation in 9. ni9u1a9s. Vectors with increased homology to the chromosome(for instance those containing ribosomal repeat DNA) did not show increased transformation frequencies(189) and integrated at non homologous sites in the chromosome, though infrequently. Conversely, strains in which the chromosomal locus for the selection marker has been completely deleted did not show an appreciable decrease in transformation frequency(194). None the less, directed integration of transforming sequences has been achieved in many cases in A. 9i9ul99s systems and this has lead to a more detailed analysis of several chromosomal loci through precise gene disruptions at specific sites in the chromosome(195-198), similar to the methodology now standard for 9. 999evisiae(199). In one case, a functional analysis of the 99991 gene cluster was examined(200). The 99991 cluster contains several genes which are highly expressed during sporulation in 9. 91991999. To determine if any of the genes in the 99991 cluster were essential for conidiation, a vector, pDC6, which contained the 9999 gene of A. 91991999(encoding ornithine carbamoyl transferase) fused between DNA fragments from the extreme ends of the SpoCl locus was used to transform 9. 91991999 to arginine prototrophy. Transformants in which a gene replacement had occurred, (i.e. elimination of the wild type SpoCl locus and 62 replacing it with the 9999 gene), were selected. It was found that a resulting 99991- deletion strain did not differ in growth or in the production of mitotic or meiotic reproductive structures compared to the wild type indicating that the 99991 deletion had no demonstrable function in sporulation or conidiation in 9. 91991999. Attempts to utilize the 9. 91991999 transformation system for the analysis of gene regulation, in a manner similar to that being currently done with 9. 9999919199(201), are being made. Several vectors which allow transcriptional fusions between specific genes of interest and the 1999 gene of 9. 9911 have been constructed(Tab1e 6, Fig. 3). By attaching the promoter region of the 9999 gene to the coding region of the 1999 gene and using a vector with this fusion to transform 9. 91991999, it was found that p-galactosidase activity could be easily assayed using either an x-gal plate assay or a fi-galactosidase enzyme assay(202). To broaden the analysis of gene expression to other promoters, a series of vectors (pAN923-4lB, pAN923-4ZB, and pAN923-43B) which allow in phase fusions of any gene to the 1999 gene were constructed(203,204). In addition to the 1999 gene, these vectors contained the 9999 gene for selection and pBR329(204). The results showed that approximately 40% of the A. 91991999 transformants contained single integrations of these vectors at the homologous 9999 site in the chromosome. Thus by screening for these homologous insertions, one could eliminate the position effect 63 variable which would be encountered in any promoter analysis if only random unlinked insertion events in the chromosome were found. Two promoter analyses using such 1999 fusions have been made. A deletion analysis of the 9999 promoter showed that seperate DNA elements were present in the upstream region controlling the level of expression and the correct initiation of transcription(ZOS). By fusing the 9999 promoter to the 1999 gene, and replacing the resident amdS gene by a two step gene replacement with this fusion, it was found that the level of p-galactosidase production by the transformant could be regulated similarly to acetamidase' expression by the wild type(206). These initial studies indicated the usefulness of the above approach to a detailed analysis of the 9. 91991999 promoters. One interesting and useful observation made with the 9. 91991999 transformation system is the fact that multiple integration events often occur in the process of the integration of the transforming vectors into the chromosome. This results in many tandemly repeated inserts at one chromosomal locus(191). This phenomenon appears to be strain specific, and no gene specifically responsible for this type of integration event has yet been identified(lQl). These studies further showed that multiple copies of the inserted gene can directly be translated into increased enzymatic activity of that gene in a dose dependent manner(191,207). This phenomenon has been taken advantage of in the examination of the genetic . 64 regulation of enzymes involved in nitrogen and carbon catabolite repression in A. 9199199§(208,209). It has been found that the introduction of multiple copies of a DNA binding element into A. 9idu1a9s can cause titration of the affected DNA binding protein, and that this effect can be reversed by the addition of multiple copies of the gene encoding the DNA binding protein. As 9. 91991999 is becoming increasingly important in the production of heterologous proteins as an alternative to 9. 9999919199, this phenomenon may allow a means to direct a high level of expression of a particular gene in a stably inherited manner by selecting recombinants with multiple copies of a desired gene. Tandem integration of transforming vectors is a type of integration event seen in many filamentous fungal systems(see below). Another common phenomenon of fungal transformation, cotransformation, was first demonstrated with 99999911199 species. When an 9. 91991995 auxotroph was transformed by a vector containing the appropriate complementing gene, it was found that a second, unselected, plasmid could also often be transfromed along with the vector that is being selected for. Southern analysis, or a second screening for the complementation of the previously unselected marker on the second plasmid often demonstrated that the unselected plasmid as well as the original vector could be integrated into the chromosome. Cotransformation was detected up to 50% of the transformants examined and was found to occur independent of the selection marker and 65 strain(132,210,211). This has been found to be a useful method to insert a gene for which there is no corresponding auxotroph, or in the cloning of genes from heterologous organsims for which there is no direct selection(212). Mitotic and meiotic analyses of 9. 91991999 integrative transformants by different investigators showed that transforming sequences are integrated into the chromosome and are stably inherited by spore progeny(186,187). However, transformants in which the vectors have integrated in multiple, tandem repeats do exhibit both mitotic and meiotic instability. The reason for this has not been determined. It may be due to a combination of effects, such as an RIP like event(see above) similar to that seen in E. 999999 or the possible inherent instability of the repeats. A further observation which has been made in transformants containing vectors which have integrated in either tandem repeats or multiple copies is the recovery of the intact vector by E. 9911 transformation. The results suggested that this is due to the precise excision from the chromosome of an intact vector and not due to autonomous replication of the vector in A. 91991999(191,194,213,214). 8. studies on the development of a shuttle vector system. Development of shuttle vectors for 99999911199 focused on the isolation of 999 elements as no endogenous 66 plasmids have been reported for A. 91991999. Furthermore, no enhancement of transformation frequency was observed using plasmids containing ribosomal repeats or a mitochondrial origin of replication(189). Ballance and Turner(132) used the vector pFBG, containing the 9994 gene of E. 999999 which complements both 9999- mutants of A. 91991999 and 9999 strains of 9. 9999919199, to attempt this. An A. 91991999 genomic library in pFBG was used to transform 9. 99999151ae to uridine prototrophy. Plasmids which transformed 9. 9e9ev15199 at a high frequency were isolated. It was found that one of these plasmids, pFBG- An2, could transform A. 91991999 at 5000 transformants per pg, compared to 50-150 transformants per pg observed with pFBG alone, and this was considered as a potential 999 containing vector. This A. 91991999 sequence with 999-like activity was designated 9991(99999911199 91991999 sequence 1). 9991 was cloned into a different vector, pDJBl, containing the 9995 gene in pBR325, to create pDJBZ. Analysis of individual transformants showed that pDJBZ had integrated into the chromosome. Southern blot of A. 91991999 genomic DNA using the 9991 sequence as a probe showed that many dispersed regions of the chromosome had homology to 9991, possibly accounting for its ability to increase the transformation frequency by homologous recombination. Further analysis of pDJBZ integration, however, found that integration at the homologous 9991 locus in the chromosome was only rarely observed. The authors also suggested that the increased transformation 67 frequency is due to the similarity of 9991 to yeast centromeres(9991 has 81% A-T, similar to element II of a yeast centromere; 119) and that the 9991 locus is closely linked to a centromere(215), but no model for the action of 9991 was postulated. Further attempts to develop an Aspergillus shuttle vector utilized mitochrondrial DNA from a ragged(999-) mutant of A9999911199 99999199991. A specific region of the wild type mitochondrial genome of A. 999991999991 undergoes an amplification, similar to the phenomenon observed in petite mutants of 9. 999991s1ae, resulting in the accumulation of two types of small circular molecules containing DNA from two different regions of the mitochondrial genome. This results in the ragged phenotype. Sequence analysis of two circular DNAs, designated 9991 and 9999 DNA, indicated that both had homology to 9. 9999919199 as well as to human mitochondrial origins of replication(216). The two circular DNAs were cloned into YIp5(see above; 105) creating pRKBl(9991) and pRKBZ(999§). Both the latter vectors were shown to transform 9. 9999919199 at a high frequency indicating that they contain 999-like elements. These 999-like sequences were recloned into pDJBl(an integrative A. 91991999 vector; described above) and transformed into A. 91991999. Southern analysis showed that the vector integrated into the chromosome at different sites and both single and multiple insertions were observed. Both vectors could be rescued in E. 9911. 68 The mitotic stability of the these transformants was reduced and this suggested the presence of tandemly integrated repeats of the vector in these transformants was the most likely cause of this recovery in 9. 9911(217) . The possibility that a centromere sequence of 9. 9999919199 would function in A. 91991999, leading to the establishment of mini-chromosomes as in yeast, has also been examined(218). The centromere of yeast chromosome 11(99911) was inserted into the vector pMAz, which contained the 9999 gene of A. 91991999 in pBR329 and this along with pMAZ was used to transform A. 91991999. The results showed that a CEN sequence inserted into pMAZ transformed at similar frequencies to pMAZ and both transformed by integration into the chromosome. No chromosomal instability was observed in such transformants, in contrast to the instability in 9. 9999919199(ll9) which is caused by the presence of two centromeres on one chromosome. These data suggested that the 9. 9999919199 centromere did not function in A. nidnlms. One of the important applications of a transformation system is the ability to use it to clone genes of interest. Genes from A. 91991999 have been isolated by complementation of both E. 9911(219) and 9. 9999919199(220) auxotrOphic mutations. However, an important limitation of this approach is the fact that many of the genes one wishes to clone may not function in these 69 organisms due to the presence of introns or due to other functional limitations. Another major limitation of most transformation systems of filamentous fungi has been the fact that fungal transforming vectors generally integrate into the chromosome of the recipient and there is no easy and efficient means of recovering complementing genes from the chromosome of transformants. Despite these barriers, the A. 91991999 transformation system has been used extensively to clone genes from both A. 91991999(see below) and from other filamentous fungi(187,212). Several methods have been used to recover vectors from A. 91991999 transformants. One of these is by marker rescue(192,212,221-223). Oakley and co-workers have used this technique to clone the 919992 and 9999 genes(222,223). A genomic library of A. 91991999 was constructed in pBR329 and used to transform riboflavin or 9999- auxotrophs to prototrophy. In this approach, to recover the complementing gene in the vector which has integrated, a restriction enzyme which does not cut within the vector or the complementing sequence is needed. Since the restriction sites in the complementing gene are not known in most instances, several enzymes are usually selected to maximize the possibility of isolating the intact complementing gene. Total DNA from a transformant is partially digested with each of the several restriction enzymes separately, the genomic DNA from this tranformant is then self-ligated and used to transform 9. 9911. Individual plasmids recovered from each pool of B. 9911 7O transformants are isolated and used to transform the same A. 91991999 919992- strain. The plasmid pool that complements the particular auxotroph contains a functionally intact complementing gene. This pool could then be subdivided and an approach similar to the sib selection procedure for 9. 999859 could then be used to isolate a single clone containing the intact gene(222). An alternative to the above approach has been to construct a cosmid library of A. 91991999 and use this to transform a specific auxotroph to prototrophy. A cosmid designated pKBYz which contains a bacteriophage lambda 999 site, the 9999+ gene of A. 91991999 for selection of ‘ fungal transformants, and ampicillin and chloramphenicol resistance genes for selection in 9. 9911 and a bacterial 991 was constructed by Timberlake and co-workers(224,225). The cosmid library was then used to transform A. 91991999 to Trp+. Transformant DNA was then be packaged into lambda and transduced into 9. 9911. Individual cosmids recovered in 9. 9911 were used to re-transform A. 91991999 to confirm that it carries the desired gene. Occasionally, direct recovery of a vector in 9. 9911 can be accomplished directly from total transformant DNA. In one case, a gene library in the vector pILJlG(pUC8 with the 9999 gene of A. 91991999) was used to transform a conidiation deficient strain(991A-) of A. 91dul9ns to Arg+. DNA from one transformant which was conidiation proficient was used to transform 9. 9911 directly and one colony was recovered which contained a genomic fragment of A. 91991999 which 71 complemented the 991A deficiency(226). One major application of the A. 91991999 vectors and selection markers is that they have been used to develop transformation systems for other fungi for which either auxotrophs or homologous selection markers have not been isolated(Table 6). As with 9. 999999, several markers have found wide use in the development of fungal transformation systems. One of the most widely used A. 91991999 vectors for transforming other fungi is p3SR2(Tab1e 6; Fig. 3), which contains the 9999(acetamidase) gene in pBR322. The 9999 gene allows growth of the fungus on acetamide as a sole nitrogen source. Transforming a fungus with pBSRz and selecting for growth on acetamide has, however, proven to be effective since many filamentous fungi are either very poor utilizers of acetamide as a nitrogen source or do not utilize it at all. Antibiotic markers, though more effective as general purpose markers, were not used in fungal transformations systems initially. This is due to the fact that fungi are often resistant to common antibiotics such as tetracycline, ampicillin, and kanamycin. Furthermore, the most well characterized antibiotic resistance markers are of bacterial origin and would probably not function in fungi unless placed downstream of a suitable fungal promoter. Once the characterization of some highly expressed fungal genes was done, it became possible to create fusions between fungal promoters and bacterial genes. This was initially 72 accomplished by two different groups using the 9. 9911 hygromycin B phosphotransferase gene(999). To construct vector pAN7-l, the 999 gene was placed between the promoter signal from the glyceraldehyde-3-phosphate dehydrogenase gene(999) and termination signals from the 9999 gene and inserted into pUC8(Fig. 3; 210). A similar construction, pDH25(Fig. 3; 227), had the 999 gene fused to both the transcription start and termination signals from the 9999 gene. Both pAN7-1 and pDH25 have been used to transform both A. 919u1ans as well as a number of other fungi(Table 6) to hygromycin resistance. In addition, oligomycin resistance(228) has been used as a selection marker. Oligomycin inhibits the activity of mitochondrial ATP synthase. pMWZ, which contains the oligomycin resistant form of the mitochondrial ATP synthase subunit 9 gene in pDJBl(see above). Transformation with pMWZ resulted in insertion events similar to those noted with other markers. c. Transformation of other 99999911199 species In the genus A9999911199, A. 91999 and A. 999999 are the most industrially important species as many useful organic acids, industrially important enzymes, and secondary metabolites are produced in abundance by these organisms(178). As with many industrially important fungi to date, however, the genetics of these two fungi is not highly developed, although many auxotrophs are available 73 and transformation systems have been described(Table 4). i-WM Once a transformation system for A. 91991999 was developed it was only logical that this would be extended to A. 91999. Transformation of A. 91999 was first demonstrated using heterologous genes from A. 91991999. Transformation with vector p3SR2(9999 gene; see above and Fig. 3), resulted in integrative transformants(211). The 9999 gene of A. 91991999 was also used(229). Vectors pDGl, constructed from the cosmid pHC79 into which the 9999 gene was cloned, and p063, a derivative of pDGl to which the L99r gene of TnS was added, both complemented the corresponding A. 91999 9999 mutant. Transformation was due to integration into the chromosome and unlinked insertion events into a variety of chromosomal locations were observed in all cases as there was little sequence homology between the selection markers of A. 91991999 and A. 91999. On the other hand, integration into the homologous site was observed when the homologous 9999 gene was used mutants of A. 91999, integration into the homologous site was observed(230,231). The 9999 cloned in the 9. 9911 vector pUN121(vector pGW613) was found to transform A. 91999 only at the homologous locus, either by gene replacement or by linked insertion(230). A corresponding 10 fold increase in the transformation frequency over transformation with the heterologous 74 1131.36 Selection markers and transformation vectors used for other Aspergillus species‘. L Selection larker vector Reference Aspergillus niger 9999: pDG3 229 amgfi pBSR2 211 EZEE pGW613 230 ' pAB4-l 231 211’ pMWBO 234 ni5n pSTAlO 232 Blane pSTAl4 237 nxrsc pAOd-Z 236 99 (adenosine triphosphate sulfurylase) pFB75 341 Dan? pBT6 233 Aspergillus orysae nxrfid pAB4-l 235 nxrfib pAO4-2 236 3:95 pILJlS 238 ' 7 181 399: 93882 ' Dian pSTA14 237 Aspergillus ficuum kph” pAN7-l 242 ms” p3SR2 - Aspergillus awasori d. 9999b pANGl 239 2:23 pUC4AP-9999 24o Aspergillus giganteus huh? pAN7-l 243 aSelection marker is from homologous organism unless noted. bfrom Aspergillus nidulans cfrom Aspergillus oryzae dfrom Aspergillus niger 9from Escherichia coli fused to A. nidulans transcription signals 9from Coprinus cinereus 75 markers(9999 and 9999) was seen, indicating that homology to the chromosome was beneficial in A. 91999. Similar results were obtained using the homologous 9999 gene cloned into pUClQ(vector pAB4-l) to transform A. 91999. Sixty percent of the transformation events were by gene replacement, while the rest were by linked insertion. In contrast, transformation with the vector pSTAlO, with the A. 91999 9199(nitrate reductase) gene cloned into pUClB showed that no gene replacement was observed. The data did suggest many linked insertion events(232). A ten fold increase in transformation frequency, (from loo-1000 transformants per pg vector DNA) was obtained when pSTAlO, linearized within the 9199 gene, was used. Integration was shown to be both by gene replacements and linked insertions in this case(233). In addition, pSTAlO was used to cotransform several unselected genes into A. 91999 and test for their expression. These include pANS- 418(containing the 1991 gene[fi-galactosidase] fused to the 999 promote of A. 91991999: Fig. 3), pNOM102(the 919A gene[p-glucuronidase] fused to the same 999 promoter; Fig. 3) and plasmid pBT6(with the tubulin-2 gene of 9. 999999; Fig. 3), which were all found to be expressed in A. 91999. An homologous oligomycin resistance transformation system has been developed(234) for A. 91999. Vector pMWBO, containing the oligomycin resistant form of mitochondrial synthase subunit 9 in pUprr(pUCl9 and the 9. 999999 9999 gene) transformed by integrating into the chromosome. The vector DNA was shown to be integrated at the homologous 76 locus in only 15% of the transformants. As with 9. c99559 and A. 91991999, all three of the types of integration events can be observed with integrative vectors. Similar to those fungi, the type of integration observed during transformation with homologous genes in A. 91999 appears to vary depending on the marker used. ii-Ammimgenm A. 999999 auxotrophs have been transformed with the 9999 gene of A. 91999(235) and A. 999999(236). With the homologous 9999 gene(vector pOA4-2, with the 9996 cloned into pUCl9), 75% of the transformation events were at the homologous locus(type II and III events: 236). About 90% of these integration were by gene replacement. The 9999 gene of A. 91999(in pAB4-1, see above) was found to transform A, 999999. Transformation was shown to be by integration at various sites in the chromosome, and in some transformants multiple integrations were seen(235). Using vector pSTA14, that contained the A. 999999 9199 gene in pUC18 for transformation, a high frequency of gene replacements and linked insertions were found(4l% and 23%, respectively). Multiple tandem integration events at the homologous locus were seen occasionally. It was also found that this A. 999999 9199 marker could transform appropriate 9199 mutants of A. 91991999, A. 91999, and 29919111199 99999999999 to 9199+(237). Vectors containing 77 the 9999(181,238) and 9999(181) genes of A. 91991999 could transform A. 999999. The vectors were found to integrate at different sites in the chromosome of individual transformants, and multiple insertions could be observed in some. Cotransformation of A. 999999 was demonstrated in several instances. Cotransformation experiments indicated that the 1999 gene of pAN5-4lB and the 919A gene of pNOM102(see above) could be expressed in A. 999999 when cotransformed with pOA4-2(236). iii. Other A9999911199 species Successful transformation of A. 9999991 was shown using pANGl, containing the 9999 gene of A. 91999 in pUCIOO using electroporation. Integration events were observed(239). A. 9w9mori has also been used in the expression of a heterologous protein(2440). Two vectors(pGRGl and pGRG3) in which the cDNA for bovine prochymosin B was fused behind the transcriptional signals of the glucoamylase gene(919A) of A. 91999 gave primarily intracellular expression of chymosin. Making a translational fusion of this cDNA behind the carboxyl terminus of the 919A protein of A. 9w9m091 resulted in a high level of secretion of chrymosin, which apparently was released autocatalytically from the 991A protein in the extracellular medium to result in an active, native sized enzyme. In addition, the aspartic proteinase aspergillopepsin A gene was cloned from A. 9999991 and 78 used to create gene disruptions of this locus(241). Transformation of A9999911199 919999 using both the 9999 gene(p3SR2) and hygromycin resistance marker(pAN7-1) has been shown to be by integration into the chromosome(242). When pAN7-1 was cotransformed with pAN5-4lB, subsequent expression of the 9. 9911 1999 gene was also demonstrated here. Transformation of A. 919999999 to hygromycin resistance using pAN7-1(Fig. 3) was shown(243). Although transformation was by integration, pAN7-1 derivatives could be recovered by using undigested DNA from the fungal transformants to transform 9. 9911. The majority of the plasmids recovered were identical and had a 2.5 kb deletion of pAN7-l, while a small number of the 9. 9911 transformants contained intact pAN7-1. 5. Other filamentous fungi In the last several years much progress has been made in the development of transformation systems for other filamentous fungi which have also been important in studying the genetic processes of lower eukaryotes. In many cases these transformation systems have been developed using the accumulated genetic data for the particular fungus of interest. However, rapid strides have been made in recent years in developing transformation systems for a variety of filamentous fungi utilizing selection markers that became available through the development of the 9. 999999 and A. 91991999 transformation systems. A finding 79 of considerable interest was the fact that in many instances, heterologous selection markers from 9999999999 or A9999911199 were shown to complement the appropriate auxotrophic mutation in a number of species of filamentouus fungi. It was also found that the transcription signals from A. 91991999 were often recognized as such in a number of other fungi examined. This eliminated the need for the isolation and detailed characterization of homologous structural gene to precede the development of a transformation system for some less well characterized fungi. A summary of transformation results and strategies used for filamentous fungi other than 9999999999 and A9999911199 which are important genetic models, plant pathogens, or of interest industrially is presented here. A-anmnm 9. 99999199 has a well characterized genetic system and has been exploited in recent years because of its usefulness in the study of the senescence phenomenon in fungi(244). The senescence phenomenon is particularly interesting with respect to the development of a transformation system as senescence in 9. 99999199 is associated with the excision of a plasmid from the mitochondrial genome of this organism during aging(245). The senescence plasmid is an intron of the cytochrome c oxidase gene of 9. 99999199 and appears similar to other 80 fungal plasmids which comprise a class of apparently mobile mitochondrial introns. Initial transformation protocols for this fungus utilized the senescence plasmid alone as the transformation vector(246). One major difficulty associated with such a vector, however, was the fact that the introduction of the senescence plasmids into the vegetative mycelium of a strain caused senescence of the transformant,i.e., the transformant began dying. Thus the use of senescence as the sole selection marker severely limited the usefulness of the senescence plasmid-based vector. Although initial studies using vector pSP17, which contained pBR322 and two copies of the senescence plasmid suggested that this vector replicated autonomously in 2. 99999199(247); however the Southern blot data could not rule out excision tandemly repeated integration into the chromosome followed by precise excision as discussed above for'5.,91991999. Further studies were done with two additional vectors each containing a different senescence plasmid. The 991:1 gene(which could suppress the 1991;; mutant) was cloned into a-pBR325(pBR325 with the a senescence plasmid), and 221-pBR325(pBR325 containing the p senescence plasmid) to create vectors pBasu4 and pBlesu4, respectively. It was shown that transformation with each of these vectors was exclusively by integration into the chromosome, in which the transforming phenotype was passed through mitotic and meiotic divisions in a stable Mendelian fashion(248). In addition these investigators concluded that the prescence 81 of the senescence DNA on the vector did not cause senescence the transformants. This difference in results of different investigators could be due to the different genotypic backgrounds of 2. 99999199 used. The 9995 gene(orotidylic acid pyrophosphorylase) of 2. 99999199 was cloned by complementation of a corresponding E. 9911 mutant and the resulting vector, pPAura5(the 9999 gene in pBR322) was used to transform 2. 99999199. The types of integration events observed in the transformants were varied. Genetic analyses indicated that unlinked insertion events are predominant, although linked insertion events were seen(249,250). Transformants with up to 10 tandemly linked copies of pPAuraS were found and an increase in the expression of the orotidylic acid pyrophosphorylase proportional to the number of copies of pPAuraS was observed in the transformants. Two of the transformants with multiple copies of pPAuraS were crossed to create recombinant strains with even greater specific activity of this enzyme(251). Another vector containing a suppressor tRNA(995;1; see above) cloned into pBR328 was used to transform 2. 99999199. Genetic analyses showed that most of the individual transformants were likely due to unlinked insertions, and a number of hotspots for integration in different chromosomes were found(252). Transformation of 2. 99999199 to benomyl resistance using pBT6, which contains the tubulin-2 gene of H~ 999999 has been accomplished in which primarily single site unlinked insertions into the chromosome are observed(253). A cosmid 82 library of 2. 99999199 was constructed starting from a transformant which contained an integrated vector linked to the mating type locus. A series of cosmids encompassing the chromosomal region surrounding the mating type locus(254). Transformation of 2. 99999199 with these several of the cosmid clones, which contained the 999 gene(suppressor tRNA) as a selection marker, resulted in a high proportion of linked insertion transformation events in transformants obtained from all cosmid clones used. Genetic analysis of the transformants with linked and unlinked insertion showed that the unlinked integration events were stable through meiosis while those carrying linked insertion events were unstable. Although the reason for the instability was not examined, the unstability seemed to be due to a RIP-like event. Attempts to isolate a centromere from 2. 99999199 for use in the development of a potential autonomous vector were not successful(255). However, this cosmid library has been used to clone two alleles(s and S) involved in heterokaryon incompatibility(256). A strain with a neutral incompatibility allele was used as a recipient for a cosmid library from an 3 allele in transformation. A process of sib selection was used to identify transformants which could then cause incompatibilty to an 8 strain to identify an 3 allele containing transformant. This allele was then used as a DNA probe to a cosmid library of an 8 allele strain of 2. 99999199 to clone the corresponding 8 allele. Recently, an autonomous vector for 2. 99999199 was 83 constructed(257). Telomeres from 19999999999 99999999119 were linked to a linearized 2. 99999199 integration vector, pPAura5(see above), resulting in vector pPATuraZ. Linearized pPATuraz was used to tranform 2. 99999199 and over 50 % of the ura+ transformants were found to be unstable by during non-selective culturing. This is in contrast to those transformants arising from transformation with pPATura2(lacking telomeres), which were all stable. This suggested the potential presence of an autonomous vector. Southern analysis of the unstable pPATuraz transformants showed the presence of an extrachromosomal band indicative of an autonomously maintained vector. The pPATuraZ transformants which were mitotically stable showed chromosomal integration of the vector only. One . interesting phenomenon was that the copy level of the linear plasmid was very low, one per 5-10 nuclei, indicating that the few nuclei with the plasmid are enough to sustain the positive phenotype of the transformants. It was reasoned that this type of low copy number maintenance is possible in coenocytic multinucleate fungi with a common cytoplasm, as opposed to single celled organisms, where each each cell must maintain the phenotype to survive. The basis for the low copy maintenance of transforming vectors in 2. 99999199 is not known at this time. 84 B-WW B. 9193991eea9us is a fungus of considerable interest since it is strongly dependent on sensory responses, including light intensity, gravity, and pressure, and as a result many behavioral mutants of this organism have been isolated(258). Both spheroplast(259-261) and liposome mediated fusion(262) transformation systems have been developed for 2. 9193991999999 . The transformation vectors used have been based on G418 resistance using the Tn903 399r gene as a selection marker without the addition of fungal transcription signa1s(260-262). Transformation of 2. 9193991999999 to G418 resistance using the 399r marker of TnS was not successful, unless a fungal promoter was inserted(259). In this case, random genomic fragments of B. 9193991999999 were ligated to the vector pVBBZ at an E99R1 site upstream of the promoterless 399r gene and used to tranform E. 9911. DNA from thirty four independent transformants was then used transform 2. 9193991999999 to G418 resistance. A range in transformation frequencies from 20 to 2909 tranformants per p 9 vector, indicating that some likely contained promoters functional in 2. 9193991999999. Analysis of conidially purified transformants from several of these plasmids indicated that the vectors were maintained in a stable form in the individual transformants, although chromosomal integration was not tested by Southern analysis. 85 Autonomous maintenance of transforming vectors in 2. 9193991999999 has been reported by two different groups, lending support to the idea that non-integrative transformation occurs in 2. 9193991999999. In the first report, pJLz, a vector in which a E. 91akesleea99s genomic DNA fragment capable of 999 activity in 9. 9999219199(9999y) was used with the Tn903 399r gene to select for G418 resistance(260). Autonomous replication of the vector in the tranformants was suggested by the fact that intact pJL2 could be recovered from the total DNA of the transformants via 9. 9911 transformation. Additonal evidence supporting autonomous replication of pJLZ was that a deletion analysis of the 2. 99193991999999 99999 element identified a specific region within the 9999y which resulted in high frequency transformation of 2. 9193991999999 in comparison to the deletions of pJL2 which did not have this region. Southern blot data to support this was not given, and whether this site in the 99991 promoted autonomous replication or more efficient integration into the chromosome was not determined. Another 999 based vector, designated pPSll, was also shown to tranform 2. 9193991999999 and the intact vector could also be recovered in B. 9911(261). Southern blot analysis of genomic digests of pPSll transformants indicated the presence of monomer length plasmids in the transformants. It was not determined, however, whether these vector sequences were maintained autonomously or integrated into the chromosome in tandem repeats, which could also account 86 . for the monomer length vectors in the hybridization analyses done. Only 2-10% of the conidia isolated from selectively grown transformants maintained the G418 resistant phenotype whereas none of the spores isolated from non-selectively grown transformants were found to be G418 resistant indicating a loss of vector consistent with autonomous replication of pPSll(260). C-Wmms 9. 9999999 is probably the most well characterized basidiomycete in terms of its' genetics and several important genetic concepts have resulted from studies with this organism, including the concept of genetic incompatibility(263). 9. 9999999 was initially transformed using the homologous 1321 gene(indole-B- glycerol phosphate synthetase) in pRK9 as the selection marker(264). The 1321 gene was isolated due to its ability to complement a 9999 mutant of 9. 9911. Transformation was shown to be by integration into the chromosome. Ullrich and coworkers used this transformation to isolate many structural genes of 9. 9999999 by complementing selected mutants with an 9. 9999999 gene library in pRK9(265) and have made many improvements in the optimization of transformation(266). Southern blotting data indicated that unlinked insertion events were common when these homologous genes were used for transformation. Genetic analysis indicated that the transforming sequences were integrated 87 into the chromosome and stably maintained through both mitosis and meiosis. This transformation system was also used to isolate two mating type alleles of 9. 9999999(267). A cosmid containing the 1321 gene for selection was constructed(pTCZO) and cosmid libraries from two strains differing in An mating type alleles(Aa4 or Aal) were made. A series of cosmid clones from each cosmid library, spanning a region of DNA 50 kb were isolated in a chromosome walk which began from the £991 gene(closely linked to the mating locus). This series of cosmids from both strains was used to transform a 9991 strain to prototrophy. Transformants were then tested in a mating assay to confirm the presence of those cosmids containing Aa alleles. 0.999111911991139” 9. 91999999, along with 9. 9999999, has been used as a model genetic system for basidiomycetes and is a particularly interesting model for the study of genetic compatibility considering the complexity of the 9. 91999999 incompatibility system(268). In one transformation system, using the homologous 999:1 gene(tryptophan synthase; 269), isolated by cross hybridization with the 1329 gene of 9. 9999919199, was used as the selection marker. The vector used, pCclOOl, contained this 999:1 gene cloned into pUC9. 88 Either mycelial fragments or oidia were used as a source for the generation of spheroplasts for transformation. Unlinked integration into the chromosome was the predominant event. Meiotic analysis of linked insertion transformants indicated that they were stable, and no RIP like phenomenon was detected. This system was also used to confirm that a gene cloned by cross hybridization to the heterologous 999D gene(isocitrate lyase) from 9. 91991999 ,was a functional 999:1 gene in 9. 9o99u9e. The vector used, pHIONAl, contained the putative 999:1 gene cloned into pUC13. Transformation to acu+ was done by cotransformation with pCclOOl and trp+ transformants were selected since selection for acu+ was toxic to the spheroplasts. Cotransformation of the 999:1 gene was found in 31 to 47% of the transformants, depending on the experiment. Transformation has been shown to be by integration and predominantly by unlinked integration events(270). Insertion of multiple copies of unlinked selection markers have been shown to occur, resulting in an increase in enzymatic activity in some of the transformants(271), ldepending on the integration site of the transforming vector. 9. 91999999 transformation was also used to examine the function of heterologous genes from other fungi in cotransformation experiments(272). It was found that the 1:21 gene of :2. 92mm and the 32:19.: gene of W 9991999999199, both basidiomycetes like 9. 91999999, could complement 999:1 mutants of the latter fungus. Seperate 89 cotransformation experiments in which the transformation vector pAK3(containing the 999:1 gene of 9. 91999999) was used along with plasmids containing the 9999 and 9999 genes of the ascomycete 9. 91991999 to transform 9. 91999999 to TRP+. It was found that the 9999 and 9999 genes of the aSCOmycete 9. 91991999 could be cotransformed with pAKB, but not expressed. In addition, the plasmid pHLl, containing the 999 gene of B. 9911 fused to a promoter of the hemibasidiomycete 99911999 999919 was also shown to be non-functional in 9. 919er9us. These results indicated that the range of applicability of fungal genes and/or promoters as selection markers may be restricted. o ' us 911999999 has also been transformed to tryptophan prototrophy using plasmid pAK3 containing the 999:1 gene of s;- mun) 3.1592299131199133: The study of the genetics of 9. 19999999 has contributed greatly to the understanding of the process of meiotic recombination(274). Transformation in this fungus has been achieved using an homologous homoserine o- transacetylase gene to complement a 9991- auxotroph(275,276). When vector H13CG1, containing the 9. 19999999 9991 gene cloned into M13, was used for transformation, the plasmid was shown to integrate either at the homologous locus(gene replacement and linked , 90 insertions) or at heterologous sites in the chromosome. The gene replacements resulted in a stable wild type phenotype through both mitosis and meiosis. The linked and unlinked insertions were unstable through meiosis, while being mitotically stable. This same pattern of stability was also found when the 9991 gene was cloned into pUC19 and pBR329(to create vectors pCGS and pCG6, respectively) and was used to transform 9. 19999999. These results suggested that the instability of the individual transformants containing a gene duplicated during transformation(linked and unlinked insertions) through meiosis may due to an RIP-like effect methylation such duplications(276). The physical state of the transforming vector also appeared to affect the type of integration event observed in 9. 19999999. A comparison was made between the type of integrative events observed using circular single stranded DNA and circular double stranded DNA using vector M13CG1(276). It was found that using single stranded N13CG1 increased the frequency of gene replacement transformation events to 65% from the 5% observed with the double stranded vector. To examine if linearizing a vector within the 9. 19999999 9991 gene similarly increased the probability of gene replacement, transformants of circular pCG5 were compared to transformants obtained with linearized pCGS. It was found that with the circular vector, only 10% of the transformation events were due to gene replacement while 85% of the transformants were of the gene replacement variety when the linearized vector was 91 used. These results suggest that gene targeting is possible in 9. 19999999 and this can be enhanced by providing either homologous double stranded DNA or a single stranded form of DNA, either of which may be intermediates in the process of homologous recombination. A further transformation study tested this methylation phenomenon using the heterologous marker 9999 on either pBSRz or pAMDZl to transform 9. 19999999 transformed to 9999+(277). It was found that 50% of the meiotic progeny containing two unlinked copies of 9999 from the unlinked transformants, and up to 90% of the meiotic progeny of transformants containing tandemly linked 9999 genes had both copies of the transforming gene inactivated. It was found that only the duplicated 9999 genes, not adjacent bacterial sequences of the tranforming vector, were methylated. This indicated that the inactivation was specific to the duplicated sequences. The methylation was found to be due to a 99 9999 cytosine methylation in the haploid dikaryon just prior to the meiotic cycle which inactivated both the duplicated genes in such transformants. In contrast the the RIP effect of 9. 999999, this methylation was reversable, as subsequent vegetative growth resulted in the loss of the methylation and the recovery of the complementing activity. 92 6. fungi of Industrial Importance 1.2213121.an This fungus produces the antibiotic penicillin during secondary metabolism. Classical mutagenesis and strain improvement techniques have resulted in strains producing extremely high levels of this antibiotic(278). Further attempts to increase production may require a better understanding of the secondary metabolic regulation of genes involved in penicillin production, which would be facilitated by the development of a transformation system. Transformation of 2. 99999999999 has been achieved by complementation of appropriate auxotrophs with vectors containing the heterologous 9991 gene of 9. 999SS9(pJD82: 279), and the 9199 genes of both 9.l91991999(vector lambdaAN8a; 280) and 9. 91999(vector pSTAlO; 280). Transformation was by integration into different sites of the chromosome with each of these vectors. Interestingly, the results of transformation with the heterologous 9199 genes indicated that the regulation of expression of these genes in 2. 99999999999 was similar to the wild type gene, indicating that the control signals of these heterologous genes were recognized in £9919111199(279). The homologous 9999 gene of 2. 99999999999 was isolated by two groups using a genomic library in to complement the 9999 mutation of E. 9911. The vector chtrpC1(9999 gene in pUC13) was 93 found to integrate into the chromosome of transformants primarily by unlinked insertion(133), while the vector pPC- 31(9999 gene in pBR328) was found to integrate primarily by linked insertion(281). Whether this difference is due to the strains used or the different vectors is not certain. Vector p3SR2(Table 6: Fig. 3) was used to transform 9. 99999999999 to acetamide utilization(282,283) and an homologous resistance marker(oligomycin, pPOLl, containing the oligomycin resistance marker in pUC9; 284). Transformation appears to be by chromosomal integration in all cases. Unlinked insertions were shown to occur in the above cases, with multiple, often tandemly repeating integrations being common. Co-transformation was found to be efficient. When pBSRz, containing the 9999 gene as the selection marker was cotransformed with pAN5-4lB(see above and Fig. 3) and amd+ transformants were selected, 90% of these transformants showed cotransformation of pAN5-4lB and expression of fi-galactosidase(282). Different transformants showed varying levels of fi-galactosidase enzyme activity indicating either a position effect due to the site of integration of pAN5-4IB or differing copies of the vector integrated into the chromosome in different transformants. Two bacterial antibiotic resistance markers have been successfully used to transform 2. 99999999999. The phleomycin resistance gene of E. 9911(919) was fused to the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of 9. 91991999 and the terminator of the 9191 94 terminator of S- 99:9y19199 in pUC8 to create pH5103(282) and used for transformation. Integrative transformation at various sites of the chromosome was found. Sulfonamide resistance has also been used as a selection marker for transformation of E. 9nzy9gg9ngm (285). B. 9hxy9999ngm transcription signals from the 9:99 gene were fused to the bacterial sulfonamide resistance gene cloned into pUC13. The resulting vectors, pMLl and pML2(pML1 with the entire 9:99 gene as an additional selection marker) were used for transformation. The results showed integration into the chromosome at various sites in both cases. Dominant fungal resistance markers have also been used. The feasability of developing an autonomous vector for B. 9hzy9999ngm was tested by constructing two vectors containing mitochrondrial DNA fragments of 2. 9h91999999m which allowed high frequency transformation of 5- 9999219199, indicating the putative presence of 9:9 elements in the mitochondrial sequences utilized. These vectors, pSP530 and pSP533(along with pSPSZS, the parental form containing a fusion of pBR325 and pACYC177 with a bacterial kgnr gene as a selection marker for E. 9nryggg9ngm transformation) were used to transform the fungus to G418 resistance. The results lead to the conclusion that pSP530 an pSP533 are maintained autonomously based on the fact that they were recovered intact by E. 9911 transformation. Not unexpectedly, the parental vector pSP525 could not be recovered by E. 9911 transformation and based on Southern blot analysis appeared 95 to be integrated into the chromosome of transformants. However, not all pSP533 and pSP53O transformants of 2. 99999999999 gave recovery of the vectors by E. 9911 transformation. Furthermore, Southern analysis of transformant DNA of pSP533 and pSPSBO transformants apparently could not distinguish between integrative and autonomous maintenance of these vectors so the conclusion of autonomous replication may be preliminary(286). 2. 99919999999 cannot produce cephalosporins. It would be useful to develop a strain which may be able to convert a naturally produced penicillin to a useful cephalosporin, 7-ADCA(7-aminodeacetoxycephalosporanic acid). A gene capable of this chemical modification, penicillin N expandase(999£), has been cloned from S§99990929es 919991199999. This was attached to the promoter from the 2. 99929999999 9999 gene(isopenicillin N synthetase) on pPSGS which contains the 9999 gene from 9. 91991999 for selection. Transformants were found which expressed both high levels of endogenous penicillin and expandase activity were found, but it was suggested that the substrate specificity of the expandase may need to be altered before production of high levels of 7-ADCA are possible(287). mmmmnim 9. 9999999199 produces the antibiotic cephalosporin C(288) which can be chemically modified to produce many clinically important antibiotics. 9. 9999999199 was 96 tranformed to hygromycin B resistance using the hygromycin B phosphotransferase gene of E. 9911 linked to the 9. 9999999199 isopenicillin N synthetase gene promoter to make the vector pP825(289). Both single and multiple integration of pPSZS into the chromosome were observed. When vector DNA was linearized within the 9. 9999999199 sequences, an increase in transformation frequency of two to three fold was obtained. Whether the increase in frequency was associated with an increased proportion of linked or gene replacement type events was not discussed. To determine whether a mitochondrial DNA fragment containing 999 activity in 9. 9999919199 promoted high frequency transformation of 9. 9999999199, two plasmid vectors, pP829(no mitochondrial insert) and pPSZl(with mitochondrial insert) were used for transformation. It was found that the mitochondrial insert did not promote a high frequency of transformation when compared to the vector without the mitochondrial insert and that both vectors transformed 9. 9999999199 by integration into the chromosome(289). To determine whether this integrative transformation system can be used to increase expression of a transformed gene, the vector pPSSG containing the 99999 gene encoding a bifunctional protein with deacetoxycephalosporin C synthase and deacetylcephalosporin C synthase, two enzyems involved in the biosynthesis of the antibiotic cephalosporin C, was used to transform 9. 9999999199 to hygromycin resistance. An increase in the levels of activity of these 97 two enzymes was observed in some of the transformants while no increase in these activities could be observed in control transformations of 9. 999emO91u9 using a vector lacking the 999EF gene(290). C- m: Wayn- n- my 9. 91991991191999 is industrially useful as a source of extracellular protease,lipases, amylases, and cellulases. In addition, this organism is of basic scientific interest since it is considered a model system for the study of dimorphic fungi(29l). The yeast ' replicative vector YRpl7, containing a 9. 91991991191999 genomic DNA library, was used to complement a M- 91991991191999 199- auxotroph(292). Two plasmids, pMCLl302 and pMCL1647, were found which complemented the 199- mutant. Southern analysis of restriction digests of DNA from individual transformants containing these plasmids showed DNA bands of hybridization corresponding to the presence of plasmid length monomers. This together with the recovery of the intact plasmid from transformant DNA by E. 9911 transformation suggested autonomous replication. Based on results seen with the other fungi discussed above, these data are also consistent with the presence of tandem insertions of the vector into the chromosome. The question of autonomous maintenance of vectors in 9. 91991991191999 was examined further with vector pMCLl302(293). Undigested transformant DNA was found to 98 have monomer length circular vectors and no hybridization to the chromosome was observed. Two different vectors, pMCLl302 and pMCL002(containing the 199 gene of 9. 91991991191999 cloned into pBR322) appeared by Southern analysis of total undigested DNA of individual transformants to be present only extrachromosomally. This indicated that there should be 999-like sequences on the chromosomal fragment containing the 199 gene were responsible for this mode of maintenance. Both vectors could be recovered intact by 9. 9911_transformation and the mitotic stability of the transformants was found to be low, also indicative of extrachromosomal maintenance of the vectors. Subcloning of the genomic insert in pMCL1302 found that the region responsible for this autonomous mainetenance could be delineated to a 4.4 kb fragment of the 10.5 kb 9. 91991991191999 insert in pMCL1302. This 4.4 kb fragment was found to contain both the 1999 gene and the 999 function, which was further localized to a 222 bp fragment upstream of the gene. Some sequence similarity in this region to the 999 consensus of 9. 9999919199 was observed, but this region did not function as an 999 in 9. gsrgxi§i§s(294)- n. Irighgggrna 912521 I. 9ees91 has been studied extensively as a model system for the analysis of fungal cellulases(295) and many of these have been cloned and sequenced(see ref. 296). 99 Transformation has been shown with both the 9999(p38R2) and 9999(pSal43) genes of A. 91991999(297). Integration of the transforming sequences into the chromosome was observed at multiple locations and occasionally in multiple copies. When pAN5-4lB was cotransformed with p3SR2, fi-galactosidase was found to be expressed in 19 to 47% of the amd+ transformants. p-galactosidase expression in these transformants was found to be independent of the carbon source, indicating that some of the regulatory signals of the 999 promoter of 9. nidu199s on pAN5-4lB were not recognized in I. 9999_1. The I. 999991 transformation system has also been used for expression of a heterologous protein, calf chymosin(298). The cDNA of calf chymosin was fused to promoter and termination signals of the cellobiohydrolase 1(9991) gene of I. 999991 in a series of vectors differing in the length of the 5' end of the 9991 gene attached to the coding region of the calf chymosin cDNA. These vectors were then cotransformed with p38R2 and amd transformants were isolated. Active calf chymosin protein was found to be expressed in all constructions, indicating that I. 999991 might be a viable system for the expression of heterologous genes. This is especially important since I. 999991 is a basidiomycete, whereas the other fungi used for expression of heterologous proteins(mainly the various 99999911199 species) are ascomycetes, and expression of genes across these lines is not always certain(272). As a further demonstration of this usefulness, a cDNA of a lignin peroxidase gene from 2919919 9991999 was fused to 100 the 9991 promoter on plasmid pMSlG for use in I. 999991 tranformants. Plasmid pMSlG was cotransformed with p38R2 and acetamidase producing transformants were selected. The lignin peroxidase gene was found to be expressed as an RNA transcript, although an antibody study found no lignin peroxidase protein either within the cell, or in the extracellular medium(94). B-WW B. 9999999999199 is the most extensively studied fungal model system for the physiological, biochemical, and molecular genetic analysis of lignin degradation(8). There have been several reports on transformation of B. 9999999999199. An heterologous 9999 gene from 9. 99mmu99 cloned into pRK9 and designated pADEz, was used to complement an ade- auxotroph(299). Similarly, the 9999 gene from 9. 9999999 was found to transform an ade- auxotroph of B. 9999999999199 to prototrophy(300). Since neither 9. 9ommu99 gene had homology to a corresponding 2. 9999999999199 gene, integration of the vectors by non- homologous recombination into various sites of the chromosome were seen exclusively. A different approach used the 999r gene of Tn903 to transform 2. 9999999999199 to G418 resistance(301). A vector containing a genomic fragment of 9. 9999999999199 cloned into YIpS showing 999 activity in 9. 9999919199(302) was modified by the addition of the 999r gene and used to transform 9. 9999999999199. 101 In this case no integration events into the chromosome were observed and the intact vector was recoverable by E. 9911 transformation indicating a possible extrachromosomal maintenance of the vector. 7. Fungal Plant Pathogens a. mum: III—2.9L!!! 9. 999919 is a corn pathogen which has been studied extensively at the genetic level for a number of years as a model for other fungal plant pathogens. It has well developed systems for both mitotic and meiotic genetic analysis(303). A PEG-CaCl2 transformation system has been developed for this fungus based on hygromycin B resistance. A heat shock promoter of 9. 999919 was fused to the coding region of the 9. 9911 hygromycin B resistance gene cloned into pUC12(pHLl; Fig. 3) and was used for transformation(304). Southern blot analysis of the transformants indicated that the vector integrated primarily at a single site in the chromosome, although some tandem repeats were observed. There were two types of integration events seen in the transformants. The predominant type of integration seen was unlinked insertion, but some linked insertion events at the heat shock promoter locus were also observed. No gene replacements were detected. Linearizing the vector inproved the transformation frequency of pHLl 20 fold. The 102 transformants were both mitotically and meiotically stable. A transformation system using the homologous £133 gene has also been described(305). The 9999 gene was isolated by transforming an 9 9911 9999 mutant with a genomic library of 9. 999919 cloned into YEpl3. Further subcloning of the 9999 gene into pBR322 resulted in the vectors pMH2002 and pMHZOO3. These vectors were linearized within the 9. 999919-chromosomal DNA and were used to transform 9. 9a9915 to prototrophy. Gene replacements were observed in up to 60% of the transformants using linearized pMH2003 whereas linked insertions were observed in 53% of the transformants using linearized pMHZOOZ. A different study analyzing the effect of DNA conformation on the type of transformation events seen has also been made(306). Vector pCM124, which contains the 9. 999919 1991 gene in pUClZ was used to transform 9. 999919 either as a circular molecule or after linearization within the 1991 gene. With the circular vector two thirds of the transformants indicated integration at sites unlinked to the 1991 gene, while the rest appeared to be gene replacements. 9. 999919 transformed with the linearized pCM124 showed a much different pattern of events. While a small fraction of the tranformants showed linked integration events, estriction digests of most of the individual tranformants(20 of 23) indicated that bands of monomer length pCM124 were present. When probed with the 1991 gene, these bands were found to be, on the average 25 times more intense than the endogenous copy of this gene. 103 Since no fast migrating species of DNA corresponding to an autonomous pCM124 was seen, it was concluded that the vectors were integrated in a large tandem repeat at non- homologous sites in the chromosome, although Southern hybridization analyses did not detect any chromosomal junction fragments expected from such an integration. The first indication that this might be an extrachromosomal concatamerization of vector DNA was that the mitotic stability of these such transformants was always much less than for those which clearly demonstrated chromosomal integration. To confirm the extrachromosomal nature of this concatamer, contour-clamped homogeneous electric field(CHEF) gels were run to seperate the 9. 999919 chromosomes in both transformants containing integrated pCM124, and those containing the putative concatamers. With integrated transformants, vector sequences could be shown by Southern analysis of the CHEF blots to associate with one of chromosome bands, whereas the concatamer transformants showed bands which did not correspond to any of the chromosomes, but to lower molecular weight regions of the gel. 2. 9911 transformation showed that only monomer length pCM124 could be recovered, even in 9999- and 99999 9999 999: strains of E. 9911. No identification of any sequences of pCM124 responsible for this unique extrachromosomal maintenance were idenfified. A one step gene disruption was shown with the homologous 9999 gene in which the coding region of the 9999 gene was disrupted with the hygromycin marker discussed 104 above. The linear DNA fragment containing the disrupted 9999 gene was then used to transform 9. 999919 and 70% of theresulting transformants were found to contain a disrupted 9999 gene(307). Disruption of a heat shock gene, 9992 was also reported by a similar procedure and this disruption was found to be lethal, indicating that the 999; gene is essential for growth(308). In addition, this transformation system has been used to clone several alleles of the 9 locus involved in both pathogenicity and sexual development in 9. 999919(309). 'An analysis of one of these alleles, 9;, by constructing various deletions and frameshift mutations and using these to replace the wild type allele in 9. 999919 by transformation, has shown that this locus is necessary for tumor induction by this fungus(310). Recently, an 999 element which apears to function similar to the yeast 999 elements was described(311). A genomic library of 9. 999919 DNA was made in pHL1(see above: Fig. 3) was used to transform 9. 999919 to hygromycin resistance. DNA from a pool of transformants was used to transform 3. 9911 and several of the plasmids which were recovered were used to retransform 9. 999919. One plasmid containing a 3.4 kb 9. 999919 DNA fragment which gave the highest transformation frequency was used for further analysis(pcn43). Southern analysis of individual transformants showed that pCM43 was maintained as a monomer extrachromosomal element, was maintained at approximately 25 copies per cell and was unstable during non selective 105 growth. A deletion analysis of pCM43 localized the minimal 999 activity to a 383 bp fragment. Consensus sequences found in either 9. 9999919199 or 9. 99999 999 elements were not seen in this 9. 999919 999-like element. In a further demonstration of autonomous plasmid maintenance in 9. 999919, a terminal inverted repeat(TIR) from a linear plasmid from 9999919 999999999999(pFSC1) was inserted into pICl9RLH, containing the hygromycin selection marker described above inserted into pIC19 to result in vector pTIRl. This vector, when transformed into 9. 999919, was found to be maintained as an autonomous element by Southen hybridization, although some transformants also contained vector integrated into the chromosome. Circular pICRLH, and linearized pTIRl also resulted in chromosomal integration in transformants studied(312). An exact identification of the putative element in the TIR allowing for autonomous maintenance was not made. Both 2:211:92 119.1191 and Listings: mum). and 99911999 91919999(314) have recently been transformed to hygromycin resistance; in each case chromosomal integration at various sites was observed. 8. 19999199 species ‘9. 991991 is an invasive pea pathogen which is of interest as a model for the penetration of plant cell wall by a pathogenic fungus(315). A transformation system based on G418 resistance has been developed for this fungus. The 106 vector used was pEMTz, which contains a promoter from cauliflower mosaic virus(3SSp) fused to a bacterial kanamycin resistance gene of Tn5 in pBR322. A spheroplast transformation system was used and all the transformants were shown to be by integration of the vector at various sites in the chromosome(316). A three fold increase in transformation frequency was observed if pEMTZ was linearized. Kolattakudy and co-workers(317) cloned the cutinase gene which is involved in the initial penetration by the fungus of the plant cell wall. The promoter region was delineated and the promoter was then fused to the bacterial hygromycin resistance gene in pUC19 to result in pCT43 which was used in both spheroplast and LiCl transformation protocols to transform E. 991991 to hygromycin resistance(318). All transformants were of the integrative type. Vector pCT43 was also successfully used to transform 9911999199199 9999199 to hygromycin resistance(318). In further studies, the cutinase gene was cloned into the hygromycin vector to result in pUS-ll. This vector was used to transform a noninvasive pathogen, 99999999999119, which then allowed hygromycin resistant transformants to become invasive, demonstrating the importance of the cutinase gene in plant cell wall penetration by the pathogen(319). Two other species of 99999199 have been transformed. Protoplasts from a cycloheximide-sensitive, non zearalonone-producing strain of 99999199 99199999 was 107 incubated with total DNA from a cycloheximide-resistant, zearalonone- producing strain of 99999199 99a919ea99m in a vectorless transformation(320). Some cycloheximide resistant transformants were found to produce zearalonone. £9999199 999999999 was transformed to hygromycin resistance with pDH25(Fig. 3). Integration of the vector into the chromosome at various sites was observed in all cases(321). Attempts to optimize transformation frequencies were made using the vector pAN301(chlorate resistance; see Fig. 3). While using original protocols, frequencies of 1-10 transformants per pg were obtained, optimizing the DNA/protoplast ratio improved this to 1-200 transformants per pg(322). An electroporation procedure was also developed for pAN301 and comparable frequencies were obtained(323). An extrachromosomally maintained transfomration vector has also been developed for 9. 999999999. A circular plasmid containing pUClz, a linear endogenous 99999199 plasmid, and a hygromycin marker(pFTl) was found to transform at a low frequency(l-lo transformants per pg). Analysis of transformant DNA found that in some cases the vector was present extrachromosomally as a linear DNA element(323). Analysis of this linear vector found that it had acquired telomere sequences from the 29999199 chromosome. Thus it was concluded that this extrachromosomal vector was the product of an in vivo rearrangement of the original vector. A similar rearrangement was found with a vector which contained no endogenous plasmid sequences. These 108 rearranged vectors were found to be able to transform 9. 939999999 and be maintained stably as a linear extrachrosomal element with no further rearrangements. This extrachromosomal vector(pFOLT4R4) was found to be lost quickly during non-selective growth. Further experiments found that pFOLT4R4 could transform both 9999919 hggmasgggsgn and erphgnggfrig paragifiga and could be maintained extrachromosomally in these fungi also. 8. Other Fungal Transformation Systems Transformation systems have recently been described for a number of other filamentous fungi which are not as well characterized as those discussed above. These are outlined in Table 5. One fact that became clear with the development of an increasing number of transformation systems for filamentous fungi. is that, barring a few exceptions, promoter signals can be recognized across a broad range of filamentous fungi. Vectors designed to transform an ascomycete such as 9. 91991999 can in many cases transform basidiomycetes or other classes of fungi. The reverse is also true, i.e. some basidiomycete genes can be expressed in ascomycetes(324). In contrast to this, some genes seem only to function within a specific class of fungi(272). The fact that promoter signals of certain genes are broadly recognized hasalso allowed the construction of fusions between these fungal promoters and bacterial 109 Transforeation Iarkers used in recently described fungal ‘ transforeatien systees‘ fungus Marker Reference Integrative Cochliobolus heterostrophus 9999 (p3SR2) 342 - ' 999 (pHIS) 329 flagnaporthe prises gran (PMAZ) 343 rulvia tulva 999 (pAN7-l) 344 Glouerella cingulata and: (P3SR2) 345 ' ' hnh (Pals) 345 Leptosphaeria maculans 999 (pAN7-1) 346 anunennonyces graninis 999 (pBT3) 347 Septoria nodorum 999 (pAN7-l) 34B Achlya ambisexualis G418 (pSVZneo-Hxac) 349 Colletotrichum trifolii 999 (pBHM-l) 350 ' ' 999 (pSVSO) 350 Gliocladiun roseum 999 (p315) 351 Gliocladiun vireus 99h (p318) 351 Pseudocercosporella herpotrichoides 999 (pAN7-l) 352 " " m (9813) 352 Botryotinia squanosa 99h (pD825) 353 Hetarhizium anisopliae 999 (pSVSO) 354 ' ' 99h (pBBNA3) 355 Sordaria nacrospora 9999 (pLLCBlO) 356 Curvularia lunata 999 (pAN7-l) 357 Colletotrichum lindenuthianun 9199 (pAN30l) 358 Beauveria bassiana ' 358 Aphanocladiun album ' 358 Nectria haematococca ' 358 Pyricularia orysae ' 358 arxphonectria parasitica 99h (pHIB,pBlS, 359 pucm, 9828, poms, pAN7-l, pELl, pP829) m (PSVSO) G418 (pEHTZ) Absidia glance 999 (pinpfll, pAnpNS) 360 lstrachroeosoeal Absidia glance 999 (pAnNGl) 330 Scytaliun flavo-brunneun 999 (pSFB-l) 359 Hectria haematococca 99h (prOLT4RQ) 323 cryphonectria parasitica ' 323 9999 - hygromycin B; 999 - benomyl: 999 - cycloheximide: 999 - neomycin; 9999 - acetamide utilization: 999 - ls-azasterol: G418 - 6418(geneticin); 9999 - ornithine transcarbamylase: 9199 - nitrate reductase I gene replacement or linked insertion; N - unlinked insertion; s - single copy: a - multiple copies: ND - data not given 110 antibiotic selection markers, as an alternative to the use of auxotrophic markers. There are several vectors in particular which have found a broad use in the tranformation of many species of fungi. These are outlined in Table 6 and shown in Figure 3. The hygromycin gene of E. 9911 has been fused to promoter signals from A. nigglgn§(pAN7-1, 210; pDH25, 227), n. m9y§1§(pHLl: 304), and Qgghligbglgg hgtgzgggzgpng§(pflls; 342) and have been used extensively in the development of new fungal transformation systems(see Table 5 for complete list of references). Three 3. grassa vectors containing the benomyl resistant p-tubulin gene(pBTB, 142; pBT6, 144; pSVSO, 161) have been used to transform various fungi to benomyl resistance(Table 5). Similarly, two A. nigglgng dominant markers(9mg§ and nng) have been used widely. The gags gene, in p38R2(188) has been used to select for scetamide utilization in those fungi which do not normally grow on acetamide as a sole nitrogen source(Tab1e 6). Vector pAN301(325), containing the nitrate reductase(nigp) gene of A. nigglgng has been used to transform many fungi to chlorate resistance(Table 6). The most extreme case of a heterologous promoter function has been the kanamycin determinant of Tn903(326) which has been used without the addition of any eukaryotic signals to tranform a range of filamentous fungi and yeasts. This gene has previously been shown to function unaltered in the yeasts fi. ss:exisiae(327) and Klnxxsrgmxsea lasti§(328) and in filamentous fungi(Table 6) such as E- 9:99:9(143), 111 Figure 3. Common transformation vectors used across species lines. The diagram is adapted from the original references describing each vector. The thick lines in each vector represent fungal DNA while the thin lines represent E. coli vector DNA(pUC18,pBR322, etc.). The location of the selection markers for both bacterial and fungal transformation are given for each vector and the approximate location is noted as a line outside of the plasmid figure. Where known, the transcriptional orientation is also denoted with an arrowhead. The number within each circle indicates the size of each vector in kilobase pairs. For the genes representing bacterial coding regions fused to fungal transcription signals the promoter(P) and terminator(T) regions are given if present(see below for abbreviations). The restriction sites noted are only those relevant to the delineation of the major functional domains of each vector and the maps shown are no; complete for these enzymes. Gene abbreviations: Ap , ampicillin: Egg. hygromycin phosphotransferase; amdS, acetamidase; gpg, glucose-6-phosphate dehydrogenase; trpc tryptophan biosynthetic gene cluster; ar B, ornithine transcarbamylase; niaD, nitrate reductase; lacZ, fi-galactosidase; uidA, B-glucuronidase: ban. benomyl; p1, promoter 1 of Cochliobolus hetergstgophus. Restriction site abbreviations: B, fiamHI; C, glal; E, EQQRI: H. fiindIII: N. Edel: 8. gall: X. 3221. Ii Ii. Ap' pBT3 % a 8 S S 22". pSVSO pNOM102 113 TABLES Vectors and selection markers used across genus/species lines for selection of transformants A) Benosyl resistance using a benomyl resistant for: of p-tubulin (bent) from Neurospora crassa Organism Vector Reference Neurospora crassa pBT3 (Pig. 3) 142 - pSVSO (Pig. 3) 161 ' pBT6, pJD21, pJD71, pJDBl, pJD82, 144 pRHlll (modified forms of p8T3) Aspergillus oryzae pBT3 237 Gaeumannomyces grammis p8T3 347 Podospora anserina pBT6 256 Aspergillus niger pBT6 233 Hetarhizium anisopliae pSVSO 354 Colletotrichium trifolii pSVSO 349 Cryphonectria parasitica pSVSO 359 Pseudocercosporella p8T3 352 herpotrichoides B) nygrosycin resistance using the hygromycin phosphotransferase gene or E. coli (92;) fused to appropriate fungal promoters Organism Vector Reference 1. Aspergillus nidulans pAN7-1 (Pig. 3) 210 Aspergillus ficuum pAN7-l 242 Fulvia fulva pAN7-1 344 Leptosphaeria maculans pAN7-1 346 Septoria nodorum pAN7-l 348 Curvularia lunata pAN7-1 357 Cryphonectria parasitica pAN7-1 359 Aspergillus giganteus pAN7-1 243 Pseudocercosporella pAN7-1 352 herpotrichoides Aspergillus nidulans pDHZS (Fig. 3) 227 Fusarium oxysporum pDfiZS 321 Borryotinia squamosa pDHZS 353 Cryphonectria parasitica pDHZS 359 3. Ustilago maydis pHLl (Fig. 3) 304 Ustilago hordei pHLl 313 Ustilago nigra pHLl 313 Cryphonectria parasitica pKLl 359 4. Cochliobolus heterostrophus pHIs (Pig. 3) 342 Colletotrichium crifolii pHIS 350 Glomerella cingulata pHIS 345 114 L Organism Vector Reference Gliocladium roseum pars 351 Gliocladium virens pHIs 351 cryphonectria parasitica pHIs 359 Oscilago violacea pUCHl 314 Cryphonectria parasitica pUCHl 359 5. tusarium solani pCT43 (F. solani promoter) 318 Colletocricbium capsici pCT45 318 C) Rcetamidase utilisation using the amds gene of Aspergillus nidulans Organism Vector Reference Aspergillus nidulans p35R2 (Fig. 3) 188 Aspergillus niger pBSR2 211 Aspergillus oryzae p3SR2 181 Aspergillus ficuum ’ _ p3SR2 242 Cochliobolus beterostropbus p3SR2 329 Tricbodermm reesei p3SR2 296 Penicillium chrysogenum p38R2 282 Penicillium chrysogenum p3SR2 283 Glomerella cingulata p3SR2 345 D) nitrate reductase (999D) gene allowing for chlorate resistance Organism Vector Reference 1. Aspergillus nidulans pAN301 (Fig. 3) 325 Fusarium oxysporum pAN301 358 Beauveria bassiana pAN301 358 Penicillium cbrysogenum pAN301 358 Apbanocladium album pAN30l 358 Nectria baematococca pAN301 358 Pyricularia oryzae pAN30l 358 flanicillium cbrysogenum lambdaANBa 280 Collecotricbium pAN301 358 lindemuthianum 2.-Aspergi11us niger pSTAlO 232 (pUC8 + A. niger 919D gene) Penicillium chrysogenum pSTA10, pSTAlZ 280 1515 3. Aspergillus oryzae pSTA14 237 (pUC18 + A. oryzae niaD gene) Aspergillus nidulans pSTA14 237 Aspergillus niger pSTAl4 237 Penicillium chrysogenum pSTAl4 237 2) Orotidine-S'-monophosphate decarboxylase (gig) Organism Vector Reference 1. Neurospora crassa pFBG 130 (pBR322 + N. crassa 21:4) Aspergillus nidulans pFBG 186 Penicillium chrysogenum pP86,pDJal,pDJ82 279 2. Aspergillus niger pAB4-1 231 (pUC19 + A. niger pygc) Aspergillus niger pGW613 230 (pUN121 + A. niger 2256) Aspergillus oryzae pAB4-1 235 Aspergillus awamori pANGl 239 P) Ornithine carbamoyl transferase (9:39 of Aspergillus nidulans) Organism Vector Reference Aspergillus nidulans pSa143,pH32,pHT201 183,214,215 pAl,pAAl,p8159 219,336 Aspergillus niger pDG3 229 Aspergillus oryzae pILJlG 238 “ - 181 Aspergillus awamori pUC4AP-9gg 240 Tricboderma reesei pSa143 296 Hagnaportbe grisea pHAZ 343 G) Isocitrate lyase (agg7 of Coprinus cinereus) Organism Vector Reference Coprinus cinereus ' pIONA-l 270 (pUC13 + C. cinereus acu7) Aspergillus nidulans pIONA-l 324 116 H) Indole-3-glycerol phosphate synthetase ($52; of Schizopbyllum commune) Organism Vector Reference Schizopbyllum commune , pAHl 265 (pRK9 + S. commune ggpl) Coprinus cinereus pScl 272 I) 6418 resistance (1593r gene of Tn903) Organism Vector Reference Neurospora crassa y p8C10 143 pEBlO Phycomyces blakesleeanus pJL2 260 ' ' pPSll 261 pPSll 262 Penicillium chrysogenum pSP525,530,533 285 Phanerocbaete chrysosporium pRR12 301 117 Ehxsemxses Dl§B§§l§§QDE§(250‘252). 22212111123 mass). and W WOW)- This particular hgnr determinant appears to be unique among prokaryotic resistance markers in that it functions unaltered in many lower eukaryotes. The sequence(s) functioning as a eukaryotic promoter in this gene has not yet been identified. In other instances, the hygromycin marker(318,329) and the kanamycin marker(259,316,330) have used to screen for fungal promoters which will drive their transcription. In this type of fusion, gene libraries of the appropriate fungus are made by inserting random fragments just upstream of a promoterless selection marker. The library is then used to transform the fungus and those transformants which arise are presumed to have promoter fragments upstream of the promoterless gene allowing for their transcription. The vector containing teh promoter funcion must then be recovered from the chromosome of an individual transformant. The promoter can then be used as a basis for the development of a vector for further transformation analyses. Two types of gene fusions between fungal promoters and bacterial reporter genes have been constructed for the analysis of regulatory signals in fungi(Fig. 3, Table 7). The glyceraldehyde-3-phosphate dehydrogenase(gpg) promoter and the 9:29 termination signal of A. nigglggg have been fused to the 1992 gene of E. ggli and this construction(pAN5-418: Fig. 3) has been used to detect p-galactosidase activity in 118 m1 Transformation vectors for expressing reporter genes from Escherichia coli A) B-galactosidase (lacZ) gene fused to promoter and termination signals of Aspergillus nidulans. Organism for expression Vector Reference Aspergillus nidulans pANS-418 (Fig. 3) 202 Aspergillus nidulans pAN923 series 203 (derivatives of pAN5-418) Aspergillus oryzae pAN5-418 236 Aspergillus ficuum pANS-418 243 Penicillium chrysogenum pAN5-418 281 Trichoderma reesei pANS-418 296 Aspergillus nidulans pCSCS-4 (gags promoter) 206 A. niger pCSCS-4 206 a promoter from glycerol-3-phosphate dehydrogenase gene except where indicated B) fi-glucuronidase (uidA) gene fused to gpg. promoter and ggpcb terminator of A. nidulans Organism for expression Vector Reference A. nidulans pNOM102 (Fig. 3) 331 A. niger pNOHlOZ 331 ' pNOK102 233 A. oryzae pNOthZ 236 ' pNOHlOZ 237 Fulvia fulva pNOMlOZ 331 aglyceraldehyde-3-phosphate dehydrogenase phosphoribosylanthranilate somerase(PRAI); indoleglycerolphosphate synthase (IGPS); glutamine aminotransferase(GAT) 1119 IABLB 8 Heterologous expression of genes of scientific, commercial and medical interest expressed in filamentous fungi Organism Gene Vector(Promoter) Reference Aspergillus nidulans nitzz (N- crassa) pNITZ (endogenous) 192 211° pNhT9 (endogenous) 212 mad; E. coli pL2 (@5159) 134 endoglucanase pGL2CENDO ($139) 132 - pALCAISENDO (915Ah) 182 interferon a2 (human) pGL2BIFN (9199) 182 - pALCAISIFN (9lgAh) 182 3.241 (human) leS9 (3:21:13) 183 Other Aspergillus species ACVS-IPNS-ACTk pcx3.2 (endogenous) 363 aspartic proteinasel pBoe1-777 (a-amylase) 181 bovine chymosin pGAMpR (glgg) 362 Heurospora crassa ACVS-IPNS-ACTk pcx3.2 (endogenous) 185 Trichoderma reesei bovine chymosin pAMHlOl-3 (cbh 1m) 298 _ pAMHlOG (con 1”) lignin peroxidasen pflSl6 94 Cephalosporium acremonium ggggfi' (C. acremonium) pPS56 (endogenous) 290 cpisatin demethylating activity of Nectria haematococca enterotoxin subunit 8 'acetamidase from Cellulomonas fimi F)Lucoamylase “alcohol dehydrogenase I tissue plasminogen activator triosephosphate isomerase kpenicillin biosynthetic cluster; 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); isopenicillin N synthetase (IPNS); acyl coenzyme Az6- aminopenicillanic acid acyltransferase (ACT) from Rhizomucor miehei mcellobiohydrolase I nfrom Phlebia radiata °deacetoxycephalosporin C synthetase(DAOCS); deaceytylcephalosporin C synthase(DACS) 120 a variety of fungi(Table 7). Similarly, the p- glucuronidase(gigg) gene of E. 9911 has been fused to the 329 promoter of A. nidglans(pNOM102; Fig. 3) has been and used to detect fi-glucuronidase enzyme activity in various fungi(Table 7). These are, of course, a prelude to the analysis of endogenous promoter elements from these fungi(331). The 1991 fusion system has already been used to anaylze the regulatory regions of A. nidulans genes(203-205). A series of vectors for this type of promoter study have also been constructed(203,204) which allow translational fusions in all three reading frames to the 1921 gene. The vectors(Fig. 3) also contained the grgg gene of A. nidulans for selection. There is a widespread interest in the use of fungal systems for the expression of heterologous proteins, as an alternative to s. cezevisigg(Table 8). The reasons for this are several. Fungi often recognize intron splice sites of genes from heterologous organisms(188) and therefore whole genes can be used for the expression of a foreign protein. High level expression with some fungi is obtainable due to a long history of selection for strains which optimize secretion of proteins(278). With the expansion of the analysis of fungal genes, many strong promoters from filamentous fungal genes have been characterized and some of these(gpg and 9mg§ of A. nigulans) can be used in the construction of the necessary gene fusions for the expression of heterologous proteins. 121 9. conclusions Rapid advances have been made in the last ten years in developing transformation systems for many yeasts and fungi of importance in medicine, agriculture, food and fermentation, and basic scientific research. Fundamental differences between yeast and fungal transformation systems are apparent in terms of the fate of the transforming vectors introduced into the organisms. In yeast there is a clear distinction between vectors that replicate autonomously and those which integrate into the chromosome. The type of transformation event also dictates the stability and recoverability of the vector. In 9. 9919219199 919 elements have been identified which, if not origins of replication 19 9199, at least mimic their action and allow vectors to be maintained autonomously Autonomous vectors are generally quite unstable, transform at high frequencies, and can be easily recovered by B. 9911 transformation. With integrative transformation, the selection vector is stably integrated(generally at homologous sites) into the chromosome and conforms to classical Mendelian inheritance patterns. These integration vectors are unrecoverable, except by artificial manipulations. Attempts to translate the principles which govern yeast transformation vectors to the development of vectors for filamentous fungi have given mixed results. There are two primary differences between yeast and filamentous fungal 122 systems. The first is that extrachromosomal replicative vectors, similar to those described for yeasts(such as the Zn or 9:9-based vectors), have been difficult to construct in fungi. Even in fungi with endogenous plasmids, it is not always possible to construct reliable extrachromosomal shuttle vectors, although isolated cases of extrachromosomal maintenance was observed. The very presence of the endogenous plasmids in filamentous fungi suggests that construction and stable maintenance in fungi of such vectors should be possible in fungi, however, investigations to this end have not been very extensive or successful to date. This may be due in part to the mitochondrial origin of most plasmids of filamentous fungi; they may not be stable in a nuclear environment. A second major difference between transformation systems of yeasts and filamentous fungi is that the integrative transformation in the latter is often by non- homologous recombination whereas in yeast homologous recombination is the most prevalent event and heterologous recombination is relatively rare. In fungi, homologous recombination events are infrequently observed even when vectors containing a homologous selection marker are employed. The types of integration events are also marker and strain dependent in many cases. This suggests that the transforming mechanisms of fungi are closer to that seen in higher eukaryotes, in which viral vectors are often seen to integrate by unlinked insertion into the chromosome(332). In some cases, such as with SV40, this may not be 123 completely random since small homologies(4-5 bp) between the vector and the chromosome have been seen at integration sites(333,334). Such a detailed analysis of integration sites in fungi has not been done. The phenomenon of head to tail tandem integration of vectors in fungi is also quite similar to the mammalian systems studied(335). The development of the molecular genetics of filamentous fungi, including the development of transformation systems for many of the most well characterized fungi has been quite rapid over the past several years. This has already been exploited in the analysis of several very interesting systems in A. . n1991999(179,180). If one looks at A. nidu1ans as a prototype of the potential for the molecular analysis of filamentous fungi using transformation systems, one can find that the development of the types of manipulations possible parallels that of 9. 9919919199 in many cases. The developments in E. 919999 and n. m9yg19 are equally impressive and offer hope that such in depth molecular genetic analysis may be possible in other systems. Construction of useful recombinant strains by creating specific gene disruptions and gene fusions, and using such strains for the detailed analysis of gene function and regulation is possible. The use of A. n19919n9 and A. 9199; as hosts for cloning and expressing foreign genes of interest has been limited to date. However, considering the close similarity between the gene structure of filamentous fungi and that of higher eukaryotic systems, this is likely 124 to become more common in the future. Based on recent investigations, filamentous fungi appear to be ideal hosts for the expression and production of medically and industrially important heterologous proteins. Transformation in filamentous fungi has presented some interesting problems, such as the difficulty in directing or controlling chromosomal integration events, the difficulty in construction of true autonomous vectors discussed above, and the RIP phenomenon in A. crgssa(177) similar related phenomena in other fungi(254,276a). These studies, none the less, have led to a better understanding of the genetics of filamentous fungi and the problems encountered do not appear to be insurmountable. In fact, some very interesting and useful properties of fungal integrative transformations have been exploited. The commonly encountered multi-copy integration of vectors in chromosomes of transformants can in part overcome the inability to develop multi-copy autonomous transformation vectors and offer some of the same advantages in increasing expression a desired gene. Such integration systems also offers the advantage of higher mitotic stability due to chromosomal integration. The RIP phenomenon can even be useful in that it may be possible to use this type of event to construct specific mutants of any cloned sequence as an alternative to gene disruptions. Further analysis of fungal transformation systems will undoubtedly uncover more scientifically interesting and biotechnologically useful properties which can be exploited in the molecular genetic 125 analysis of individual fungal systems. 10. 11. 12. 126 REFERENCES Zeikus, J. G. 1983. Lignin metabolism and the carbon cycle: polymer biosynthesis, biodegradation, and environmental recalcitrance. in A929n999_1n_u19999191 3991991, vol. 5, ed. M. Alexander, pp. 211-43. Plenum Press, New York Buswell, J.A., E. Odier. 1987. Lignin biodegradation. C39 9219,39v.§19technol. 6: 1-60 Crawford. R.L. 1981. L19n1n_Degradation_anQ Ezgngfgzmggign, John Wiley & Sons, Inc., New York. 154pp Kirk, T.K. 1984. Degradation of lignin. in B12shemistrx_21.nisrohial_negradation. ed D.T. Gibson, pp. 399-437. 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Identification of cDNA clones for ligninase from 2222212222222 2211222221122 using synthetic oligonucleotide probes. 21999999 2122212_222122221 137: 649-56 de Boer, H.A., y.z. Zhang, C. Collins, C.A. Reddy. 1987. Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, 29a9e1999aete ch1ys999o1ium. Gene 60: 93-102 Tien, M., C.-P. Tu. 1987. Cloning and 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 132 sequencing of a cDNA for a ligninase from 2212222221122. 222212 325: 520-23 Zhang, Y. Z. PhD Thesis. Michigan State University, 1987. Holzbaur, E.L.F., M. Tien. 1988. Structure and regulation of a lignin peroxidase gene from 2222212222222 2212222221122. ' BiQEh¥§1BE§2QQEE1 1553 526-33 Brown, A., P.F.G. Sims, U. Raeder, P. Broda. 1988. Multiple ligninase-related genes from 2222212222222 2212222221122. 2222 73: 77-85 Smith, T.L., H. Schalch, J. Gaskell, S. Covert, D. Cullen. 1988. Nucleotide sequence of a ligninase gene from 2222212222222 2212222221122 2221212_A2122_2221 16: 1219 Asada, Y., Y. Kimura, M. Kuwahara, A. Tsukamoto, K. Koide, A. Oka, M. Takanami. 1988. Cloning and sequencing of a ligninase gene from a lignin degrading basidiomycete. 2222212222222 1 O O O O 29: 469-473 Walther, I., M. Kalin, J. Reiser, F. Suter, B. Fritsche, M. Saloheimo, M. Leisola, T. Teeri, J.K.C. Knowles, A. Fiechter. 1988. Molecular analysis of a 2222212222222 2212222221122 lignin peroxidase gene. 9999 70: 127-37 Schalch, K., J. Gaskell, T.L. Smith, D. Cullen. 1989. Molecular cloning and sequences of lignin peroxidase genes of 2222212222222 2212222221122. 22112211121211 9: 2743-47 Andrawis, A., E.A. Pease, I-c. Kuan, E. Holzbaur, M. Tien. 1989. Characterization of two lignin peroxidase clones from 2222212222222 2212222221122- 2122222121222221222122221 162: 673-80 Pribnow, D. M.B. Mayfield, V.J. Nipper, J.A. Brown, and M.H. Gold. 1989. 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Genetic factors influencing lignin peroxidase activity in 2222212222222 2212222221122 ME446- 22112121221211 3: 919-24 Tien, M., P.J. Kersten and T.K. Kirk. 1987. Selection and improvement of lignin-degrading microorganisms: potential strategy based on lignin model-amino acid adducts. 9991129911999 2121221212 53: 242-45 Kirk, T.K. 1985. New horizons for biotechnological utilization of the forest 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 134 resource. u21222.2211222212_22222122221 21221 2 1-27 Bumpus, J.A., M. Tien, D. Wright, S.D. Aust. 1985. Oxidation of persistent environmental pollutants by a white rot fungus. 9919999 228: 1434-36 Bumpus, J. A., S. D. Aust. 1987. Biodegradation of DDT[1,1,1-Tri chloro-2 ,2-bis(4-chlorophenyl)ethane] by the white rot fungus 2222212222222 2212222221122. 22211122112212121221211 53: 2001-08 Mileski, G. J., J. A. Bumpus, M. A. Jurek, S. D. Aust. 1988. Biodegradation of pentachlorophenol by the white rot fungus 2222212222222 2212222221122. 22211 222112212121221211 54: 2885-89 Johnsrud, S.C., K.-E. Eriksson. 1985. Cross-breeding of selected and mutated homokaryotic strains of 2222212222222 2212222221122 K-3: New cellulase deficient strains with increased ability to degrade lignin- A2211212122121121222222211.21: 320-27 Kirk, T.K., M. Tien, S.C. Johnsrud,K.-E. Eriksson. 1985. Lignin-degrading activity of 2222212222222 2212222221122 Burds.: Comparison of cellulase-negative and other strains. 221222.212122112222211 3: 75'30 Raeder, U., P. Broda. 1984. Comparison of the lignin-degrading white rot fungi 2999919999999 ' 2212212122222 2212222221122 and 222122112222 at the DNA level. 99119999999 8: 499-506 Rao, T.R., C.A. Reddy. 1984. DNA sequences from a ligninolytic filamentous fungus 2999919999999 9911999991199 capable of autonomous replication in yeast. 2122222121222221222122221 118: 821227 Zhang, 2.2., C.A. Reddy. 1988. Use of synthetic oligonucleotide probes for identifying ligninase cDNA clones. 9999129919911 161: 228-37 Raeder, U., P. Broda. 1988. Preparation and characterization of DNA from lignin-degrading fungi. 9999129919911 161: 211-20 Ballance, D.J. 1986. Sequences important for gene expression in filamentous fungi. 29999 2: 229-36 Saloheimo, M., V. Barajas, M.- -L. Niku-Paavola, J. K. C. Knowles. 1989. A lignin peroxidase-encoding cDNA from the white-rot fungus 2919919 1991999: characterization and expression in 11199999199 122221. 2222 85: 343-51 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 135 Esser, K., J. Kamper. 1988. Transformation systems in Yeasts: Fundamentals and applications in biotechnology. BIREQEE.§12£D§E~ 35' '41 Parent, S.A., C.M. Fenimore, K.A. Bostian. 1985. Vector systems for the expression, analysis and cloning of DNA sequences in 9, 9919119199 'Xfiflfii 1: 83-138 Cabib, E., R. Roberts. 1982. Synthesis of the yeast cell wall and its regulation. 999129112199999. 51: 763-93 Hanahan, D. 1983. Studies on transformation of 2211 with plasmids. 1122112121. 166: 557-30 Ito, H., Y. Fukuda, K. Murata, A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. 9,29cte1191. 153: 163-68 Becher, D., B. Conrad, F. Béttcher. 1982. Genetic transfer mediated by isolated nuclei in m e 2212212122. 2211122222- 6: 163-65 Gyuris, J., E. G. Duda. 1986. High-efficiency transformation of 9999991991999 2212212122 cells by bacterial minicell protoplast fusion. 2211221112121- 6: 3295-97 Johnston, S., P.Q. Anziano, K. Shark, J.G. Sanford, R.A. Butow. 1988. Mitochrondrial transformation in yeast by bombardment with microprojectiles. 9919999 240: 1538-41 Armeleo, D., G.-N. Ye, T.M. Klein, K.B. Shark, J.C. Sanford, S.A. Johnston. 1990. Biolistic nuclear transformation of 2222221222222 2212212122 and other fungi. 99111999999 17: 97-103 Hinnen, A., J.B. Hicks, G.R. Fink. 1978. Transformation of Yeast. 2122122211222212211222 75: 1929-33 Struhl, K., D.T. Stinchcomb, S. Scherer, R.W. Davis. 1979. High-frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. 2122122211222212211222.76: 1035-39 Orr-Weaver., T. L., J. W. Szostak, R. J. Rothstein. 1981. Yeast transformation: A model system for the study of recombination. 2122122211222212211222 78: 6354-58 107. 108. 109. 110. 111. 112. 113. 114. 115. 117. 118. 119. 136 Stinchcomb, D.T., M. Thomas, J. Kelly, E. Selker, R.W. Davis. 1980. Eukaryotic DNA segments capable of autonomous replication in yeast. EIQ§1HQEl1AQQQ1§£11Q§A 77: 4559-53 Broach, J.R. 1983. Construction of high copy yeast vectors using Z-pm circle sequences. 222212212221. 101: 307-25 Futcher, A. 8., B. S. Cox. 1983. Maintenance of the 22m circle plasmid in populations of 9999991991999 2212212122 11222221121 154: 612-22 Beggs, J.D. 1978. Transformation of yeast by a replicating hybrid plasmid. 999919 275: 104-08 Stinchcomb, D.T., K. Struhl, R.W. Davis. 1979. Isolation and characterization of a yeast chromosomal replicator. 999919 282: 39-43 Williamson, D.R. 1985. The yeast ARS element, six years on: A progress report. 19999 1: 1-14 Maundrell, J., A.P.H. Wright, M. Piper, S. Shall. 1985. Evaluation of heterologous ARS activity in 2222221222222 2212212122 using cloned DNA from 2221122222221222222M 2221212_A2122_22_- 13: 3711-22 Murray, A.W., J.W. Szostak. 1983. Pedigree analysis of plasmid segregation in yeast. 9911 34: 961-70 Som, T., K.A. Armstrong, F.C. Volkert, J.R. Broach. 1988. Autoregulation of 22m circle gene expression provides a model for maintenance of stable plasmid copy levels. 9911 52: 27-37 Dobson, M.J., A.B. Futcher, 8.8. Cox. 1980. Control of recombination within and between DNA plasmids of 2212212122. 221112222.- 2: 193-200 Zakian, V.A., J.F. Scott. 1982. Construction, replication, and chromatin structure of TRPI R1 circle, a multiple-copy synthetic plasmid derived 2212212122 from 9999991991999 chromosomal DNA. 2211921112121 2: 221-32 Blackburn, E.E., J.W. Szostak. 1984. The molecular structure of centromeres and telomeres. 222122212122222. 53: 163-94 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 137 Clarke, L., J. Carbon. 1980. Isolation of the centromere-linked 99910 gene by complementation in yeast. 2122122211222212211222 77: 2173-77 Murray, A.W., J.W. Szostak. 1983. Construction of artificial chromosomes in yeast. 999919 305: 189-93 Riethman, H.C. R.K. Moyzis, J. Meyne, D.T. Burke, M.V. Olsen. 1989. Cloning human telomeric DNA fragments into 2222221222222 2212212122 using a yeast-artificial-chromosome vector. 2199,Nat1,Acad. 9911299 86: 6240-44 Johnstone, I.L. 1985. Transformation of 99991911199 21221222. 21212212112211 2: 307-11 Hynes, M.J. 1986. Transformation of filamentous fungi. EZE1!¥§2_- 10: 1’3 Rambosek, J., J. Leach. 1987. Recombinant DNA in filamentous fungi: Progress and prospects. 929 211212221212122121 6: 357-93 Leong, S.A. 1988. Recombinant DNA research in phytopathogenic fungi. 991121999_299991. 6: 1-26 Fincham, J.R.S. 1989. Transformation in fungi.° 2121221211222. 53: 148-70 Farkas, V. 1979. Biosynthesis of cell walls of fungi. 212122121122_- 43: 117-44 Ferrer, S., D. Rambn, J. Salom, E. Vicente, 1985. Protoplasts from 299999919 99991199: Isolation, purification, and transformation. 221112121221_1. 12: 301-06 Buxton, F.P., A. Radford. 1984. The transformation of mycelial spheroplasts of 9991999919 919999 and the attempted isolation of an autonomous replicator. 2211222122222. 196: 339-44 Mishra, N. C., E. L. Tatum. 1972. Effect of L-sorbose on polysaccharide synthetases of 9991999919 919999. 2122122211222212211222 60: 313- 17 Ballance, D. J., G. Turner. 1985. Development of a high-frequency transforming vector for 99991911199 21221222. 2222 36: 321- 31 Sanchez, P., M. Lozano, V. Rubio, M. A. Penalva. 1987. Transformation in 22212111122 22122222222 9999 51: 97-102 F . Uruburu . 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 138 Dhawale, S.S., J.V. Paietta, G. Marzluf. 1984. A new, rapid and efficient transformation procedure for HQBIQéEQIQ- 99:2222222- 3: 77'79 Radford, A., 8. Pope, A. Sazci, M.J. Fraser, J.H. Parish. 1981. Liposome-mediated genetic transformation 0f HQBIQEEQIQ Elfifiéfio M2129§D2§2fl§§~ 134: 557-59 Barratt, R.W. 1974. 9991999919 919999. in Handbook of Genetics, vol.1, ed. R.C. King, pp. 511-29. Plenum Press. Shamoian, C. A., A. Canzanelli, J. Melrose. 1961. Back-mutation of a 9991999919 919999 mutant by a nucleic acid complex from the wild strain. 212221212122222222_2- 47: 208-11 Shockley, T. E., E. L. Tatum. 1962. A search for genetic transformation in Neu19s9o19 919999. .21222121212222212212 61: 567- 72 Mishra, N. C., E. L. Tatum. 1973. Non-mendelian inheritance of DNA-induced inositol independence in 2221222212 2122122111222212211222 70: 3875-79 Mishra, N.C., M.C. Niu, E.L. Tatum. 1975. Induction by RNA of inositol independence in 9991999919 919999. 2122122111222212211222 72: 642-45 Mishra, N. C. 1979. DNA-mediated genetic changes in HEEIQ_EQI§ Qléééio l.§§fl1fli_222121- 113' 255' 59 Orbach, M.J., E.E. Porro, C. Yanofsky. 1986. Cloning and characterization of the gene for p-tubulin from a benomyl-resistant mutant of 9991999919 919999 and its use as a dominant selection marker. 9911991119191. 6: 2452-61 Bull, J.R., J.C. Wootton. 1984. Heavily methylated amplified DNA in transformants of 9991999919 212222. HQEEIQ 310: 701-04 McClung, C.R., Phillips, J.D., Orbach, M.J., Dunlap, J.C. 1989. New cloning vectors using benomyl resistance as a dominant marker for selection in 9991999919 919999 and in other filamentous fungi. 9991_9y9911 13: 299-302 Case, M.B.,M. Schweizer, S.R. Kushner, N.H. Giles. 1979. Efficient transformation of 9991999919 919999 utilizing hybrid plasmid DNA. EIQ§2E2512AQQQ1EQI2H§A 75: 5259-53 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 139' Kinsey, J.A., J.A. Rambosek. 1984. Transformation of 919999 with the cloned 99(glutamate 3222959922 dehydrogenase) gene. 9911991119191. 4: 117-22 Akins, R.A., A.M. Lambowitz. 1985. General method for cloning 9991999919 919999 nuclear genes by complementation of mutants. 9911991119191. 5:2272-78 Kinniard, J.H. M.A. Keighren, J.A. Kinsey, M. Eaton, J.R.S. Fincham. 1982. Cloning of the 99(glutamate dehydrogenase) gene of 9991999919 919999 through the use of a synthetic DNA probe. 9999 20: 387-96 Paietta, J.V., G.A. Marzluf. 1985. Gene disruption by transformation in 9991999919 919999. H911Q§1122121- 5: 1554-59 Rossier, C. A. Pugin, G. Turian. 1985. Genetic analysis of transformation in a microconidiating strain of H§HIQ§EQI§ EIQEEQ- 2211222221. 10: 313’20 Dhawale, S.S., G.A. Marzluf. 1985. Transformation of 9991999919 919999 with circular and linear DNA and analysis of the fate of the transforming DNA. QBII1QQDQS- 10: 205.12 Kim, S.Y., G.A. Marzluf. 1988. Transformation of s 919999 with the 119-1 Gene and the effect of host strain upon the fate of the transforming DNA. 9911199991. 13: 65-70 Asch, D.K., J.A. Kinsey. 1990. Relationship of vector insert size to homologous integration during transformation of 9991999919 919999 with the cloned 99(GDH) gene. 99119991999911 221: 37-43 Paietta, J., G.A. Marzluf. 1985. Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Hfifllflfiflgli 212222- QEII1§§D§E~ 93 383-83 Hughes, K., M.E. Case, R.Geever, D. Vapneck, N.H. Giles. 1983. Chimeric plasmid that replicates autonomously in both 99999119919 9911 and Wm-W 80: 1053-57 Hughes, K., M.E. Case, R. Geever, D. Vapneck, N.H. Giles. 1983. 2199199111999919911999 80: 7678 Stewart, V., S.J. Vollmer. 1986. Molecular cloning of 911-2, a regulatory gene required for nitrogen metabolite repression in 9991999919 919999. 5999 46: 291-95 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 140 Fu, Y.-H., G.A. Marzluf. 1987. Characterization of 911-2, the major nitrogen regulatory gene of 2221222212 212222. 2211221112121. 7: 1691-96 Paietta, J.V., R.A. Akins, A.M. Lambowitz, G.A. Marzluf. 1987. Molecular cloning and characterization of the 919-3 regulatory gene of 9991999919 919ssa. 2212221112121. 7: 2506-11 Ish-Horowitz, D. D. F. Burke. 1981. Rapid and efficient cosmid cloning. 9991919_99199_999. 9: 2989-98 Vollmer, S. J., C. Yanofsky. 1986. Efficient cloning of genes of 2221222212 212222 2122122111222212212 222 33: 4869-73 Glass, N.L., S.J. Vollmer, C. Staben, J. Grotenlueschen, R.L. Metzenberg, C. Yanofsky, 1988. DNAs of the two mating-type alleles of 9991999919 919999 are highly dissimilar. 9919999 241: 570-73 Austin, 8., B.M. Tyler. 1990. Strategies for High- efficiency cotransformation of 9991999919 9199_9. EXE2M¥2211 14: 9’17 Nelson, M.A., G. Morelli, A. Carattoli, N. Romano, G. Macino. 1989. Molecular cloning of a 9991999919 91aSS9 carotenoid biosynthetic gene(albino-B) regulated by blue light and the products of the white genes. 9911991119191. 9: 1271-76 Orbach, M. J., W. P. Schneider, C. Yanofsky. 1988. Cloning of methylated transforming DNA from 2221222212 212222 in 22222112212 2211 22112211121_1. 8: 2211- 13 Natvig, D.C., G. May, J.W. Taylor. 1984. Distribution and evolutionary significance of mitochondrial plasmids in 9991999919 spp. 21229222121- 159: 283-93 Nargang, E.E., J.B. Bell, L.L. Stohl, A.M. Lambowitz. 1984. The DNA sequence and genetic organization of a 9991999919 mitochondrial plasmid suggest a relationship to introns and mobile elements. 9911 38: 441-53 Lambowitz, A.M. 1989. Infectious introns. 9911 56: 323-26 Stohl, L.L., A.M. Lambowitz. 1983. Construction of a shuttle vector for the filamentous fungus 212222. 2122122111222212211222 80: 1058-62 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 141 Stohl, L.L., R.A. Akins, A.M. Lambowitz. 1984. Characterization of deletion derivatives of an autonomously replicating 9991999919 plasmid. 2221212_A2122_B22. 12: 6169-78 Grant, D.M., A.M. Lambowitz, J.A. Rambosek, J.A. Kinsey. 1984. Transformation of 9991999919 919999 with recombinant plasmids containing the cloned glutamate dehydrogenase(99) gene: Evidence for autonomous replication of the transforming plasmid. H911Q§ll22191- 4: 2041-51 Kuiper, M.T.R., H. de Vries. 1985. A recombinant plasmid carrying the mitochondrial plasmid sequence of o 1919199919 LaBelle yields new H§B£Q§E_£§ plasmid derivatives in 9991999919 919999 transformants. 1985. 99111999_1. 9: 471-77 Fehér, 2., M. Schalablik, A. Kiss, A. z§indely, G. Szabo. 1986. Characterization of 191 transformants of 9991999919 919999 obtained with a recombinant cosmid pool. 9911199991. 11: 131-37 Selker, E.U., P.W. Garrett. 1988. DNA sequence duplications trigger gene inactivation in 2221222212 212222. 2122222111222222211222 85: 6870-74 Selker, E.U., E.B. Cambareri, B.C. Jensen, K.R. Haack. 1987. Rearrangement of duplicated DNA in specialized cells of 9991999919. 9911 51: 741-52 Cambareri, E.B., B.C. Jensen, E. Schabtach, E.U. Selker. 1989. Repeat-induced G-C to A-T mutations in 2221222212. 2212222 244: 1571-75 Fincham, J.R.S., I.F. Connerton, E. Notarianni, Harrington, K. 1989. Premeiotic disruption of duplicated and triplicated copies of the 9991999919 919999 99(glutamate dehydrogenase) gene. EDII1QQDEL- 15: 327'34 Barbesgaard, P. (1977). Industrial enzymes produced by members of the genus 22221211122. in 22221122 222_R222121222_2f_22221211122. ed. J.R. Smith and J.A. Pateman, Academic Press, London. Davis, M.A., M.J. Hynes. 1989. Regulatory genes in 22221211122 2122122.. 112222.22222- 5: 1-4 Timberlake, W.E., M.A. Marshall. 1988. Genetic regulation of development in 99991911199 91991999. 112292.92223- 4: 162-69 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 142 Christiansen, T., H. Woeldike, E. Boel, S.B. Mortensen, K. Hjortshoej, L. Thim, M. T. Hansen. 1988. High level expression of recombinant genes in 22221211122 212222 21211222221222 6: 1419-22 Gwynne, D.I., F.P. Buxton, S.A. Williams, S. Garven, R.W. Davies. 1987. Genetically engineered secretion of active human interferon and a bacterial endoglucanase from 22221211122 2122122.. 21211222221222 5: 713-19 Upshall, A., A.A. Kumar, M.C. Bailey, M.D. Parker, M.A. Favreau, K.P. Lewison, M.L. Joseph, J.M. Maraganore, G.L. McKnight. 1988. Secretion of active human tissue plasminogen activator from the filamentous fungus 99991911199 919u1a9s. fliozlec99o1ogy 5: 1301-04 Turnbull, I.F., Rand, K., Willets, N.S., Hynes, M.J. 1989. Expression of the Esc9erichi9 9011 enterotoxin subunit B gene in 99991911199 9id919ns by the 9998 promoter. 91911999991999 7: 169-73 Smith, D.J., M.K.R. Burnham, J. Edwards, A.J. Earl, G. Turner. 1990. Cloning and heterologous expression of the penicillin biosynthetic gene cluster from 22212111122 22122222222 21211222221222 8: 39-41 Ballance, D.J., F.P. Buxton, G. Turner. 1983. Transformation of 9S9ergi11us 91dul99s by the orotidine-5’-phosphate decarboxylase gene of 2221222212 212222 212222212122222122212222 112: 284-89 Davig, M.A., Hynes, M.J. 1987. Complementation of 919A regulatory gene mutations of AS9er911199 919u1ans by the heterologous regulatory gene 911-2 of 2221222212 212222. 2122122111222212212222 84: 3753-57 Connerton, I.F., J.R.S. Fincham, R.A. Sandeman, M. J. Hynes. 1990. Comparison and cross-species expression of the acetyl-CoA synthetase genes of the ascomycete fungi. 22221211122 21221222 and 2221222212 212222. 2211212122121 4: 451- 460 Tilburn, J., C. Scazzocchio, G. G. Taylor, J. H. Zabicky-Zissman, R. A. Lockington, R. W. Davies. 1983. Transformation by integration in 99991911199 21921222- 922. 26: 205 21 Hynes, M.J., C.M. Corrick, J.A. King. 1983. Isolation of genomic clones containing the 9998 gene of 99991911199 91991999 and their use in the analysis of 191. 192.. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 143 structural and regulatory mutations. 9919991119191. 3: 1430-39 Werners, K., T. Goosen, L.M.J. Wennekes, J. Visser, C.J. Bos, H. W. J. van den Broek, R.F.M. van Gorcom, C. A. M. J. J. van den Hondel, P. H. Pouwels. 1985. Gene amplification in 59991911199 by transformation with vectors containing the 9998 gene. 99911999_9. 9: 361-68 Yelton, M.M., J.E. Hamer, W.E. Timberlake. 1984. Transformation of Asp9r9111us 91991999 by using a 212C plasmid- 2122222111222212212222 81: 1470-74 Miller, B. L., K. Y. Miller, and W. E. Timberlake. 1985. Direct and indirect gene replacements in 22221211122.21221222 22122211121_1. 5: 1714- -21 Barnes, D. E. D. W. MacDonald. 1986. Behaviour of recombinant plasmids in 59991911199 91991999: structure and stability. 9929199999.10:767-75 Dunne, P.W., and B.R. Oakley. 1988. Mitotic gene conversion, reciprocal recombination and gene replacement at the 9992, beta-tubulin, locus of 22221211122 21221222 2211.22122222- 213: 339- 45 May, G.S., J. Gambino, J.A. Weatherbee, N.R. Morris. 1985. Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in 22221211122 21221222. 2122112 9191. 101: 712-19 Jones, I. G., H. M. Sealy-Lewis. 1989. Chromosomal mapping and gene disruption of the 999111 gene in 22222911122 21221222 2211.22221 15: 135- 42 Jones, I. G., H. M. Sealy-Lewis. 1990. Chromosomal mapping of an 9199 disruption with respect to 9995 in 22221211122 21221222 22111222222 17:81-83 Rothstein, R.J. 1983. One-step gene disruption in Yeast- 2222222_2222221. 101: 202-11 Aramayo, R., Adams, T.K., Timberlake, W.E. 1989. A large cluster of highly expressed genes is dispensable for growth and development in 22221211122 21221222. 22222122 122: 65-71 Casadaban, M., Martinez-Arias, A., Shapina, S., Chow, J. 1983. Beta-galactosidase gene fusions for analyzing gene expression in ' 22222112212 and yeast. M999999_Enzxm91. 100: 293—308 van Gorcom, R.F.M., P.H. Pouwels, T. Goosen, J. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 144 Visser, H. W. J. van den Broek, J. E. Hamer, W.E. Timberlake, C. A. M. J. J. van den Hondel.1985. Expression of an 99999919919 9911_p-galactosidase fusion in 5S9e99111us 91991999. gene 40: 99-106 van Gorcom, R.P.M., Punt, P.J., Powels, P.H., van den Hondel, C.A.M.J.J. 1986. A system for the analysis of expression signals in 59999911199. £999 48: 211-17 van Gorcom, R. F. M., van den Hondel, C. A. M. J. L 1988. Expression analysis vectors for 5s 9999111us 91991a95. 2991919 5c 19$ Res. 16: 9052 Hamer, J.E., Timberlake, W.E. 1987. Functional organization of the 22221211122 21221222 112C promoter. 9919991119191 7: 2352-59 Davis, M.A., Cobbett, c.s., Hynes, M.J. 1988. An 9998-1992 fusion for studying gene regulation in 5999991119_. Ge99 63: 199-212 Kelly, J. M., M. J. Hynes. 1987. Multiple copies of the 2228 gene of 22221211122 21221222 cause titration of trans-acting regulatory proteins. £2112§§2§_- 12: 21'31 Andrianopoulos, A., M. J. Hynes. 1988. Cloning and analysis of the positively acting regulatory gene 222R from 22221211122 2212221122121 8: Hynes, M.J., C.M. Corrick, J.M. Kelly, T.G. Littlejohn. 1988. Identification of the sites of action for regulatory genes controlling the 9m mdS gene of 22221211122 21221222 2212221122121 8: 2589-96 3532-41 Punt, P.J., R.P. Oliver, M.A. Dingemanse, P.H. Pouwels, C.A.M.J.J. van den Hondel. 1987. Transformation of 59999911199 91991999 based on the hygromycin B resistance marker from 39999919919 . £999 56: 117-24 Kelly, J. M, M. J. Hynes. 1985. Transformation of 22221211122 21221 by the 2228 Gene of 22221211122 21221222 2222.2 4: 475-79 Weltring, K.-M., B.G. Turgeon, O.C. Yoder, H.D. VanEtten. 1988. Isolation of a phytoalexin- detoxification gene from the plant pathogenic fungus 9999919 99emato99cca by detecting its expression in 22221211122 2122122.. 2222 68: 335-44 Wernars, K., T. Goosen, K. Swart, H.W.J. van den Broek. 1986. Genetic Analysis of 59999911199 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 145 21221222 2228 Transformants. 2211222122221- gos: 312- 17 Upshall, A. 1986. Genetig and molecular characterization of 9993 transformants of 22221211122 21221222 2211122221 10: 593-99 Cullen, D., L.J. Wilson, G.L. Grey, D.J. Henner, D. J. Ballance. 1987. Sequence and centromere proximal location of a transformation enhancing fragment 9991 from 22221211122 21221222 2221212_22122_B22. 15: 9163-75 Lazarus, C.M., Kfintzel, H. 1981. Anatomy of amplified mitochondrial DNA in "ragged" mutants of 99999911199 99999199991: excision points within protein genes and a common 215 bp segment containing a possible origin of replication. Q999,Ge9e9. 4: 99-107 Beri, R.K., E.L. Lewis, G. Turner. 1988. Behaviour of a replicating mitochondrial DNA sequence from 22221211122 22212122221 in 2222221222222 2212212122 and 22221211122 21221222- 921112222_- 13: 479- -86 Boylan, M.T., M.J. Holland, W.E. Timberlake. 1986. 9999999999999 c99evisia9 centromere CEN11 does not induce chromosome instability when integrated into the 22221211122 21221222 genome- H911§§ll1fiiglo 6: 3621-25 Yelton, M. M., J. E. Hamer, E. R. de Souza, E. J Mullaney, W. E. Timberlake. 1983. Developmental regulation of the 22221211122 21221222 112C gene- 2122122111222212211222 80: 7576-80 McKnight, 6.1., H. Kato, A. Upshall, M.D. Parker, P.J. O'Hara. 1985. Identification and molecular analysis of a third 22221211122 21221222 alcohol dehydrogenase gene. £999_9. 8: 2093-99 Oakley, B.R., Rinehart, J.E., Mitchell, E.L., Oakley, C.E., Carmona, C. Gray, G.L., May, 6.8. 1987. Cloning, mapping and molecular analysis of the 999G(orotidine-5'-phosphate decarboxylase) gene of 22221211122 21221222 2222 61: 385-99 Oakley, C. E., C. F. Weil, P. L. Kretz, B. R. Oakley. 1987. Cloning of the 91993 Locus of 99999911199 21221222. 2222 53: 293-98 Ballance, D. J., Turner, G. 1986. Gene cloning in 59999911199 91991999: isolation of the isocitrate lyaae gene(222D). 2211222122221- 202: 271-75 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 146 Timberlake, W.E., Boylan, M.T., Cooley, M.B., Mirabito, P.H., O’Hara, E.B., Willett, C.E. 1985. Rapid identification of mutation-complementing restriction fragments from 59999911199 91991999 cosmids. 999199991. 9: 351-55 Yelton, M. M., Timberlake, W. E., van den Hondel, C. A. M. J. J. 1985. A cosmid for selecting genes by complementation in 59999911199 selection of the developmentally regulated 25 locus. 21221221112222.2211222 82: 834- 38 Johnstone, I. L., Hughes, 8. G., Clutterbuck, A. J. 1985. Cloning an 59999911199 91991999 developmental gene by transformation. £999_1. 4: 1307- 11 Cullen, D., 8.2. Leong, L.J. Wilson, D.J. Henner. 1987. Transformation of 59999911199 9i9u199s with the hygromycin-resistance gene, 999. £999 57: 21-26 Ward, M., B. Wilkinson, G. Turner. 1986. Transformation of 22221211122 21221222 with a cloned. oligomycin-resistant ATP synthase subunit 9 gene. 99119991999_9. 202: 265-70 Buxton, F.P., D.I. Gwynne, R.W. Davies. 1985. Transformation of 59999911199 91999 using the 2128 gene of 22221211122 21221222. 9999 37: 207-14 Goosen, T., G. Bloemheuvel, C. Gysler, D.A. deBie, H.W.J. van den Broek, K. Swart. 1987. Transformation of 59999911199 91999 using the homologous orotidine-S'-phosphate-decarboxylase gene. 9999199999. 11: 499-503 van Hartingsveldt, W., I.E. Mattern, C.M.J. van Zeijl, P. H. Pouwels, C. A. M. J. J. van den Hondel.1987. Development of a homologus transformation system for 22221211122 21221 based on the 2x16 gene- 2211222122221 206: 71-75 Unkles, S.B., E.I. Campbell, D. Carrez, C. Grieve, R. Contreras, W. Piers, C.A.M.J.J. Van den Hondel, J.R. Kinghorn. 1989. Transformation of 59999911199 91999 with the homologous nitrate reductase gene. £999 78: 157-66 Campbell, E.I., S.E. Unkles, J.A. Macro, C. van den Hondel, R. Contreras, J. R. Kinghorn. 1989. Improved transformation efficiency of 59999911199 91999 using the homologous 919D gene for nitrate reductase. 9999199999.16: 53-56 234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 147 Ward, M., L. J. Wilson, C. L. Carmona, G. Turner. 1988. The 911C3 gene of 59999911199,n1999: Isolation, sequence and use as a selectable marker for transformation. 9999199999. 14: 37-42 Mattern, I.E., S. Unkles, J. R. Kinghorn, P.H. Pouwels, C. A. M. J. J. van den Honde1.1987. Transformation of 59999911199 991999 using the 2 21221 2216 gene- 22112221_2221. 210: 460- 61 de Ruiter-Jacobs, Y.M.J.T., M. Broekhuijsen, S.B. Unkles, R.I. Campbell, J.R. Kinghorn, P.H. Powels, C.A.M.J.J. van den Hondel. 1989. A gene transfer system based on the homologous 999G gene and efficient expression of bacterial genes in 5s9e99111us 09929 . Q999,Ge9e . 16: 159-63 Unkles, S.B., E.I. Campbell, Y.M.J.T. de Ruiter-Jacobs, M. Broekhuijsen, J.A. Macro, D. Carrez, R. Contreras, C. A. M. J. J. van den Hondel, J. R. Kinghorn. 1989. The development of a homologous transformation system for 59999911199 based on the nitrate assimilation pathway: A convenient and general selection system for filamentous fungal transformation. 99119991999991 218: 99-104 Hahm, Y. T., C. A. Batt. 1988. Genetic transformation of an 9998 mutant of 59999911199m m. 2221122211221212122121 54: 1610-11 Ward, M., K. H. Kodama, L. J. Wilson. 1989. Transformation of 59999911199 9999991 and 5. 91999 by electroporation. £99_99991.13: 289-93 Ward, M., L. J. Wilson, K. H. Kodama, M. W. Rey, R. M. Berka. 1990. Improved production of chymosin in 59999911199 by expression as a glucoamylase-chymosin fusion. 21211222221221 8: 435- 40 Berka, R.M., M. Ward, L.J. Wilson, R.J. Hayenga, K.H. Kodama, L.P. Carlomango, S.A. Thompson. 1990. Molecular cloning and deletion of the gene encoding asvergillopepsin A from 22221211122 2222211. 2222 35: 153-162 Mullaney, E. J., P. J. Punt, C. A. M. J. J. van den Hondel. 1988. DNA mediated transformation of 22221211122 112222 2221121212212112121222221- 28: 451- 54 Wendt, S., M. Jacobs, U. Stahl. 1990. Transformation of 59999911199 919999999 to hygromycin B resistance. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 148 922215222§2 17: 21'24 Stahl, U., P.A. Lemke, P. Tudzynski, U. Kfick, K. Esser. 1978. Evidence for plasmid like DNA in a filamentous fungus, the ascomycete 299oS9o9a 99999199. 901,9e9,G9ne9. 162: 341-43 Osiewacz, H.D., K. Esser. 1984. The mitochondrial plasmid of 299999999 99999199: A mobile intron of a mitochondrial gene. 9999199999. 8: 299-305 Tudzynski, P., U. Stahl, K. Esser. 1980. Transformation to senescence with plasmid like DNA in the ascomycete 222222212 22221122. 221112222.- 2: 181-84 Stahl, U., P. Tudzynski, U. Kfick, K. Esser. 1982. Replication and expression of a bacterial- mitochondrial hybrid plasmid in the fungus 299os9o9a 2222112.. 2122122211222212211222 79: 3641-45 Sainsard-Chanet, A., 0. Begel. 1986. Transformation of yeast and 20d059or9: Innocuity of senescence- specific DNAs. 99119991999_9. 204: 443-51 J. Bégueret, V. Razanamparany, M. Perrot, C. Barreau. 1984. Cloning gene 9995 for the orotidylic acid pyrophosphate of the filamentous fungus 222222212 22221122- 2222 32: 487-92 Turcg, B., J. Begueret. 1987. The 9995 gene of the filamentous fungus 299999999 99999199: Nucleotide sequence and expression in transformed strains. 9999 53: 201-09 Razanamparany, V., J. Begueret. 1986. Positive screening and transformation of 9995 mutants in the fungus 222222212 22221122: Characterization of the transformants. QBIILQQDEL- 10: 311-17 Brygoo, Y., R. Debuchy. 1985. Transformation by integration in 299999999 99999199: 1. Methodology and phenomenology. 9911999199999. 200: 128-31 Fernéndez-Larrea, J, U. Stahl. 1989. Transformation of 299999999 99999199 with a dominant resistance gene. Q9991_§99_9. 16: 57-60 Picard, M., R. Debuchy, J. Julien, Y. Brygoo. 1987. Transformation by integration in 29doS9o99 99999199. II. Targeting to the resident locus with cosmids and instability of the transformants. 221122212222_- 210: 129‘34 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 149 Debuchy, R., E. Coppin-Raynal, D. Le Coze, Y. Brygoo. 1988. Chromosome walking towards a centromere in the filamentous fungus 299999999 99999199: Cloning of a sequence lethal at a two-copy state. 99991999_9. 13: 105-11 Turcq, B., M. Denayrolles, J. Begueret. 1990. Isolation of the two allelic incompatibility genes 9 and S of the fungus 222222212 22221122 2211122222. 17: 297- 303 Perrot, M., C. Barreau, J. Bégueret. 1987. Nonintegrative transformation in the filamentous fungus 299999999 99999199: Stabilization of a linear vector by the chromosomal ends of 19999999999 12212222112. 2211221112121. 7: 1725-30 Cerda-Olmedo, E. 1977. Behavioral genetics of Phycomyces. 22212221212122121- 31: 535-47 Arnau, J., P.J. Murillo, S. Torres-Martinez. 1988. Expression of Tn5-derived kanamycin resistance in the fungus 2121221222222- 2211222122221. 212: 375-77 Revuelta, J.L., M. Jayaram. 1987. Transformation of 299com99e9 9199991999999 to G418 resistance by an autonomous replicating plasmid. 299c.9991. 222212211222 83: 7344-47 Suarez, T., A.P. Eslava. 1988. Transformation of 2999999999 with a bacterial gene for kanamycin resistance. 9911999199999. 212: 120-23 Arnau, J., A. Ortiz, J.C. Gomez-Fernandez, F.J. Murillo, S. Torres-Martinez. 1988. Liposome- protoplaet fusion in 2222222222 2122221222222- EEM212121221211L222. 51: 37-40 Raper, C. A. 1983. Controls for development and differentiation of the dikaryon in basidiomycetes. in 222222212.2212221122_222_211121221121122_12_22221. ed. J. W. Bennet, and A. Ciegler, pp. 195-238. Dekker, New York. Froeliger, E.H., A.M. Munoz-Rivas, C.A. Specht, R.C. Ullrich, C.P. Novotny. 1987. The isolation of specific genes from the basidiomycete 9991999991199 EQEEQDQ- QBIILQQDEE- 123 547'54 Munoz-Rivas, A., C.A. Specht, B.J. Drummond, E. Froeliger, C.P. Novotny, R.C. Ullrich. 1986. Transformation of the basidiomycete, 9991999991199 £2EEBD§o QBIILQEBEE' 205: 103'05 266. 267. 268. 269. 270. 271. 272. 273. 274. 275. 276. 150 Specht, C.A., A. Munoz-Rivas, C.P. Novotny, R.C. Ullrich. 1988. Transformation of 2991999991199 9999999: An analysis of parameters for improving transformation frequencies. 2991999911 12: 357-366 Giasson, L., C.A. Specht, C. Milgrim, C.P. Novotny, R. C. Ullrich. 1989. Cloning and comparison of An mating-type alleles of the basidiomycete 2221222221122 2222222 2211222122221. 218: 72-77 Casselton, C. A. 1978. Dikaryon formation in higher basidiomyceteS- in 122.21122221222_22221-. vol. 3 ed. J. E. Smith, and D. R. Berry, pp. 275- 314. Edward Arnold, London. Binninger, D.M., C.Skrzynia, P.J. Pukkila, L.A. Casselton. 1987. DNA-mediated transformation of the basidiomycete Co9ri9us 9ine999 . E929_9. 6: 835-40 Mellon, F.M., P.F.R. Little, L.A. Casselton. 1987. Gene cloning and transformatin in the basidiomycete fungus 99991999 91999999: Isolation and expression of the isocitrate lyase gene(222-7)- 2211222122221. 210: 352-57 Mellon, F.M., L.A. Casselton. 1988. Transformation as a method of increasing gene copy number and gene expression in the basidiomycete fungus 21221222. 2211122221. 14: 451-56 Casselton, L.A., A. de la Fuente Herce. 1989. Heterologous gene expression in the basidiomycete fungus 22211222 21221222. 2211122222. 16: 35-40 Burrows, D.M., T.J. Elliott, L.A. Casselton. 1990. DNA-mediated transformation of the secondarily homothallic basidiomycete 99991999 911999999 99991999991 17: 175-177 Rossignol, J.-L., A. Nicolas, H. Hamza, T. Langin. 1984. The origin of gene conversion and reciprocal exchange in 222222122. 2212.221122_221221_22221 22221121211 49: 13-21. Faugeron, G., C. Goyon, A. Gregoire. 1989. Stable allele replacement and unstable non-homologous integration events during transformation of 222222122 12221222. 2222 76: 109-19 Goyon, C., G. Faugeron. 1989. Targeted transformation of 599999199 19999999 and de novo methylation of the resulting duplicated DNA sequences. 9911991112191. 9: 2818-27 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. 287. 151 Faugeron, G., L. Rhounim, J.-L. Rossignol. 1990. How does the cell count the number of ectopic copies of a gene in the premeiotic inactivation process acting in 222222122 12221222. 22221122 124: 585-591 Pirt, S. J. 1987. Microbial physiology in the penicillin fermentation. 199999_9199999991. 5: CantoraL J. M., B. Diez, J. L. Barredo, E. Alvarez, J. F. Martin. 1987. High-frequency transformation of 22212111122 221222222_2. 21211222221222 5: 494- 97 69-72 Whitehead, M.P., S.B. Unkles, M. Ramsden, E.I. Campbell, S.J. Gurr, D. Spence, C. Van den Hondel, R. Contreras, J.R. Kinghorn. 1989. Transformation of a nitrate reductase deficient mutant of 29919111i9m ch99sogenu9 with the corresponding 22221211122 21221 and 2- 21221222 2120 genes. 901,999,6996 . 216: 408-11 Picknett, T. M., G. Saunders, P. Ford, G. Holt. 1987. Development of a gene transfer system for 22212111122 22122222222 22111222.;- 12: 449- 55 Kolar, M., P.J. Punt, C.A.M.J.J. van den Hondel, H. Schwab. 1988. Transformation of 29919111199 99999999999 using dominant selection markers and expression of an 22221112212 2211 1222 fusion gene. 9999 62: 127- 34 Beri, R. K., G. Turner. 198L Transformation of 29919111199c c999sogenu9 using the 222.1211122.21221222 222$ gene as a dominant selective marker. 9999199999. 11: 639-41 Bull, J.H., D.J.-Smith, G. Turner. 1988. Transformation of 2e91c1111um 99999999999 with a dominant selectable marker. 9999199999. 13: 377-82 Carramolino, L., M. Lozano, A. Perez-Aranda, V. Rubio, F. SAnchez. 1989. Transformation of 29919111199 99999999999 to sulfonamide resistance. 9999 77: 31- -38 StahL U., E. Leitner, K. Esser. 1987. Transformation of 22212111122 22122222222 by a vector containing a mitochondrial origin of replication. 599119199991911 2121222221. 26: 237 41 Cantwell, C.A., R.J. Beckmann, J.E. Dotzlaf, D.L. Fisher, P.L. Skatrud, W.-K. Yeh, S.W. Queener. 1990. 288. 289. 290. 291. 292. 293. 294. 295. 296. 297. 298. 152 Cloning and expression of a hybrid 599999999999 212221122122 2212 gene in 22212111122 22122222222 92111222221 17: 213-221 Bunnell, C. A., W. D. Luke, F. M. Perry. 1986 Industrial manufacture of cephalosporins. in fi999_199999 A2212122122_f21_21121221_222. ed- 8 F. Queener. J. A. Webber, and S. W. Queener, pp. 255- 278. Marcel Dekker, New York. Skatrud, P. L., S. W. Queener, L. G. Carr, D. L. Fisher. 1987. Efficient integrative transformation of 22222122221122 2212222122-2211122221 12: 337-48 Skatrud, P.L., A.J. Tietz, T.D. Ingolia, C.A. Cantwell, D. L. Fisher, J. L. Chapman, S. W. Queener. 1989. Use of recombinant DNA to improve production of cephalosporin C by 22222122221122 2212222122 21221222221222 7: 477- 85 Somkuti, G.A. 1974. Synthesis of cellulase by 99999 22211122 and 22221 222221. 112221212122121- 81: 1-6 van Heeswijck, R., M.T.G. Roncero. 1984. High frequency transformation of 99999 with recombinant plasmid DNA. 221122212_B221222222- 49: 691-702 van Heeswijck, R. 1986. Autonomous replication of plasmids in 99999 transformants. 99999999_B999 99991 51: 433-43 Roncero, M. I. G., L. P. Jepsen, P. Stroman, R. van Heeswijck. 1989. Characterization of a 1999 gene and an 222 element from 22221 21121221121222 9999 84: 335-43 Monteoourt B S 1983 11122222122 122221 cellulases. I99999_fl199999991. 1: 156- -61 Van Arsdell, J.N., S. Kwok, V.L. Schweickart, M.B. Ladner, D.R. Gelfand, M.A. Innis. 1987. Cloning, characterization, and expression in 9999999999999 2212212122 of endogluoanase I from 11122222122 122221. 21221222221222 5: 60-64 Penttilé, M., H. Nevalainen, M. Rattd, E. Salminen, J. Knowles. 1987. A versatile transformation system for the cellulolytic filamentous fungus 19199999999 999991. 9999 61: 155-64 Harrki, A., J. Uusitalo, M. Bailey, M. Penttilé, J.R.C. Knowles. 1989. A novel fungal expression system: Secretion of active calf chymosin from the filamentous fungus 11122222122 122221- 299. 300. 301. 302.- 303. 304. 305. 306. 307. 308. 309. 310. 153 21221222221222 7: 596-603 Alic, M., J.R. Kornegay, D. Pribnow, M.H. Gold. 198L Transformation by complementation of an adenine auxotroph of the lignin degrading basidiomycete 2222212222212 2212222221122 222112221122 919999191. 55: 406- -11 Alic, M., R.K. Clark, J.R. Kornegay, M.H. Gold. 1990. Transformation of 2222212222212 221222221122 and 999999 with adenine biosynthetic genes 2221222212 from 2221222221122 22222221 2211122221. 17: 305-311 Randall, T.A., T.R. Rao, C.A. Reddy. 1989. Use of a shuttle vector for the transformation of the white-rot basidiomycete 2999999999999 2212222221122. 212222212122222122212222. 161: 720-25 Rao, T.R., Reddy, C.A. 1984. DNA sequences from a ligninolytic filamentous fungus 29ane9ocha999 9999999999199 capable of autonomous replication in Yeast. 2122222121222221222122__ 118: 821- 27 Holliday. R- 1974. 22111222 222212. in 22222222 9fi_§9999199, ed. R.C. King, pp. 575-95. Plenum, New York. Wang, J., D.W. Holden, S.A. Leong. 1988. Gene transfer system for the phytopathogenic fungus 99911999 22221.. 2122122111222212211222 85: 865-69 Banks, G. R., S. Y. Taylor. 1988. Cloning of the PYR3 gene of 99911999 999919 and its use in DNA transformation. 9919991112191. 8: 5417-24 Fotheringham, S., W.K. Holloman. 1990. Pathways of tranformation in 99911999 999919 determined by DNA conformation. 99999199 124: 833-843 Kronstad, J.W., J. Wang, S.F. Covert, D.W. Holden, G.L. McKnight, S.A. Leong. 1989. Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus 99911999 22221.. 2222 79: 97-106 Holden, D.W., J.W. Kronstad, S.A. Leong. 1989. Mutation in a heat-regulated hsp70 gene of 99911999 22221., EMEQ_J2 331927'34 Kronstad, J. W., S. A. Leong. 198L Isolation of two alleles of the b locus of 99911999 999919. EIQQ1H2111222212211H§A 86: 978-82 Schulz, B., F. Banuett, M. Dahl, R. Schlesinger, W. 311. 312. 313. 314. 315. 316. 317. 318. 319. 320. 154 Schéfer, T. Martin, I. Herskowitz, R. Kahmann. 1990. The 9 alleles of 9. 999919, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif. 9911 60: 295- 306 Tsukuda, T., S. Carleton, S. Fotheringham, W.K. Holloman. 1989. Isolation and characterization of an autonomously replicating sequence from 22111222 222212 22112211121_1. 8: 3703-09 Samac, D.A., Leong, S.A. 1989. Characterization of the termini of linear plasmids from 9ect919 999999999999 and their use in construction of an autonomously replicating transformation vector. 2211122221. 16: 137-94 Holden, D. W., J. Wang, S. A. Leong. 1988. DNA- mediated transformation of 99911999 999991 and 22111222 21212 22221211221121221_221221- 33: 235- -39 Bej, A.K., M.H. Perlin. 1989. A high efficiency transformation system for the basidiomycete 99911999 919199ea employing hygromycin resistance and lithium-acetate treatment. 9999 80: 171-76 Kolattukudy, P.E. 1985. Enzymatic penetration of the plant cuticle by fungal pathogens. 22213221222122212211.23: 223-50- Marek, E.T., Schardl, C.L., Smith, D.A. 1989. Molecular transformation of 29999199 991991 with an antibiotic resistance marker having no fungal DNA homology. 9999199999. 15: 421-28 Soliday, C.L., W.H. Flurkey, T.W. Okita, P.E. Kolattukudy. 1984. Cloning and structure determination of cDNA for cutinase, an enzyme involved in fungal penetration of plants. £1.21H221152222fisi1flfib 31: 3939-43- Soliday, C.L., M.B. Dickman, P.E. Kolattukudy. 1989. Structure of the cutinase gene and detection of promoter activity in the 5'-flanking region by fungal transformation. 91999999191. 171: 1942-51 Dickman, M.B., G.K. Podila, P.E. Kolattukudy. 1989. Insertion of cutinase gene into a would pathogen enables it to infect intact host. 999999 342: 446-48 Madhosingh, C., W. Orr. 1985. Zearalenone production in 29999199 99199999 after transformation with DNA of 1- 21221222122. 21221.221221. 34: 402-07 321. 322. 323. 324. 325. 326. 327. 328. 329. 330. 331. 332. 155 Kistler, H.C., D.R. Benny. 1988. Genetic transformation of the fungal plant wilt pathogen, 12221122 222222122- 2211122221. 13: 145-49 Langin, T., M.J. Daboussi, C. Gerlinger, Y. Brygoo. 1990. Influence of biological parameters and gene transfer technique on transformation of 29999199 232222222- 9221192222- 173 313-319 Powell, W.A., H.C. Kistler. 1990. In vivo rearrangement of foreign DNA by 29999199 9999999999 produces linear self-replicating plasmids. 99999999191. 172: 3163-3171 Hynes, M. J. 1989. Complementation of an 99999911199 91991999 mutation by a gene from the basidiomycete .222122_ 21222222- EKE1MYEQl- 133 195 98 Malardier, L., M.J. Daboussi, J. Julien, F. Roussel, C. Scazzocchio, Y. Brygoo. 1989. Cloning of the nitrate reductase gene(919D) of 99999911199 91991999 and its use for transformation of 22221122 22222212.. 2222 78:147-56 Grindley, N.D.F., C.M. Joyce. 1980. Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903. 2999199911999919911999 77: 7176-80 Jimenez, A., J. Davies. 1980. Expression of a transposable antibiotic resistance element in 2222221222222. 221212 287: 869-71 Sreekrishna, K., T.D. Webster, R.C. Dickman. 1984. Transformation of 9199999999999 199919 with the kanamycin(G418) resistance gene of Tn903. 9999 28: 73-81 Turgeon, B.G., R.C. Garber, O.C. Yoder. 1987. Development of a fungal transformation system based on selection of sequences with promoter activity. 99119911191_1. 7: 3297-305 Wéstemeyer, J., A. Burmester, C. Weigel. 1987. Neomycin resistance as a dominantly selectable marker for transformation of the zygomyceter 9991919 919999. QQII1QQQQL- 123'625-27 Roberts, I. N., Oliver, R. P., Punt, P. J., van den Hondel, C. A. M. J. J. 1989. Expression of the 99999919919 9911 p-glucuronidase gene in industrial and phytopathogenic filamentous fungi. Qur9,999e . 15: 177-80 Botchan, M., J. Stringer, T. Mitchison, J. Sambrook. 1980. Integration and excision of SV40 333. 334. 335. 336. 337. 338. 339. 340. 341. 342. 343. 156 DNA from the chromosome of a transformed cell. 9911 20: 143-52 Gutai, M.W., D. Nathans. 1978. Evolutionary variants of simian virus 40: Cellular DNA sequences and sequences at recombinant joints of substituted variants. 9199119191. 126: 275-88 Bullock, 2., w. Forrester, M. Botchan. 1984. DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells. I1M2112121- 1743 55-84 Folger, K.R., E.A. Wong, G. Wahl, M.R. Capecchi. 1982. Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules. 991.Ce1l.9101. 2: 1372-87 Avalos, J., R.P. Geever, M.E. Case. 1989. Bialophos resistance as a dominant selectable marker in HQBIQfiEQIQ 212222. QQEELQEDELL 15: 369’72 John, M. A., J. F. Peberdy. 1984. Transformation of A2221211122.21221222 using the 2128 Gene- 2222222_2121221I222221 6: 386- 89 Da Silva, A.J.F, H. Whittington, J. Clements, C. Roberts, A. R. Hawkins. 1986. Sequence analysis and transformation by the catabolic3 3-dehydroquinase (2213) gene From 22221211122 21221222- 919999911. 240: 481-88 Durrens, P., P.M. Green, H.N. Arst, Jr., C. Scazzocchio. 1986. Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in 99999911199 9199199_. 991,G99,Ge9e . 203: 544-49 de Graff, L., H. van den Broek, J. Visser. 1988. Isolation and transformation of the pyruvate kinase gene of 82221211122 21221222. 2211122221- 13:315-21 Buxton, F.P., D.I. Gwynne, R.W. Davies. 1989. Cloning of a new bidirectionally selectable marker for 99999911199 strains. 9999 84: 329-34 Turgeon, B.G., R.C. Garber, D.C. Yoder. 1985. Transformation of the fungal maize pathogen 99c9110bolus heterostr09h9s using the A2221211122.21221222 2228 gene- M21_§22_§222§ 2013 450- 53 Parsons, K.A., F.G. Chumley, B. Valent. 1987. Genetic transformation of the fungal pathogen responsible 344. 345. 346. 347. 348. 349. 350. 351. 352. 353. 354. 157 for rice blast disease. 2122122111222212211222 84: 4161-65 Oliver, R.P., I.N. Roberts, R. Harling, L. Kenyon, P.J. Punt, M.A. Dingemanse, C.A.M.J.J. van den Hondel. 1987. Transformation of £91919 99199, a fungal pathogen of tomato, to hygromycin B resistance. 9999199999. 12: 231-33 Rodriguez, R.J., O.C. Yoder. 1987. Selectable genes for transformation of the fungal plant pathogen 212221212 f 82- 2122212112 (22112121112222 11222221212222). 2222 54: 73-81 Farman, M.L., R.P. Oliver. 1988. The transformation of protoplasts of 1221222222112 22221222 to hygromycin B resistance. 9999199999. 13: 327-30 Henson, J.M., N.K. Blake, A.L. Pilgeram. 1988. Transformation of 22222222222222 21221212 to benomyl resistance. 9999199999. 14: 113-17 Cooley, R.N., R.K. Shaw, F.C.H. Franklin, C.E. Caten. 1988. Transformation of the phytopathogenic fungus 22212112 2222122 to hygromycin B resistance. 9999199999. 13: 383-89 Manavathu, E. K., K. Suryanarayana, S. E. Hasnain, M. Leung, Y. F. Lau, W.-C. Leung. 1987. Expression of 999999 9199199 virus thymidine kinase gene in aquatic filamentous fungus A99199,999199x99119. 9999 57: 53- 59 Dickman, M. B. 1988. Whole cell transformation of the alfalfa fungal pathogen 22112121112222 11112111 2211122221 14: 241-46 Thomas, M. D., Kenerly, C.M. 1989. Transformation of the myoovarasite 21122122122 2211122221 15: 415- -20 Blakemore, E.J.A., M.J. Dobson, M.J. Hocart, J.A. Lucas, J.E. Peberdy. 1989. Transformation of 2222222212222212112 221221112221222 using two heterologous genes. 99991_99999. 16:177-80 Huang, D., Bhairi, S., Staples, R.C. 1989. A transformation procedure for 99999991919 99999999. QHII1_§2222- 153411-14 Bernier, L., Cooper, R.M., Charnley, A.K., Clarkson, J.M. 1989. Transformation of the entomopathogenic fungus 99999919199 9919991199 to benomyl resistance. EEH§_M192221211_L§§21 603261-56 355. 356. 357. 358. 359. 360. 361. 362. 363. 2 158 Goettel, M.S., R. J. S. Leger, S. Bhairi, M. K. Jung, B. R. Oakley, D. W. Roberts, R. C. Staples. 1990. Pathogenicity and growth of 99999919199 9919991199 stably transformed to benomyl resistance. 9999,699et. 17: 129-132 Le Chevanton, L, G. Leblon, S. Lebilcot. 1989. Duplications created by transformation in 99999919 9999999999 are not inactivated during meiosis. 2211222122221. 218: 390'96 Osiewacz, H.D., A. Weber. 1989. DNA mediated transformation of the filamentous fungus 9999919919 199999 using a dominant selectable marker. 2221121222121121212222_1- 30: 375-80 Daboussi, M., Djeballi, A., Gerlinger, C., Blaiseau, P.L., Bouvier, I., Cassan, M., Lebrun, M.H., Parisot, D., Brygoo, Y. 1989. Transformation of seven species of filamentous fungi using the nitrate reductase gene of 22221211122 21221222. 2211122221. 15: 453-56 Churchill, A.C.L., L.M. Ciuffetti, D.R. Hansen, H.D. Van Etten, N.K. Van Alfen. 1990. Transformation of the fungal pathogen 2122222221112 2212211122 with a variety of heterologous plasmids. 99991999991 17: 25-31 Burmester, A., A. wostemeyer, J. wostmeyer. 1990. Integrative transformation of a zygomycete, 9991919 919999 with vectors containing repetitive DNA. £9991§9999. 17: 155-161 Caprioglio, D.R., L.W. Parks. 1989. Temporal expression of transcription and relative copy number of plasmid pSFB-l in 22212112122 11222221222222- 11999999191. 171: 4876-80 Ward, M., L.J. Wilson, K.H. Kodama, M.W. Rey, R.M. Berka. 1990. Improved production of chymosin in 99999911199 by expression as a glucoamylase-chymosin fusion. 91941999991991 8: 435-40 Smith, D.J., M.K.R. Burnham, J. Edwards, A.J. Earl, G. Turner. 1990. Cloning and heterologous expression of the penicillin biosynthetic gene cluster from 22212111122 22122222222 21211222221221 8: 39-41 159 CHAPTER 1 A Novel Extrachromosomally Maintained Transformation Vector for the Lignin Degrading Basidiomycete 22222122222122211222221122 in review, Molecular and Cellular Biology 160 ABSTRACT A stable extrachromosomally maintained transformation vector (pGlZ-l) for the lignin-degrading filamentous fungus 2999999999999 9999999999199 is described. The vector is 6.3 kb and contains a 1599r marker, pBR322 991, and a 2.2 kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of 9. 9999s059991um. Vector pGlz-l was able to transform 2. 9999999999199 to G418 resistance and was readily .and consistently recoverable from the total DNA of transformants via 59999919919 9911 transformation. Southern blot analyses indicated that pGlz-l is maintained at a low copy number in the fungal transformants. The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction. Q99 methylation and exonuclease III analyses, respectively, indicated that pGlZ-l undergoes replication in B. 9999999999199 and that it is maintained extrachromosomally in a circular form. The vector is stably maintained in the transformants even after long term non-selective growth. There is no evidence for integration of the vector into the chromosome at any stage. 161 INTRODUCTION In recent years, research has intensified worldwide on the ligninolytic fungus 2999999999999 9999999999199 because of the industrial potential of this organism in biopulping, in the conversion of lignin and lignocellulosic materials to feeds, fuels, and chemicals(22,47), and in detoxifying recalcitrant environmental pollutants such as dioxins, PCBs, and benzo(a)pyrenes(10,11). Lignin peroxidases and manganese peroxidases, two families of extracellular, glycosylated, heme-proteins involved in lignin degradation by B. 9999999999199, have been isolated and characterized(16,22, 47,48). Genetic studies with this organism have been limited, but auxotrophic mutants(15,26,27) and mutants lacking various secondary metabolic activities have been isolated(7,21,24). Both cDNA(13,49,53,54) and genomic clones(3,5,8,20,41,42,52, Y.Z. Zhang, and C.A. Reddy, 1988. Abstr.Annu.Mtg.Am.Soc. Microbiol. H51) for lignin peroxidases and cDNA clones for manganese peroxidases(31,34) have been isolated and sequenced. We previously described vector pRR12 which was shown to transform 2. 9999999999199 to G418 resistance and was rescued in 9. 9911 from the total DNA of pRR12 transformants(35). Alic et al.(1,2) have described an integrative transformation system in which a plasmid vector carrying either an heterologous 2.222 or 2225 gene from 2221222221122 2222222 was used to complement the appropriate adenine auxotroph of 2. 162 9999999999199 . We describe here a novel tranformation vector for 2. 9999999999199 that is maintained stably in the transformants in an extrachromosomal circular form and is readily recoverable from the total DNA of transformants. MATERIALS AND METHODS Strains and Media. 2. 9999999999199 strain ME446(ATCC 34541) was maintained as previously described(21). E. 9911 DHSa (F' 99921 222R17trk- 111;] 999244 991-1 999A1 999A96 121AII212F' 19QZYA]U169 809992-15 lambda"), was used for the maintenance of plasmid vectors. Gold’s medium B(14) supplemented with 10% sorbose and 3% agar (Difco, Detroit, MI) was used for the regeneration of B. 9999999999199 spheroplasts. Golds' medium B, without sorbose or deoxycholate and supplemented with 100 pg per ml G418 (Gibco, Grand Island, NY) was used for the mitotic stability assay . DNA Isolation: and Manipulations. Plasmid DNA was isolated from E. 9911 by the method of Birnboim and Doly(6). Molecular biological techniques were performed according to Maniatis et al.(25), except where indicated. Restriction enzymes were used per manufacturers’ specifications. DNA probes were labeled according to specifications in the Random Primer labeling kit (BMB Biochemicals, Indianapolis, IN). 2. 9999999999199 DNA isolation and the high stringency Southern hybridization conditions have been outlined previously(36). 163 Transformations. Transformation of B. 9999999999199 was carried out according to previously described methods(35). 9. 9911 transformation was done by the method of Hanahan(18). Polymerase Chain Raaction(PCR) analysis. Amplification by PCR was carried out as follows: 2 pg of A991I digested genomic DNA was resuspended in 10 pl of TE(10 mM Tris pH 7.4, 1 mM EDTA). Individual reactions were performed using a GeneAmp DNA Amplification Kit (Perkin Elmer Cetus, Norwalk, Ct.) using 199 polymerase. 20 mer primers (5'-ATGTGGTGATTTTGAACTTT-3' and 5’-GTTGGTGATTTTGAACTTTT-3' from Research Genetics, Inc., Huntsville, Ala.) corresponding to the terminal ends of the kgnr determinant as defined by the A9911 sites at positions 945 and 2146 of the published DNA sequence(29) were used to prime the synthesis of a 1201 bp DNA fragment at 100 pmol per reaction. To determine the optimum amplification conditions, + titration was carried out and a final concentration of a Mg+ 2.1 mM MgCl2 was found to be optimal. Individual reactions were carried out using a Perkin Elmer Cetus DNA Thermal Cycler (source as given above). After an initial denaturation by boiling for 5 min, 30 amplification cycles were done as follows: 1 min at 94°C (denaturation); 2 min at 55°C (annealing); and 2 min at 72°C (extension). Individual samples were then phenol/ chloroform-isoamyl alcohol extracted, ethanol precipitated and resuspended in 20 p1 TE for agarose gel electrophoresis and Southern blotting(25). A 520 bp 9199111-3991 DNA fragment of the 393’ determinant internal to the amplified region was used as the hybridization probe. 164 Rxonuclease III Analysis. Total DNA (5 pg) from fungal transformants was incubated with 4 U of Exonuclease III according to the manufacturers' specifications (Bethesda Research Laboratories, Bethesda, Md.) at 37°C for 2 hrs. The DNA was then phenol-chloroform-isoamyl alcohol extracted, ethanol precipitated, and incubated in 20 pl of $1 buffer with 10 U of 81 nuclease at 37°C for 2 hrs. This DNA along with an equal aliquot of untreated transformant DNA was electrophoresed on a 0.7% agarose gel and stained with ethidium bromide. A linear DNA sample (lambda 9199III DNA marker, BRL, Gaithersburg, Md.) and a circular plasmid (pUC18) served, respectively, as positive and negative controls for the Exonuclease III and $1 nuclease treatments. Isolation of basidiospores. A mycelial plug from individual pG12-1 transformants was placed across from a similar plug from 2. 9999999999199 ME446 on an agar plate containing Kirks low N medium supplemented with 2% agar. Growth and anastomosis of hyphae was allowed to occur, and after 7-10 days basidiospores were ejected to the lid of the inverted Petri dish. Basidiospores were collected in H20, diluted on Golds medium B and transferred to the same medium containing 100 pg/ml G418 for selection of G418 resistant progeny which ‘were used for further analyses. Stability analyses. To determine the stability of vector pG12-l during vegetative growth without G418-selection, fungal transformants were subcultured on malt extract medium (2% malt extract. 2% glucose, 0.1% peptone, pH 4.5) without G418. For 165 each subculture, a small mycelial plug was transferred to a malt extract slant and was incubated at 37°C for 2-3 days when a dense, white growth of new mycelium covered the slant. A mycelial plug from the latter was used to inoculate a new slant and the process was repeated for 10 such transfers.i After 1, 5, and 10 such non-selective transfers, a mycelial plug was transferred to malt extract slants containing 200 pg/ml G418 to determine if the transformants were still resistant to G418. Total DNA from each of the three above mentioned non-selectively maintained cultures, grown in malt extract broth was isolated and was subjected to PCR analysis as described above to detect the presence of vector sequences. This DNA was also used for recovery of pG12-1 by E. 9911 transformation as further confirmations for the presence of pGlZ-l in transformants maintained under non-selective conditions. RESULTS Isolation and characterization of plasmid pGiz-l. Vector pRR12 (Fig. 1A), previously used for transformation of 2. 9999999999199 (35: Appendix 1), consists of the plasmid YIp5(46) into which the kgnr determinant of Tn903(17) and a genomic fragment of B. 9999999999199 having 999 activity in 9999999999999 9999919199(36) were inserted. In the course of characterizing pRR12 transformants, a derivative of pRR12, designated pG12- 166 Figure 1. A schematic illustration of plasmid vectors pRR12(A) and pG12-1(B). The vectors are presented in a linear form for ease of comparison. Symbols: thin line, pBR322 sequences, including or1gin of replication (ori), andrampicillin resistance (amp) marker: thick line, 999 determinant; cross hatched box, 999 of 2. ch99sos9oriu9(36): double line, 9393 sequences of 9. cerevis19e; the dashed box (designated ME-l), the region of DNA that hybridizes to extrachromosomal DNA of g. 9999999999199 ME-446(see text, and Fig. 2). Restriction site abbreviations: A: A9911, B: EQEHI, 39: 9911, Bs: 999HII: E: E99R1, H: H19dIII, P: 999-1, Pv; 291.111, 3: $911: Sm: 91191: x: 25991. e: Tm: .58. . .HIJH lllllllll J 1 ]l.]....l1|l] . e. m o 3. Eu mmgm m mm< 5%: <8 5 m 55.. m5. ES. «55 0% [LI ax com 3.3.55 . __ .J _ 4 1w. mm >a_ Mr < x. Em: <3? Em .1 mm m «Ema 168 1 (Fig. 1B), was recovered by E. 9911 transformation from the total DNA of one of the transformants. A comparison of the restriction map of pG12-1 to that of pRR12 (Fig. 1) and Southern blot analyses showed that pG12-1 is an extensively rearranged version of pRR12. Vector pG12-1 contains homology to all the major domains of pRR12 except that the 9393 gene and the 999 of B. 9999999999199 appear to have been deleted. The intact coding region of the 999r gene (internal to the 999HII fragment) was present. Furthermore, pG12-1 contained a new DNA fragment designated ME-l (Fig. 1B) that is not originally a part of pRR12 and appears to have been acquired from an endogenous extrachromosomal element of 2. 9999999999199 (see below). To determine the origin of ME-l, a Southern blot of total DNA of 2. 9999999999199 was probed with 32P-labeled ME-l(991 1-299 11 fragment of pG12-1 as shown in Fig. 2A (lane a). To determine the position of the chromosomal DNA, the same blot was stripped of ME-l and reprobed with lignin peroxidase cDNA CLGS (Fig. 2A, lane b: 53), a chromosome specific probe, to show the relative migration of the chromosomal DNA. The results show that ME-l hybridizes to extrachromosomal bands of DNA presumably representing open circular and covalently closed circular forms of a circular DNA element. To purify this endogenous plasmid with ME-l homology, hereafter designated pME, we ran large samples of 2. 9999999999199 genomic DNA (~ 1 mg) in a two step CsCl-ethidium bromide gradient(25). We were not able to see a plasmid band 169 a b a b c KB _ S ‘ '- 1 . 2131-; -231 12 1- 9_4- . 10.1- -9.4 5-5' 9.0- ~ _ -6.6 8.0 ° . 4.3- . _o- -4 3 523- 4.0“ _2.3 2.3- 3.0- '2.0 2.0- 2.0- Figure 2. Southern hybridization of 2. c999sos9o91um total genomic DNA to the ME-l fragment of pG12-1. In Fig. 2A, total DNA was electrophoresed through a 0.7% agarose gel and transferred to a nitrocellulose filter. he a shows the hybridization pattern when probed with a P—labeled 9911- 29911 fragment of pGlZ-l containing ME-l (see Fig. 1). The ME-l probe was then stripped off and this filter was re-probed with a chromosome-specific lignin peroxidase cDNA (CLG5;53) and this is shown in lane b. Fig. 28, lane a contains an exonuclease III digest of pME: lane b contains a lambda exonuclease digest of pME, and lane c containsasn E99R1 digest of pME. This Southern blot was probed with a P-labeled 919d III digest of pME. The "S" on the left refers to supercoiled plasmid DNA marker and the "L" refers to the linear DNA marker. The sizes of the markers are in kilobases. Fig. 2C is a photograph of a 0.7% agarose gel after staining with ethidium bromide. Lane a contains plasmid DNA isolated from a CsCl-EtBr gradient containing 2. c999sos9o91um ME446 genomic DNA and lane b contains plasmid DNA isolated from a CsCl-EtBr gradient containing 3. 9h99sos9o91u9 999_§;§ genomic DNA. The arrows indicate the migration of the plasmid bands. 170 below the main chromosomal band, but were able to isolate a circular plasmid from a fraction below the main chromosomal band (Fig. 28 and C). The plasmid isolated (Fig. 28) was digested with either exonuclease III or lambda-exonuclease, Southern blotted, and probed with total pME. The results showed that neither exonuclease III nor lambda exonuclease affected the migration of the plasmid DNA, indicating that it was circular (Fig. 2B, lanes a and b). The E99R1 digest of the plasmid (Fig. 23, lane c) indicated that this circular element was resolved to a linear form of 8.5 kb. This extrachromosomal circular DNA element, which is the source of ME-l sequences, is present at a relatively low copy number since it is not visible on ethidium bromide stained agarose gels. Furthermore, from 500 pg - 1 mg of 2. 9999999999199 genomic DNA, we can purify only about 0.5 - 1.0 pg of pME. Only after purification of such a quantity of DNA through a CsCl-EtBr gradient can enough plasmid be isolated to observe on an agarose gel (Fig. 2C). A further characterization of pME, including a basic restriction map, cloning, and transcript analysis is underway. PCR analysis of pG12-1 transformant DNA. To demonstrate the presence of transforming DNA, total DNA from individual G418 resistant meiotic progeny (basidiospores) of pGlz-l transformants of g 9999999999199 was analyzed. When this DNA was probed with the labeled 999r marker (Fig.3) no band(s) of hybridization were detected (Fig. 3, lanes b,d,f, and h) suggesting that the 171 h n'r ‘— 'v. ab 0 d 9.1-9. 2.3 F 1.9 r 1.3 ( H. a . 1.21 "~1w Figure 3. Southern blot analysis of polymerase chain reaction (PCR)-amplified and unamplified DNA from G418 resistant basidiospore progeny of pG12-1 transformants. Lane a contained A99II digested, genomic DNA from wild type 2. 9999999999199 ME446 amplified by PCR. Lanes b,d,f, and h contained unamplified A99II-digested DNA while lanes c,e,g, and 1 contained PCR amplified DNA. ne j contained 50 ng of the 1.2 kb A9911 fragment of the 999 degsrminant (positive control). The blot was probed with the P-labeled 0.5 kb 9199 III-x99 1 DNA fragment internal to the amplified A9911 999 determinant. 172 vector sequences are maintained at a low concentration, below the normal detectability limit (usually 0.1 pg of linear or circular DNA) of the Southern hybridization procedure. Amplification by polymerase chain reaction (PCR: 40) is a sensitive procedure for visualizing DNA present at a very low concentration. For this analysis, we used total DNA from G418 resistant basidiospore progeny of pG12-1 transformants and amplified the 999r sequences by using the appropriate primers (see Materials and Methods). Positive hybridization of the 999r probe to this amplified DNA from the G418 resistant progeny of pG12-1 transformants (Fig. 3, lanes c, e, g, and i), but not to amplified DNA from the wild type 2. 9999999999199 strain (lane a) or to that from unamplified total DNA from the same G418 resistant basidiospore progeny (lanes b, d, f and h). These results indicate that the vector is indeed present in a low copy number in the fungal transformants and that amplifying the vector sequences using the PCR procedure is necessary for detecting the vector in 2. 9999999999199 transformants by Southern analysis. The above data also indicate that recovery of pG12-1 from the total DNA of fungal transformants does not result from excision of pG12- 1 from the chromosome. If excision from the chromosome were the case, hybridization of the 999r probe to the unamplified genomic DNA of the basidiospore progeny of the G418 resistant transformants would have been observed. Similar amplification by PCR was needed to detect pG12-1 in conidial progeny also (data not shown). Furthermore, pG12-1 173 could be rescued by E. 9911 transformation from the genomic DNA of G418 resistant basidiospore or conidial progeny while no vector was rescued from the total DNA of G418 sensitive conidial progeny, indicating a positive correlation between the presence of pG12-1 and G418 resistance. When conidia were isolated from the G418-resistant meiotic progeny tested in Fig. 3, 14 to 27% of these conidia were G418-resistant. These data showing a low transmission of the G418-resistance marker through mitosis also indicate an extrachromosomal maintenance of pGlZ-l. Low copy maintenance of the pG12-1 is further supported by the fact that pG12-1 can be recovered from the DNA of fungal transformants by E. 9911 transformation only when a high transformation frequency (>1x108/pg DNA) is obtained. We routinely obtain transformation frequencies of 4-9 x 108 transformants per ug of pUC18 plasmid DNA. Ideally, at these transformation frequencies, one should be able to rescue in E. 9911 as little as 1-10 fg of transforming DNA. Such high sensitivity, which is generally beyond the detectability range of Southern hybridization analysis, appears to be necessary to detect a transformation vector that is maintained at a low copy number. Replication and exonuclease III assays of pG12-1 transformants. A 999 methylation assay(19,23) was used to confirm that pG12-1 undergoes replication in 2. 9999999999199 transformants. The restriction enzyme 999 1 cuts at the sequence GATC when the adenine residue in this sequence is 174 6 position while 999 1 cuts at this site unmethylated at the N only when the adenine is methylated. Plasmids which pass through 999+ E. 9911 strains acquire an N6 methylated adenine at GATC sites due to 999 methylation(12) and are resistant to digestion by 999 1. E. 9999999999199 has no comparable 999 methylation activity and its DNA has previously been shown to be susceptible to 999 1 digestion(34) and resistant to 999 1 digestion. Hence, pG12-1 DNA recovered from B. 9999999999199 should be unmethylated and completely digestible by 999 1 if it replicates in the fungus. Our results showed this to be the case since pG12-1 could not be recovered by E. 9911 transformation (Table 1) if the total DNA from primary pG12-1 transformants or their G418 resistant basidiospore progeny was digested with 999 1 whereas pG12-1 could be rescued from 999 l-digested DNA of the same fungal transformants. These results indicate that pG12-1 undergoes replication in 2. 21111595222139- An exonuclease assay was used to confirm that pG12-1 is maintained as an extrachromosomal circular DNA. Exonuclease III, which digests linear DNA but not circular DNA, completely degraded B. 9999999999199 chromosomal DNA (Fig. 4). An equal aliquot of the undigested and £99 111 digested transformant DNA samples used in Fig. 4 were employed to transform E. 9911 DHSa to kanr and individual colonies arising on the plates were counted: miniprep DNA was then prepared from selected kanamycin resistant colonies of 9. 9911 to confirm the presence of pGlz-l (Table 2). The recovery of pG12-l in 9. 175 TABLE 1. Transformation of z. 9911 with 9991 and 9991 digested DNA from G318-resistant pG12-1 transformants of 2. 9999soS9091um Total No. of Transformation No. Treatment DNA19Q) Tr9nsform9995 £9eg9en99(£gug) l 9991 2.0 63 31.5 9991 2.0 0 O 2 9991 2.0 51 25.5 9991 2.0 O O 3 9991 2.0 112 56 9991 2.0 O 0 aAliquots of 999 l and 999 l digested DNA from four individual G418 resistant pG12-1 transformants of B. 9999999999199 was used to transform E. 9911 to kanamycin resistance. 1 and 2 were DNA from primary pGlz-l transformants, while 3 was from a G418 resistant basidiospore purified progeny of 1. The total number of transformants obtained as well as the no. of transformants per pg DNA are indicated. 8The E. 9011 transformation frequency in this experiment was 2.7x10 transformants per pg of pUC19 (positive control) DNA. 176 ablcd kb 23.1 9.4 Figure 4. Exonuclease-Sl nuclease digestion of total DNA from two individual 2. c999sos9o9199 transformants. DNA(2.5 yg) from two G418 resistant pGlz-l transformants(l and 2 of Table 1) was digested with exonuclease III and $1 nuclease as described in Materials and Methods. Lanes a and c contained undigested DNA while lanes b and d contained DNA digested with exonuclease III and $1. 177 TABLE 2. Transformation of E. 9911 with exonuclease III- Sl nuclease treated genomic DNA of G418-5esistant pGlZ-l transformants of E. ch995959091u9 . Number of E. 9911 transformants recovered Transformant u9919es9ed DEA ExoLII-S1 d1gested DNA 1 41 60 2 25 34 a2.5 pg of DNA from two individual G418 resistant pG12-1 transformants of B. 9999soS9or1um, either undigested or digested with exonuclease 111 and $1 nuclease, was used for transforming 3. 9911 DHSa to kanamycin resistance. The total number of 3. 9011 transformants obtained with 2.5 pg of transformant DNA are presented. 178 9911 transformed with exonuclease III-digested total DNA of individual pG12-1 transformants of 2. 9999999999199 was comparable to that obtained with undigested total DNA from the fungal transformants indicating that pG12-1 is maintained in 2. 9999999999199 transformants in a circular, extrachromosomal form. Maintenance of pGiz-l in transformants through non-selective growth. One means of examining the mitotic stability of the transforming vector is to monitor its maintenance through vegetative growth in non-selective media. For those fungal systems for which extrachromosomally maintained vectors have been described, such a vector is generally lost quickly during non-selective growth(50,51). In the case of single celled fungi, such as 9. 9999919199 this is easily measured by determining the doubling time of the organism and examining the stability as a function of the number of generations of growth(28). This approach is impractical for filamentous fungi and stability can be expressed as a function of the number of transfers to new slants of solid media, although each transfer actually encompases many nuclear divisions. We transferred several of our pG12-l transformants through ten transfers and examined whether or not pG12-1 could be idemonstrated in the vegetative mycelium after 1, 5, and 10 such non-selective transfers. The results showed the presence of pG12-1 in the transformants after 1,5, and 10 subcultures in the non- 179 selective medium based on three lines of evidence: growth when transferred to selective slants (G418-supplemented malt extract slants): rescue of pG12-1 from the total DNA of the non- selective subcultures by E. 9911 transformation: and demonstration of vector sequences by PCR analysis of the total DNA samples from the non-selectively grown subcultures (Table 3). A representative PCR analysis (Fig. 5) confirmed the presence of pG12-1 after 10 subcultures in non-selective medium. The results show that the 1599r marker, as defined by the 1.2 kb A9911 restriction fragment, was present in the amplified DNA of these cultures (lane d), and was identical to the 1599r containing A9911 fragment of the same pG12-1 (lane e). The 1599r marker was not detected in unamplified DNA from the 10th non-selective transfer of this pG12-1 transformant (lane c) nor in unamplified or amplified DNA from untransformed g. 9999999999199 (lanes a and b, respectively). These data indicate that pG12-1 is maintained at a low copy level in transformants after long term non-selective growth. 180 TABLE 3. Stability of pG12-1 through vegetative growth under non-selective conditions. Subcultures a Recovery inb G418r c PCR detecaion E. coli of pGlZ-l 1 79 + + 5 62 + ' n.d. 10 23 + + a A G418-resistant pGlz-l transformant of g. s s o um was . b subcultured non-selectively as described in Materials and Methods. DNA isolated from the transformant after 1,5, and 10 transfers on non-selective medium was used to transform E. 9911 and Egg colonies were counted. The 38 9911 transformation frequency in this experiment was 2.2x10 transformants per pg of pUC19 (positive control) DNA. A fragment of mycelium was inoculated onto a malt extract(ME) slant containing G418 and growth or no growth was indicated as "+" or "-" respectively. Untransformed 2. gnxysgspgrigm did not grow d in these ME+G418 slants. PCR analysis was carried out as described in Materials and Methods. n.d. - not determined. ‘ 181 a be d e kb mpqp TTTT 41 - 3.:— 20- l3- Figure 5. Southern blot analysis of PCR amplified DNA from G418-resistant pGlZ-l transformants of B. ghgysospogigm after 10 subcultures on non-selective medium. DNA samples were electrophoresed in a 0.7% agarose gel and transferred to nitrocellulose. Lanes a and b contained total DNA from untransformed B. chrysgsporium M3446 digested with AEQII. Lanes c and d contained total DNA from a non-selectively grown G418-resistant pGlZ-l transformant digested with 52311. The DNA in lanes a and c was unamplified while that in lanes b and d was subjected to the PCR amplification protocol described in Materials and Methods. Lane e containgzgel purified Kan fragmentf The blot was probed with a P-labeled fiind III- xggl Kan gene fragment. 182 DISCUSSION The results of this study show that pGlz-l is stably maintained in a low copy, extrachromosomal form in B. ghzysggpgzigm transformants even under non-selective conditions and that the amplification by PCR is necessary to visualize the vector sequences in the total DNA of the transformants. The vector can be rescued from the DNA of individual fungal tranformants by E. coli transformation. Treatment of the total DNA of fungal transformants with exonuclease III-Sl nuclease resulted in complete digestion of the chromosomal DNA whereas intact pGlz-l was rescued from such treated DNA by E. coli transformation, indicating that the vector is maintained in a circular form in the transformants (Table 2). Sensitivity of the transforming DNA to Mpg 1 digestion but not to Q23 1 digestion indicates that the vector pGlz-l replicates in the fungus (Table 1). PCR analysis of the DNA from the basidiospore progeny of the individual G418-resistant fungal transformants confirmed that pGlz-l is present in a low copy extrachromosomal form (Fig. 3). These results, plus the fact that no hybridization of pGlZ-l DNA with chromosomal DNA (unamplified or amplified) of individual basidiospore progeny was ever observed argues against the possibility that pGlz-l integrates into the chromosome and that the recovery of the vector is due to its precise excision from the chromosome. No evidence for integration of the vector sequences into the 183 chromosome was found even after two years of maintenance of pGlZ-l transformants under selective conditions. Furthermore, the low copy extrachromosomal maintenance of pGlZ-l in B. ghrysgfipgxinm transformants was observed even after 10 successive transfers of these transformants during non- selective conditions. This stable maintenance of pGlZ-l in transformants at a low copy level, and during non-selective growth conditions is quite novel and distinguishes it from previously described fungal extrachromosomal vectors(32,39,44,45,50,51). Extrachromosomal maintenance of pGlz-l in B. ghzygggpgxigm appears to be unique to this vector since Alic et al.(l,2) recently demonstrated an integrative transformation system for 2. ghxygggpgzinm in which a plasmid vector carrying either an heterologous AQEZ gene or 5235 gene from Sghizgphyllgm commune was used to complement different adenine auxotrophies. The results indicate that the ME-l component of pGlz-l originated from a low copy extrachromosomal plasmid (pME) of 2. gnzyggspgzigm. As the ME-l component is common to both pGlZ-l and pME, it appears possible that the ME-l sequences acquired by pGlz-l might be responsible for the stability of this vector and its low copy maintenance, which is similar to that of pME. Further studies are needed to do a detailed characterization of pME and to determine the mechanism allowing for the stability and extrachromosomal maintenance of pGlz-l in B. gnxygggpggigm. A similar report of an autonomously replicating vector which underwent an in vivo 184 rearrangement was recently reported for Engaginm oxyspgznm(33). In this case, a circular vector containing sequences from a Engaginn linear plasmid, pUC12, and a hygromycin selection marker was found in some instances to acquire chromosomal telomere sequences which allowed the resulting rearrangement to be maintained autonomously as a linear plasmid. Previous reports on circular, extrachromosomal vectors in other fungi indicated that such vectors were either transiently circular, preceding integration into the chromosome(30), or were due to excision of either intact or altered vectors from the chromosome(23,37). . Stohl and Lambowitz(4l,42) described a transforming vector containing mitochondrial plasmid sequences of Ngnzgsngzn grassa and. showed that the original vector, along with a consistently recovered deletion derivative, is maintained extrachromosomally in N. crassa, and is recovered by B. 9211 transformation. In Egggsngzn gnggzinn, on the other hand, the attachment of telomeres from Igtzgnyngnn to the ends of a linear plasmid resulted in extrachromosomal maintenance of this plasmid(32) as an unstable extrachromosomal element in approximately half of the transformants studied. High copy autonomous vectors for ngtilngg nnyg1§(50) and Mung; giggingllgidg§(39,50) with features similar to those of Saggnnrgnyggs ggzggiging autonomous vectors have also been described. It is of interest that pGlz-l, even at a low copy number, 185 confers adequate G418 resistance to E. ghrysgsngrinn transformants. Low copy level maintenance of extrachromosomal vectors (one copy per 5-10 nuclei) has also been reported for B. ansg:1na(32). It is possible that the coenocytial nature of filamentous fungi allows for sufficient cross-feeding to allow survival of transformants under selective conditions. The kgnr determinant of Tn903 (encoding an aminoglycoside phosphotransferase which confers resistance to kanamycin and G418: 17), was chosen as a selection marker in this study as it has been shown to be expressed in several filamentous fungal systems without the addition of eukaryotic promoter and termination signals(9,35,38,43). This property appears to be unique to the Knnr from Tn903 as other knnr determinants, such as that from Tn5, require appropriate eukaryotic transcription signals for similar function(4). The pGlZ-l based transformation system described here should be of considerable value for molecular genetic analysis of the slignin biodegradation system and for studying the regulation of secondary metabolism in B. gnzysggpgzinm, if we can improve the copy number of this vector. Furthermore, this system should serve as a useful model for the analysis of low copy extrachromosomal vectors in filamentous fungi. 186 ACKNO'LBDGIMBNTS. We would like to thank Novo Industries for supplying samples of Novozyme 234; Dr. William Holben for assistance with the PCR analysis; and Drs. Barbara Sears and Shree Dhawale for a critical review of the manuscript. This research was supported by the Agricultural Experiment Station, Michigan State University and by grants from the National Institute of Health(1 ROI GM39032-01),-and the Department of Energy (DE- FGOZ-BSER13369). 10. 187 LITERATURE CITED Alic, M., J.R. Kornegay, D. Pribnow, and M.B. Gold. 1989. 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Identification of cDNA Clones for ligninase from Phanergchaete chrxeesnerium using synthetic oligonucleotide probes. Biochem.Biophys. Res.Comm. 137: 649-656. Zhang, Y.Z., and C.A. Reddy. 1988. Use of synthetic oligonucleotide probes for identifying ligninase cDNA clones. Methods Enzymol. 161: 211-220. 192 CHAPTER 2 The Nature of Extrachromosomal Maintenance of Transformation Vectors in the Filamentous Basidicmycete W chases-gem 193 Summary. The nature of extrachromosomal maintenance of the transforming vector p12-6 in finenezeeneete enzyeeeperinn was studied. Our results indicate that the transforming vector is maintained in the fungal transformants extrachromosomally as part of a larger endogenous plasmid of E. enzyeeepeginm. Using total DNA of p12-6 fungal transformants, not only p12-6, but also a larger plasmid, p511, were recovered in zeeA- E. e911 strains while only p12-6 was recovered in zeeA+ g. e911 strains. The 1292 gene cloned into p12-6 was shuttled through 2. Eh12§2§22112m transformants. Introduction In view of the ecological and industrial importance of lignin biodegradation there has been a great increase in research on the lignin degrading white rot basidiomycete, BDQDEIQQDQQSQ enryeeeperinn (Kirk and Farrell 1987: Tien 1987). This organism is known to degrade lignin more rapidly and completely than most other organisms. Integrative DNA transformation systems using heterologous adenine genes from fien1eepnyllnn eennnne to complement appropriate adenine auxotrophs of B. enzyeeepeginn have been described recently (Alic et a1. 1989: Alic et a1. 1990). A vector, pG12-1, which transforms 2. engyeeener1nn to G418-resistance has also been described recently by 194 Randall et al.(l989 and 1990). In contrast to the integrative system of Alic et al., (1989 and 1990), vector pGlZ-l is maintained in the fungal transformants extrachromosomally in a circular form in both primary transformants and their basidiospore progeny. The plasmid occurs in a low copy number in both cases and polymerase chain reaction (PCR) amplification of the vector sequences is necessary to demonstrate its presence in the total DNA of transformants. Extrachromosomal maintenance of transforming plasmids is unusual in filamentous fungi (Fincham 1989), and so far appears unique to a few filamentous fungi (Tsukuda et al., 1988: Roncero, et al., 1989: Randall et al., 1990) We were, therefore, interested in studying further the nature of extrachromosomal maintenance of transforming plasmid(s) in 2. engyeeenex1nn. In this study, we investigated the following questions: 1) Are the transforming plasmids (pG12-1 and p12-6) maintained in a true autonomous form in B. QhI¥§Q§DQI1BE or are they maintained as part of a larger circular element? 2) Does the :eeA allele of the E. 9911 strain which is used to rescue the transforming plasmid from the total DNA of fungal transformants have an effect on the structure of the plasmid recovered in E. 9911? and 3) Is plasmid p12-6 potentially useful as a shuttle vector? 195 Materials and methods W. R. W strain ME446 (ATCC 34541) was used in this study. The various 5. celi strains used, and their source, are listed in Table 1. 2. ehryeeeperinn and B. ee11 were maintained as previously described (Kelley et al. 1986, Maniatis et a1. 1982). We B-Wim DNA was isolated by the method of Rao and Reddy(1984) and the transformation of 2. ehgysosporinm spheroplasts was performed as described by Randall et al. (1989). Plasmid DNA isolation and all other molecular biological techniques were performed according to Maniatis et al.(1982) unless specified otherwise. All DNA fragments used as probes were twice purified through low melting agarose (SeaPlaque, FMC Bioproducts, Rockland, ME) and were labeled using the Random Primer labelling kit(BMB Biochemicals, Indianapolis, IN). E. ee11 transformation was done according to the method of Hanahan(1983) and the transformants were plated either on LM medium (Hanahan 1983) supplemented with 50 pg/ml kanamycin or ampicillin. LM medium was supplemented with 40 pg/ml x-gal (5-bromo-4-chloro-3-indolyl-fi-D— galactoside: BRL, Gaithersburg, MD). 196 Results We have previously shown that plasmid pG12-1 (6.3 kb: Fig. 1A) transformed B. enzyeeenezinn, was maintained in a low copy, circular, extrachromosomal form, and that it could be readily rescued from fungal DNA by E. 9911 transformation (Randall et a1. 1990). Further studies showed that in addition to pGlz-l a second smaller class (3.1 kb) of plasmids was also rescued from pG12-1 transformants. Generally, 10 to 20% of plasmids rescued in E. e911 from pGlz-l transformants were of this class. The fact that these smaller plasmids had identical restriction maps suggested that they were probably generated by some consistent deletion or alteration of the parental pG12-1 vector. A comparison of the restriction map (Fig. 1A) of a representive of this class of small plasmids, designated p12-6, to the parental pG12-1 showed that p12-6 has major domains of homology to pBR322, the kenr selection marker, and an endogenous plasmid of R. enryeeepezinn ME446 designated pME (Fig. 18: Randall et a1. 1990). Plasmid p12- 6 showed homology to the 9:1 (origin of replication) of pBR322, as defined by a 473 bp heel-fleeII-fleeII region flanked by pME sequences designated ME-2 and ME-3. Southern hybridization of ME-2 to total DNA from 197 Figure 1. A) Restriction map of plasmids pGlz-l, p12-6, and p511. The plasmids are presented in a linear form for ease of comparison. The designation of all sequences is based on Southern analysis with the appropriate fragments prepared as described in Materia}s and Methods. Sequences hybridizing to the Len gene are indicated by a thick line: The open boxes indicate the regions of DNA in each plasmid with pME homology. These are designated ME-l to ME-6. Regions of ME-S in p511 which showed hybridization to either ME-l or ME-2 are also shown. Other fragments of pME (ME-1 to ME-6) have no apparent cross-hybridization to each other. The thin line in each plasmid designates sequences of pBR322, with the region containing the E. 9911 e11 shown in each case. B) Southern blot of undigested total DNA of B. engyeeenerinn ME446. Panel 1 is probed with labeled fieeHII-Aeel fragment of p12-6, designated ME- . 2 (Fig. 1A). In panel 2 the same blot is reprobed with a lignin peroxidase cDNA to show the position of the chromosomal DNA band. Restriciton site abbreviations: A: MIL Ac: 8291 8: Benin. 139: 5.911. 88: Bs_sHII. E: EeeRl, H: 31nd III, Ha: neeII, K; Knnl, P: 22311, S; Sell, Sc; £231, Sp; fipnl, Ss: §§§II, X: znel, Xb: K291- 198 e: _ 9m: _ ems. :0 «-ms. Tms. L. 53. 7m: 1H 1,4 L. - ”1 . u q 1 4411'. «J ‘41 m was cm 9.13 am 33:8. :8 x : mm amim ad Ex 8. od :9. mm: to 9m: :9. . a _ "Il1II]II-J mm1xm—:I > _.u< _u m SEE _ _ _ Noom r6 .2me mm m 1 . M3 E m< so .8 3m; . .354 o: .38.... mm mm . .u<_> :maxiimmwo Fm. 2.. we .8 .834 ammo: :mé 225 Figure 3. Restriction map of pAN7-1 and its derivatives rescued in E. ee11 from the total DNA of pAN7-1 transformants. The plasmids are presented in a linear form for ease of comparison. The designation of all sequences is based on Southern analysis with the appropriate fragments prepared as described in Materials and Methods. The single thin line represents sequences of pBR322: the single cross-hatched boxes indicate sequences from A. nidulens and open boxes indicate regions containing homology to pME of E. ennyeeepez1nn. Restriction site abbreviations: A: AerI, B: BenHl, Bg: Egll, E: EeeRl, Er: EQQRV, H: Hind III, N: 11251: P; M1: PV; MIL SC; $.21, 8m; ml: 85: 95:1, 882: SEEII, X: thl. P is the promoter from the glycerol-3-gggsphate dehydrogenase gene of A. n1gn1en§ and T is the terminator from the tzng gene clustegrgg A. n1gnlene. momma Mm mam one . .... Moran. m 63:. a QUE TU: "nut“ 03‘ o):— I m 1 mm mama _ Em _ u no? . #6.... c.“ z 6.6.... UQUQ Emu mat 3m-m . o>z~ .H. t< mo 2mmmt< w t . . 3 c 4. "I . _ H.— A..“ xv 227 1A and B). These results suggested that the vector sequences are maintained at a low copy number in the transformants similar to pG12-1 described by Randall et al. (1990). In an attempt to recover pAN7-1 from 2. enzyeeepez1nn transformants, total DNA from the latter was used to transform E. e211. Two classes of plasmids (pANl and pAN2) were recovered from these E. 9911 transformants, neither of which was identical to pAN7-1 (Fig. 3). Each of the two plasmid classes, however, contained some residual pAN7-l sequences. Plasmid pANl had homology to the gen region of pAN7-1 while pAN2 had homology to the tan region. The restriction maps of even these two regions did not correspond to those of the original pAN7-1, suggesting that these regions may have also been rearranged.t Neither pANl, nor pAN2 contained any homology to the npn marker but both contained sequences homologous to pME (not shown). Vector pAN2 contained sequences which cross-hybridized to ME- 1 (Randall et al. 1990) whereas pANl had homology to regions I designated ME-7 and ME-8 (Randall and Reddy 1990). Total DNA of YIpS-Jsenr transformants yielded in E. e911 two classes of plasmids both of which are different from YIps-kenr and were found to be identical to pGlZ-l and p12- 6 previously described (Randall et al. 1990: Randall and Reddy 1990). The recovery of plasmids identical to pG12-1 and p12-6, though unexpected, may be explained by the fact 228 that pG12-1 and YIpS-Jsenr are both based on the yeast vector YIpS (Struhl et al. 1979). The fact that identical types of plasmids weere rescued in E. e911 from the total DNA of either pGlZ-l or YIpS-Jsenr transformants of B. enryeeeper1nn suggested that sequences common to both YIpS- kenr and pG12-1 are involved in recombination with pME. The PCR data (Fig. 18) showed that the nnn selection marker(identified by the amplified fieel-EeeRl fragment) is present in the total DNA from pAN7-1 transformants. However, pANl and pAN2, the plasmids rescued in E. ee11 from the total DNA of pAN7-1 transformants, did not carry the hygromycin selection marker, suggesting that they are altered forms of the pAN7-1 vector. This is analagous to the situation found with p12-6 transformation of B. enryeeenen1nm (Randall and Reddy 1990). We found that we could recover two types of plasmids in E. 9911, p12-6 and p511. Both of these plasmids were smaller than the circular vector which we could see in Southern blots of p12-6 transformants, suggesting that p12-6 and p511 were altered forms of that plasmid which we could observe in the genomic DNA of p12-6 transformants. Both YIpS-kenr and pAN7-1 were maintained in E. engyeeepez1nn transformants at a low copy level. With both YIps-henr and pAN7-1 E. enzyeeener1nn transformants, we found the recovery in E. e911 of rearranged forms of these two vectors each with pME sequences. This indicated that each original vector recombined with the pME. These results 229 lead us to conclude that the recombination event with pME is a general phenomenon common to a number of plasmids that have been used to transform 2. enzyeeener1nn. Leknegleggenenge. This work was supported in part by the Agricultural Experimental Station of Michigan State University, grants DE-F602-85ER 13369 from the U.S. Department of Energy, 1-ROl-GM39032-01A1 from NIH and a REF grant from Michigan State University. 230 References Fincham JRS (1989) Microbiol Revs 53:148-170 Grindley NDF, Joyce CM (1980) Proc Natl Acad Sci USA 77:7176-7180 Hanahan D (1983) J Mol Biol 166:557-580 Kelley RL, Ramasamy K, Reddy CA (1986) Arch Microbiol 144:254-257 Kirk TK, Farrell RL (1987) Ann Rev Microbiol 41:465-505 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor New York Punt PJ, Oliver RP, Dingesmanse MA, Pouwels PH, van den Hondel CAMJJ (1987) Gene 56:117-124 Randall TA, Rao TR, Reddy CA (1989) Biochem Biophys Res Comm 161:720-725 Randall TA, Reddy CA, Boominathan K (1990) Mol Cell Biol (submitted) Randall TA, Reddy (1990) Curr Genet(in prep) Rao TR, Reddy CA (1984) Biochem Biophys Res Comm 118:821- 827 Rao TR, Reddy CA (1986) Nucleic Acids Res 14:7504 Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76: 1035- 1039 Tien M (1987) CRC Crit_Rev Microbiol 15:141-168 231 CHAPTER 4 An Improved Transformation Vector for the Lignin-degrading White Rot Basidiomycete Were chaseeperium submitted to Gene 232 SUMMARY In this study, a lignin peroxidase(LIP) gene of Ehenereeheege enryeeepez1nn was disrupted by inserting into its coding region the KmR determinant from Tn903. The resulting recombinant plasmid, pUGLGl:kan, was transformed into 2. enzyeeenex1nn with the expectation that the disrupted gene might replace the homologous L12 gene in the chromosome. However, the results showed that pUGLGl:kan sequences do not integrate into the chromosome: instead, the plasmid is maintained intact in the transformants in an extrachromosomal state. Our data also show that pUGLGl:kan undergoes replication in E. enryeeenex1nn, is maintainted as a circular element, is stable through meiosis, and can be recovered intact by Eeener1en1e e911 transformation. These results indicate that the genomic clone £191 contains as yet unidentified sequences that allow autonomous replication of pUGLGl:kan in B. enzyeeener1nm transformants. These sequences should potentially be useful for constructing a stable high copy number tranformation vector for 2. enzyeeepez1nn. 233 INTRODUCTION There has been much recent interest in lignin biodegradation by fungi because of the ecological and industrial importance of this process(Kirk and Farrell, 1987: Tien, 1987). Most of these studies focused on the filamentous basidiomycete finenezeeneeee enzyeeepez1nn since it has been shown to degrade lignin more rapidly and completely than most other organisms(Kirk and Farrell, 1987). 2. engyeeenen1nn produces two families of important extracellular, glycosylated H20 requiring heme 2 proteins, designated lignin peroxidases(LIP) and manganese peroxidases(MNP) that appear to play a key role in lignin degradation(Tien and Kirk, 1984: Wariishi, et al., 1988). These enzymes are produced in response to nutrient starvation only during secondary metabolism. At least six major LIP isozymes (arbitrarily designated H1, H2, H6, H7, H8, and H10) and four MNP isozymes have been described(Kirk and Farrell, 1987: Tien, 1987: Wariishii, et al. 1988). Both cDNA and genomic clones of LIP isozymes have been isolated and sequenced (Tien and Tu, 1987: de Boer, et al., 1987: Brown, et al., 1988: Walther, et al., 1988: Schalch, et al., 1989: Zhang, et al., 1990). We have recently described a DNA transformation system for 2. enryeeeper1nn in which the circular DNA vector was shown to be maintained in an extrachromosomal, low copy form which 234 can only be observed after amplification by polymerase chain reaction. The vector can be recovered by E. 9911 transformation (Randall, et al. 1989: 1990). Integrative DNA transformation systems (Alic et al., 1989: 1990) in which adenine auxotrophs of E. EDIX§Q§RQI12E was complemented with the corresponding heterologous adenine genes of Een1eennyllnn eennnne have also been described. Because of the presence of multiple LIPs in B. QDIYEQ§PQI1QE. the isolation of a mutant lacking a specific LIP isoenzyme using traditional mutagenic approaches has been difficult (Boominathan, et al., 1990). Mutants lacking specific LIP isozymes will be useful in determining the relative contributions of each LIP isozyme to lignin degradation as well as to other biological activities known to be catalyzed by LIP isozymes. Hence, we initiated this study to construct mutants lacking Hz, a major L12 isozyme, by employing a gene disruption technique. To accomplish this, we transformed E. enzyeeeper1nn with a vector carrying a disrupted L122 gene that encodes H However, 2. we did not observe either the expected disruption of the LIE gene or the integration of the vector sequences into the chromosome. Instead, we made the important finding that the transforming vector is maintained extrachromosomally in a circular form at a relatively high copy number, as compared to the pG12-1 vector previously described by us (Randall, et al., 1990). 235 MATERIALS AND METHODS (a) Strains and media 3. enzyeeener1nm strain BKM-F (ATCC 24725) was maintained as previously described (Kelley, et al., 1986). n. 9211 D858: (F_, ean, hede7trk'.mk+]. ean44, 1111-1, reeAl, mA96, 311114113112 leeZYA]Ul69, 80,71392-15, '), was used for the maintenance of plasmid vectors(Maniatis, et al., 1982). Gold’s medium B (Gold, et al, 1978) supplemented with 10% sorbose and 3% agar (Difco, Detroit, MI) was used for the regeneration of B. enxyeeepez1nm spheroplasts. Golds medium B, without sorbose or deoxycholate and supplemented with 100 u g per ml of G418 was used for the mitotic stability assay (see below). (b) DNA Isolations and other manipulations Plasmid DNA was isolated from E. ee11 by the alkaline lysis method of Maniatis et al.,(l982). Most molecular biological techniques were performed according to Maniatis et.al, (1982). Restriction enzymes were used per manufacturers' specifications. DNA probes were labelled according to specifications in the Random Primer labelling kit (Boeringer Mannheim Biochemicals, Indianapolis, IN). 2. enzyeeeper1nn DNA was isolated by the method of Rao and . 236 Reddy(1984). DNA fragments used as probes were purified twice through low melting agarose (SeaPlaque, FMC Bioproducts, Rockland, ME) before labelling. Transformation of 2. ennyeeenez1nn spheroplasts was performed as previously described (Randall, et al., 1989). E. e911 transformation was done according to the method of Hanahan(1983). RESULTS AND DISCUSSION (a) Insertion of the KmR determinant into the L122 gene in clone GLG1. Our initial objective was to inactivate a specific L12 gene of B. EDI¥§Q§QQI1BE and construct strain lacking that specific L12 enzyme so that the relative contribution of each of the LIE enzymes to overall lignin degradation by B. enzyeeeper1nm could be evaluated. For this, we first needed to determine whether a disrupted L12 gene introduced into E. enzyeeenez1nn would replace the endogenous L12 gene by integrating into a homologous and/or heterologous site(s) in the chromosome as is common in a number of fungi, including 2. enxyeeepez1nn (Fincham, 1989: Alic et al., 1989). The E. enzyeeepez1nn BKM-F genomic insert in clone GLGI (a 4.1 kb Een H1 genomic fragment) contains the L122 237 Figure 1. Restriction map of pUGLG1:kan. The LIP2 gene was carried on a 4.1 kb EenHl fragment designated §L§l isolated from a E. chgysospoziun BKM-F genomic library in YRp12(Zhang, et al., 1990). §L§1 was R recloned into pUC19 and designated pUGLGl. The Km gene of Tn903 was inserted as a 1.7 kb EanI fragment into the single, blunt ended EeEI site within the coding region of the LIP2 gene. The resulting recombinant plasmid is designated pUGLG1:kan. Only the §L§1:kan segment of pUGLGl:kan is shown in detail. The thick line represents the approximate coding region of the LIP2 gene and the arrow indicates its transcriptional orientation(Zhang, et al., 1990). The thin line represents chromosomal DNA flanking the L122 gene. Restriction site abbreviations: A: AyeII, Al: m1: B: Eamfil, H: H.1ndIII, K: 132111, P: 2511: Pv: EanI, Sm: Smel, Sp: Snnl, SII: SetII, X: Engl: Xb: Ebel. m3 3 P. > A I 2 x > m< . 9d :6 xo: 9: mm L a; to 238 Orbs f‘. mCQ—umrrm: Sb :5 239 gene based on the fact that the sequence of the coding region of this gene is identical to that of the cDNA clone QL§4 which is known to encode L12 protein H2 (de Boer, et al., 1987: Naidu and Reddy, 1989). The choice of L122 is based on the fact that the enzyme encoded by this gene is readily identifiable in FPLC profile of extracellular fluid (Dass and Reddy, 1990: Tien and Kirk, 1988) and that L122 shows only faint hybridization with the other L12 genomic clones of B. ennyeeeneg1nn (Zhang, et al., 1990). The §L§1 genomic fragment contains the L122 gene flanked by 1.3 kb of genomic DNA upstream on the 5' end and 1.05 kb of genomic DNA on the 3' end (Fig. l). The KmR determinant from Tn903 (Grindley and Joyce, 1980) was inserted as a blunt ended, 1.7 kb EanI fragment into an unique fieeI site within the L122 coding region to disrupt the L122 gene. The resulting plasmid (i.e. pUC19, plus §L§1 with the inserted KmR fragment) was designated pUGLGl:kan and was used to transform 2. EDIYEQEPQI1BE BKM-F to G418 resistance. (b) Analysis of the DNA of transformants Undigested total genomic DNA from individual G418- resistant fungal transformants was electrophoresed through an agarose gel and the Southern blot was probed with a fi1ndIII-xnel fragment internal to the KmR gene (see Fig. 1) 240 abcdefgh abcdefgh kb . kb 0 6.4: 6.4— 5.7: O 5.7 :3: 13 - 2:: 13.7- 3.7 2.3- .. 2.3 1.9- 1.9 Figure 2. Southern analysis of genomic DNA from pUGLGl:kan transformants. Uncut genomic DNA (10 pg/lane) was electrophoresed on a 0.7% agarose gel, stained with ethidium bromide and transferred to nitrocellulose. In the leftRpanel, the Southern blot is probed with the 1.7 kb EanI Km fragment. I the right panel, this same blot was then stripped of the Km probe and reprobed with the LIEE gene the LLB gene to determine the position of the chromosomal DNA in this blot. Lane a contained genomic DNA from untransformed 2. QBIY§2§EQ£12E BKM-F and lanes b through h contained total genomic DNA from individual GLGl:kan transformants of BKM-F. 241 to determine if the transforming sequences integrated into the E. ennyeeenez1nn chromosome (Fig. 2A). Our results showed that the probe exhibited intense hybridization with extrachromosomal DNA but not to the chromosomal DNA of E. enzyeeeper1nn. In addition, undigested genomic DNA isolated from seven different G418 resistant transformants exhibited identical hybridization patterns with the KmR probe (Fig. 2A, lanes b-h). In contrast, total DNA from untransformed 2. QDI¥§Q§PQ£1BE gave no chromosomal or extrachromosomal hybridization with the KmR probe (Fig. 2A, lane a). This blot was then stripped of the KmR probe and reprobed with the chromosome-specific L12 cDNA CLGS (de Boer, et al., 1987) to demonstrate the position of the chromosomal DNA in both transformants and wild type 2. enzyeeenex1nn (Fig. 28). These results indicate that the KmR probe is hybridizing to extrachromosomal DNA of the transformants. c. Intact transforming plasmid(pUGLGl:kan) is maintained extrachromosomally Southern blot analyses indicated that the extrachromosomal DNA seen in Fig. 2 contained intact pUGLGl:kan plasmid. Southern blots of undigested and EenHl digested total DNA from three of the transformants were probed with the 4.1 kb §L§1 insert (Fig. 3A). Identical patterns of hybridization were observed with the DNA of all .A B (3 PANEL 1 PANEL 2 [a tfli: die f] a b c c! .a b c d e f a :31; kb kb kb - 211 - (P ‘ - g . ‘ 9.4 - . 8.5 - . - ‘ '1 - 6.6 - 6,‘ - 8.5 - ... - . 2'7- . - - " 4.3 ' 3' 54- 4.3“ . a — 57.. 3.7- :13- - -..' . 1 : - 23 - . 23' 31 — 20 - - 1.9- 2.3- O 1.3- 11:: . 12‘ 1:2- - . 1 I «.4 Figure 3. Restriction and Southern blot analysis of transformant DNA. (A) Total DNA from three transformants(labelled 1, 2, and 3) were electrophoresed on a 0.7% agarose gel and transferred onto nitrocellulose. Lanes a,c, and e contain uncut DNA: lanes b,d, and f contain EenHl digests of total DNA. (B) Total DNA from one G418-resistant transformant(#1 from gig. 3A) was probed with the E1ndIII-Enel fragment of the Km fragment. Lane a contains 3 pg uncut DNA: lanes b and c contain, respectively, Knel and AzeII digests of the same transformant DNA. Lane d contained 50 ng AerI digested pUGLGl:kan DNA. (C) Panel 1. Total DNA from untransformed B. chnysosoogigm and a G418 resistant transformant was probed with Km fragment. Lanes a-c in panel 1 contained 2. ehrxeeseerium DNA. uncut. Eamfll cut. and AerI cut, respectively. Lanes d-f in panel 1 contain genomic DNA from a G418-resistant transformant, uncut, EenHl cut, and AyeII cut, respectively. The Southern blot of panel 1 was stripped and reprobed with pUC19 for panel 2. Lanes a-c of panel 2 shows the part of the blot in panel 1(lanes d-f) containing the transformant DNA. 243 the three transformants. In the lanes containing digested DNA (b,d, and f) a 5.8 kb band of hybridization (corresponding to the sum of the 4.1 kb EenHl fragment containing the §L§1 insert, and the 1.7 kb KmR fragment) and a 4.1 kb band (corresponding to the endogenous GLGl locus of B. 9hI1§Q§QQI122: see also Fig. 5, lane a) were observed. The faint 8.5 kb band in lane f is due to partial digestion of pUGLGl:kan. In the lanes containing undigested transformant DNA (Fig. 3A, lanes a, c, and e) the largest band corresponds to the position of the chromosomal DNA, while the smaller extrachromosomal bands of hybridization are due to the transforming plasmid, pUGLG1:kan. Additional analyses of these data also indicated that approximately one or two copies of pUGLGl:kan can be maintained per haploid nucleus in E. engyeeeper1nn. Xth digestion of the total DNA from one of the transformants (Fig. 3B, lane b) showed that the size of the extrachromosomal element (8.5 kb) is identical to that of the original pUGLGl:kan vector. An AyeII digest of total transformant DNA (Fig. 3B, lane c) resulted in a 1.2 kb band of hybridization, identical to that of the 1.2 kb Aye II KmR fragment of pUGLGl:kan (Fig. 3B, lane d) showing that the KmR fragment is intact. Our results also showed that both the GLG1:kan and the pUC19 portions of pUGLGl:kan are present intact in the transformants. The KmR probe showed no hybridization to the total DNA from untransformed 2. enzyeeener1nn (Fig. 3C, panel 1, lane a-c), while the EenHl 244 and AyeII digests of the transformant DNA, respectively, showed 5.8 kb (representing the GLGl:kan insert in pUGLGl:kan: panel 1, lane e) and 1.2 kb (the 1.2 kb AyeII fragment of KmR in pUGLGl:kan: panel 1, lane f) bands of hybridization. When the same blot was reprobed with pUC19, the undigested DNA (Fig. 3C, panel 2, lane a) showed bands of hybridization representing the extrachromosomal element, while in lane b a 2.7 kb band of hybridization (representing the size of the pUC19 portion of pUGLGl:kan) was seen indicating that pUC19 is a part of the extrachromosomal element in the transformant. Two small AgeII bands (Fig. 3C, lane c) are observed which represent EgeII fragments of pUGLGl:kan containing both pUC19 and GLG1 homology. In summary the above data suggest that the intact pUGLGl:kan is being maintained extrachromosomally in the B. enzyeeeper1nn transformants. (d) pUGLGl:kan is maintained in a circular form An exonuclease III (Exo III) assay was used to demonstrate that the vector DNA is maintained in a circular form in the transformant. Exo III digests linear DNA but leaves circular DNA intact and has been used to assay the physical state of plasmid elements (Kistler and Leong, 1986). Exo III treated transformant DNA showed extrachromosomal bands of hybridization to the KmR probe (Fig. 4, lane c) identical to the extrachromosomal bands 24S Figure 4. Confirmation of circular and fungal origin of the transforming plasmid in a G418-resisfiant E. ennyeeepeg1nn transformant. Fragment purified Km gene was used as hybridization probe. Lane a contained 3 pg of untransformed BKM-F DNA: lane b-e contained 3 pg, respectively, undigested, exonuclease III digested, M221 digested, and anl digested transformant DNA: lanes f-i contained, respectively, 10 ng of undigested, exonuclease III digested, M221 digested, and pnnl digested pUGLGl:kan plasmid DNA. Restriction digestions were performed per manufacturers specifications. 246 observed with the undigested transformant DNA (lane b) as well as with the undigested and Exo III digested pUGLGl:kan DNA isolated from E. 9911 (lanes f and g). These results indicate that pUGLGl:kan exists in a circular form in the transformant DNA. (e) pUGLGl:kan replicates in 2. 9991999999199 transformants A Dem methylation assay was performed to confirm that the pUGLG:kan sequences undergo replication in the fungus. M991 and Epnl are isoschizomers which recognize GATC sites. 99n1 specifically digests DNA that is methylated at the N6 position of the adenine residue, while M991 digests only when the adenine residue at these sites is unmethylated (Holst, et al., 1988). DNA which has passed through a 999+ strain of E. 9911 will have a N6-methylated adenine at GATC sites in the plasmid whereas DNA which has replicated in B. enzy9999911nn is not known to have the methylated adenine at GATC site(s). The KmR gene of Tn903 contains a number of M99 1 sites which digest the gene into fragments of several hundred base pairs. Therefore, if pUGLGl:kan replicates in B. 999y9999991nn it will be digested by M991 but not by npnl. The data in Fig. 4 also illustrates the specificity of M991 and ppnl. The pUGLGl:kan DNA isolated from E. 9911 DHSa (9991) was resistant to digestion with M991 (lane h) but was 247 susceptible to digestion by 99n1 (lane i). In contrast, when 2. 9999999999199 transformant DNA was digested with M991 (lane d), pUGLGl:kan bands characteristic of complete digestion at GATC sites were seen to hybridize to the KmR probe as in Fig. 4, lane i. When the same transformant DNA was digested with Dnnl (lane e), the pUGLGl:kan bands characteristic of undigested plasmid were observed (see lanes b and c). These results indicate that pUGLGl:kan has undergone replication in B. 9999999999199 transformants. (f) pUGLG1:kan is passed intact through meiosis and mitosis in E. 9992999999199 transformants Total DNA from basidiospore progeny of a pUGLGl:kan transformant was analyzed by Southern blot hybridization to determine if pUGLGl:kan is stably inherited by basidiospore progeny. Using the 4.1 kb 9L91 fragment as the probe, two of the basidiospore progeny (Fig. 5, lanes 2 and 3) showed a 5.8 kb band of hybridization (representing the GLGl:kan insert of pUGLGl:kan) indicating the presence of pUGLGl:kan in these progeny. Reprobing this blot with the KmR gene also showed the same 5.8 kb band in lanes 2 and 3, whereas reprobing with pUC19 showed a 2.7 kb band representing the linear size of pUC19 (similar to Fig. 3C, panel 2, lane b: data not shown). The above two basidiospore progeny also grow well on G418 containing media. In contrast, three of the basidiospore progeny 24.8 7 BKM 1 2 3 ‘4 5 kb [f “3:114:31; , an. Figure 5. Analysis of individual basidiospore progeny of a pUGLGl:kan transformant of g. ghzysosporium. Basidiospores were isolated as described(Alic et al., 1989). Lane BKM contained total DNA from untransformed 2. chgysospozium BKM-F. Lanes 2 and 3 contained DNA from G418-resistant basidiospore progeny of a pUGLGl:kan transformant, whereas lanes 1, 4, and 5 contained G418-sensitive basidiospore progeny of the same transformant. All DNA samples were digested to completion with figmfil and the Southern blot was probed with the labelled GLGl:kan insert shown in Fig. 1. 249 examined (Fig. 5, lanes 1,4, and 5) which were G418 sensitive did not show the 5.8 kb band of hybridization. Note that the B99Hl digests of all the basidiospore progeny as well as that of undigested E. 9991999999199 BKM-F DNA gave one major band of hybridization (4.1 kb) representing the GLGl locus in the chromosome and two minor bands of hybridization which are most probably one or two other 912 genes of B. 9992999999199 related to the 9122 gene. Upon further Southern analysis of a larger number of basidiospore progeny pUGLGl:kan was found to be transmitted to about 12% of the basidiospore progeny of the pUGLGl:kan transformants tested. These data indicated that the vector is transferred to the progeny at a relatively low rate as would be expected for an extrachromosomally maintained vector (van Heeswijck, 1986; Tsukuda, et al., 1988). A mitotic stability assay using conidia isolated from G418-resistant and G418-sensitive basidiospore progeny (lanes 3 and 5 of Fig. 5, respectively), was then performed to determine how well pUGLGl:kan is passed to conidial progeny. Only 3% of the conidia derived from a G418-resistant strain grew on G418 media, whereas none of the conidia derived from a G418-sensitive strain grew on the G418 plates. These results indicate that pUGLGl:kan could be transferred to conidial progeny, although at a low frequency. 250 (9) Recovery of pUGLGl:kan in 9. 9911 Further evidence for the presence of pUGLGl:kan in GAIB-resistant 2. 9991999999199 transformants as well as in the G418-resistant basidiospore progeny was obtained by using total DNA of these strains to transform 3. 9911 to either Km or A9 resistance, and by recovering intact pUGLGl:kan from the E. 9911 transformants. In contrast, total DNA from wild type B. 9991999999199 and G418- R sensitive basidiospore progeny did not yield either Km or ApR transformants of E. 9911. (h) Conclusions 1) The results of this study show that vector pUGLGl:kan containing the 2. 9991999999199 9122 gene, disrupted by the KmR gene and flanked on either side by approximately 1 kb of genomic sequences functions as a relatively high copy number vector for transforming 2. 9991999999199 BKM-F to G418 resistance. 2) Vector pUGLGl:kan is maintained intact as an autonomous, extrachromosomal element in a relatively high copy number compared to the previously described pG12-1 vector plasmid(Randall et al., 1990) which was found to be maintained in too low a copy number to be detected by standard Southern hybridization procedure and could be demonstrated in the total DNA of fungal transformants only after amplification by polymerase chain reaction (PCR). 251 Thus, pUGLGl:kan should be a particularly useful improvement as the basis for a shuttle vector over our previously described vector, pG12-1. 3) Vector pUGLGl:kan could be passed through basidiospore and conidial progeny of the transformants at a relatively low rate. This is quite similar to the maintenance of high copy, unstable, extrachromosomal vectors in both 99911999 991919 (Tsukuda, 1988) and 99999 91991991191999 (van Heeswijck, 1986) 4) Intact pUGLGl:kan could be recovered by transforming E. 9911 using total DNA from either the initial G418 resistant fungal transformants or from G418 resistant basidiospore progeny of these transformants. The general behavior of pUGLGl:kan in B. 9991999999199 transformants is quite similar to 999 vectors of 9. 9999119199 (Williamson, 1985) and of filamentous fungi (Tsukuda, et al., 1988: van Heeswijck, 1986: Roncero, et al., 1990). Specific 999 elements have been identified for both 9. 991919 (Tsukuda, 1988) and 9. 91991991191999 (Roncero, et al., 1990) and these are apparently analagous to those of 9. 9999119199 (Williamson, 1985). Identification of the DNA sequences responsible for the autonomous replication of pUGLGl:kan in 2. 9991999999199 is an important future goal in our laboratory as it should be very useful in further developing a shuttle vector which can be used to isolate 2. 9991999999199 genes directly by complementation of appropriate mutants. 252 ACKNOWLEDGEMENTS This work was supported in part by the Agricultural Experimental Station of Michigan State University, grants DE-FG02-85ER 13369 from the U.S. Department of Energy, 1-R01-GM39032- 01A1 from NIH and a REF grant from Michigan State University. 253 REFERENCES Alic, M., Letzring, C., and Gold, M. H.: Mating system and basidiospore formation in the lignin-degrading basidiomycete Ehsngr22h§2_§ Appl. Environ. Microbiol. 53 (1987) 1464- 1469. Alic, M., Kornegay, J.R., Pribnow, D., and Gold, M.H.: -Transformation by complementation of an adenine auxotroph of the lignin-degrading basidiomycete 2999999999999 8 ' . Appl. Environ. Microbiol. 55 (1989) 406-411. Alic, M., Clark, E.K., Kornegay, J.R., and Gold, M.H.: Tranformation of 2999erocha9t9 9991sos9091u9 and 9999999999 999999 with adenine biosynthetic genes from 9991999911199 90m u99. Curr. Genet. 17 (1990) 305- 311. Boominathan, K., Dass, S.B., Randall, T.A., Kelley, R.L., and Reddy, C.A.: Lignin peroxidase-negative mutant of the white-rot basidiomycete 2999999999999 9991999999199 J. Bacteriol. 172 (1990) 260-265. Brown, A., Sims, P.F.G., Raeder, U., and Broda, P.: Multiple ligninase-related genes from 2999999999999 9991999999199. Gene 73 (1988) 77-85. Dass, S.B., and Reddy, C.A.: Characterization of extracellular peroxidases produced by acetate-buffered cultures of the lignin-degrading basidiomycete 9991999999199. FEMS Microbiol. Letters (in press). de Boer, H.A., Zhang, Y.Z., Collins, C., and Reddy, C.A.: Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Ehgngrgghagsg 9h1259§£2rium- Gene 60 (1987) 93-102. Fincham, J.R.S.: Transformation in fungi. Microbiol. Rev. 53 (1989) 148-170. Gold, M.H., and Cheng, T.M.: Induction of colonial growth and replica plating of the white rot basidiomycete Ehangrgghggsg Appl. Environ. Microbiol. 35 (1978) 1223- 1225. Grindley, N.D.F., and Joyce, C.M.: Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903. Proc. Natl. Acad. Sci. USA 77 (1980) 7176-7180. 254 'Hanahan, D.: Studies on transformation of 29999919919 9911 with plasmids. J. Mol. Biol. 166 (1983) 557-580. Holst, A., Muller, F., Zastrow, G., Zentgraf, H., Schwender, S., Dinkl, E., and Grummt, F.: Murine genomic DNA sequences replicating autonomously in mouse L cells. Cell 52 (1988) 355-365. Kelley, R.L. Ramasamy, K., and Reddy, C.A.: Characterization of glucose oxidase-negative mutants of a lignin degrading basidiomycete Ehzngrgshzgtg £h:¥§2§22:i§9- Arch. Microbiol. 144 (1986) 254-257. Kirk, T.K., and Farrell, R.L.: Enzymatic "Combustion": The microbial degradation of lignin. Ann. Rev. Microbiol. 41 (1987) 465-505. Kistler, H. C., and Leong, S. A.: Linear plasmid-like DNA in the plant pathogenic fungus 29999199 991999999 f. sp. 999919919999. J. BacterioL 167 (1986) 587-593. Kuiper, M.T.R., and de Vries, H.: A recombinant plasmid carrying the mitochondrial plasmid sequence of N9u9oS9999 1999999919 LaBelle yields new plasmid derivatives in 9999999999 c99s s9 transformants. Curr. Genet. 9 (1985) 471- 477. Maniatis, T., Fritsch, E.F., and Sambrook, J.: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, NY, 1982. Padmavathy, S.N., and Reddy, C.A.: Analysis of nucleotide sequence of a lignin peroxidase gene from a white-rot basidiomycete. Engngrgshgggg ghzxggéngrinm BKM-F. Astr. Annu. Mtg. Am. Soc. Microbiol. # H70 (1989). Randall, T.A., Rao, T.R., and Reddy, C.A.: Use of a shuttle vector for the transformation of the white rot basidiomycete. Ehgngrgghagng ghzxgggngrinm- Biochem. Biophys. Res. Comm. 161 (1989) 720-725. Randall, T.A., Reddy, C.A., and Boominathan, K.: A novel extrachromosomally maintained transformation vector for the lignin-degrading basdiomycete 2999999999999 9991999999199. Mol. Cell. Biol. (submitted). Raeder, U., Thompson, W., and Broda, P.: RFLP-based genetic map of Ehangrgghggtg 9hr1§2§221129 ME446: lignin peroxidase genes occur in clusters. Mol. Microbiol. 3 (1989) 911-918. . 255 Rao, T.R. and Reddy, C.A.: DNA sequences from a ligninolytic filamentous fungus 2999999999999 9991999999199 capable of autonomous replication in yeast. Biochem. Biophys. Res. Comm. 118 (1984) 821-827. Roncero, M.I.G., Jepsen, L.P., Stroman, P., van Heeswijk, R.: Characterization of a 199A gene and an 929 element from 99999 9199199119199_. Gene 84 (1989) 335-343. Schalch, B., Gaskell, J., Smith, T.L., and Cullen, D.: Molecular cloning and sequences of lignin peroxidase genes of EDQDEIQQDQQEQ 2hrx§2§22rium- Mol. Cell. Biol. 9 (1989) 2743-2747. Tien, M.: Properties of ligninase from 2999999999999 9991999999199 and their possible applications. C.R.C. Crit. Rev. Microbiol. 15 (1987) 141-168. Tien, M., and Kirk, T.K.: Lignin-degrading enzyme from Ehanergshgege 2912929221139: Purification. characterization and catalytic properties of a unique H O -requiring enzyme. Proc. Natl. Acad. Sci. USA 83 ?1984) 2280-2284. Tien, M., and Kirk, T.K.: Lignin peroxidase of c c991soS9o9199. Methods Enzymol. 161 (1988) 238-249. Tien, M., and Tu, C.-P.: Cloning and sequencing of a cDNA for a ligninase from 2999999999999 9991999999199. Nature 326 (1987) 520-523. Tsukuda, T., Carleton, S., Fotheringham, S., and Holloman, W.K.: Isolation and characterization of an autonomously replicating sequence from 99911999 991919. Mol. Cell. Biol. 8 (1988) 3703-3709. van Heeswijck, R.: Autonomous replication of plasmids in 99999 transformants. Carlsberg Res. Comm. 51 (1986) 433-443. Walther, I., Kélin, M., Reiser, J., Suter, F., Fritsche, B., Saloheimo, M., Leisola, M., Teeri, T., Knowles, J.K.C., Fiechter, A.: Molecular analysis of a Ehanergghgese 2hr2§2§291129 lignin peroxidase gene. Gene 70 (1988) 127-137. Wariishi, H., Akileswaran, L., Gold, M.H.: Manganese peroxidase from the basidiomycete 2999999999999 9991soS9or199: spectral characterization of the oxidized states and the catalytic cycle. Biochemistry 27 (1988) 5365-5370. 256 Williamson, D.R.: The yeast ARS element, six years on: A progress report. Yeast 1 (1985) 1-14. Zhang, Y.Z., and Reddy, C.A.: Cloning of several lignin peroxidase(LIP)-encoding genes and sequence analysis of L129 gene from the white-rot basidiomycete, 2999999999999 c s s o ' . Gene (in review). 257 APPENDIX A USE OF A SHUTTLE VECTOR FOR THE TRANSFORMATION OF THE THE WHITE ROT BASIDIOMYCETE EEAEEBQQEAEIE QEBXfiQfiEQBIHM BY Thomas Randall, T.R. Rao, and C.A. Reddy PUBLISHED In Biochem. Biophys. Res. Comm. 161:720-725 (1989) 258 Vol. 161, No. 2. 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 15, 1989 Pages 720-725 USEOPASHIHTLEVECTORFORMTRAMSPORMATIOMOFfl-IEHMIEROT BASIDIMCEI'E, Phanerochaete chgsosmrim+ I Thomas Randall, T. Rajeswara Rao, and C. Adinarayana Reddy Department of Microbiology and Public Health, Michigan State University, East Lansing, MI ABBZH-IIOI Received April 25, 1989 A novel shuttle vector based spheroplast transformation system for the lignin degrading filamentous fungus g. chrysosporium is described. The transformation vector, designated pRR12, consists of the yeast integration plasmid YIpS, a putative autonomous replication sequence (a_rs_) of 2. chrysosporiug, and a 2.2 kb mil fragment carrying k_ar_:" determinant from plasmid pNG3S, which confers resistance against both kanamycin and the related antibiotic GIIIB. Two different strains of g. chrysosgrium (MEIIlIb and BKM-F) were transformed to GIIIB resistance using vector pRR12. Approximtely 20 transformants per ug of vector DNA were obtained. The transforming vector pRR12 could be recovered from the total DNA of transformants by 9. coli transformation, albeit at a low frequency. e 1989 Academic mu. Inc. Lignin is a highly complex, amorphous, aromatic polymer, which is a major component of woody plants, and is the second most abundant renewable organic resource on the earth (1). Research has intensified worldwide on ligninolytic microorganisms and their enzymes because of their potential industrial applications in biopulping and in the conversion of lignin and lignocellulosic materials to useful chemicals (1,2). Phanerochaete chrysosmrium, a white-rot basidiomycete, is a rapid lignin degrader and is the most widely studied organism from both the basic and applied standpoints. Genetic analysis of this organism has been limited, but recently auxotrophic mtants and mutants lacking various secondary metabolic activities have been isolated (3-6) and a protoplast fusion system has been described (7.8). One of the long range objectives of research in our laboratory is to develop a molecular genetic approach to the analysis of lignin degradation by 2. chrysosmrium and to study the regulation of secondary metabolism in this organism. To this end, cDNAs coding for lignin peroxidases, H202 dependent extracellular heme proteins expressed during secondary metabolism, and genomic clones encoding these proteins have been isolated and sequenced (9-13; Zhang, Y. Z. and Reddy, C. A. 1988. Abstr. Annu. Mtg. Am. Soc. Microbial. M51). tJournal article no. 13031 from the Michigan Agricultural Experiment Station. "to whom correspondence should be addressed. mos-291x189 $1.50 Copyright 0 1989 by Academic Press. Inc. Autwflhsqfnquudhamulfilanyfiwwlnumnnd. 720 259 Vol. 161. No. 2. 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS An integrative transformation system for 2. chrysosmrium has also recently been described (III). Ne describe here the development of a novel trans- formation system for 2. chrysosmrium in which the transforming vector can be isolated intact from the genomic DNA of M18 resistant fungal transformants by 2. co_li_ transformation. MATERIALS AND METHODS Strains and Media. 2. chrysosgrium strains MEIIIIG (ATCC 3115141) and strain BKM-F (ATCC 2A725) were maintained on malt extract slants as previously described (2). 2. coli DHSo [F-,endA1,_h__st17(rk-,mk+),§_9£lIlI,thi-1,re__c_AI, gy_rA96,relA1, A(_rgF- l_a_c2YA)0169, P80d_l__acz AM15, 1.], used for the maintenance of plasmid vectors, was maintained as described (15). Gold' s medium E (16) supplemented with 101 sorbose and 3 agar (Difco) was used for regeneration of 2. chrysosporium spheroplasts. A sterile aqueous solution of 61118 (Gibco Laboratories, Chagrin Falls, OH) was added to the autoclaved, cooled medium to a final concentration of 100 ug/ml. 2. coli was grown in L broth (15) supple- mented with 50 ug/ml of kanamycin. DNA Isolations and Manialgtions. Plasmid DNA was isolated from 2. coli by the method of Holmes and Quigley (17) and was purified through a two step CsCl-Ethidium bromide gradient (18) for use in transformation. 2. chryso— smrium DNA was isolated by the method of Rao and Reddy (19). All molecular biological techniques were performed according to Maniatis et al. (15). Construction of 9RR12. A 5. 8 kb derivative of plasmid pRR2, which contains a putative ar__s_ of 2. chrysosgrium inserted into Ylp5, has previously been described (19). This plasmid was further modified by the addition of the 2.2 kb 299' determinant from pNG35 (20) and was_designated pRR12 (Fig.1). Transfomtion of P. Email. Conidial suspensions (2 x 108 ml") were prepared from malt extract plate cultures of 2. chrysosporium as described (2) and used to inoculate 100 ml of malt extract broth in 250 ml foam plugged Erlenmeyer flasks, which were then incubated with shaking (150 Figgre I. Construction of vector pRR12. A 5. 8 kb derivative of pRR2 (16) which contains a putative autonomous replication sequence (a_r_s) of 2. chryso- rium was cut with PvuII and was ligated to a 2. 2 kb Pvuil fragment of M35 520)mll which contains the —ka_n" determinant of Tn903. Legend: Thick line within the m1! sites is the ka—n" determinant, with the arrows indicating inverted repeat regions. 8: E__coRi; M: Rindlll; P: 2_vu_ll; Ps: 2991; 5: 3.921; X: x__hoi. 721 260 Vol. 161. No. 2. 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS rpm) at 30°C until small germ tubes were visible microscopically on 'a small fraction of the conidia (approximately 8 hrs). The conidia were then spun down in a clinical centrimge, rinsed in sterile H20, resuspended in 10 ml of 1 M sorbitol containing 5 mg/ml novozyme 2311 (Novo Industries, Copenhagen, Denmark) and incubated with shaking (150 rpm) for 80 min at 30°C. Spheroplast formation was monitored by the addition of 20 ul of 101 sodium dodecylsulfate to 20 ul of the above spheroplast suspension and checking for lysis under a phase contrast microscope. The spheroplasts were then spun down at low speed in a clinical centrifuge and were washed twice in 1 M sorbitol, once in SMC [1 M sorbitol, 10 mM MOPS(3-(N-Morpholino) propanesulfonic acid), 50 mM CaClz, pH 6.3] and resuspended in 0.11 ml SMC plus 0.1 ml PMC {'40} PEG (Polyethylene glycol, BDH Chemicals Ltd., Poole, England), 10 mM MOPS, 50 mM CaClz] and 10 ul dimethyl sulfoxide. An equal volume of spheroplasts were added to each DNA sample, preincubated in 80 ul of 5 mg/ml heparin in 10 mM MOPS for 30 min, and to a negative control containing no DNA. The tubes were incubated on ice for a Nrther 30 min, vortexed after adding 2.5 ml PMC and incubated at room temperature for 20 min. Each sample was then split into two aliquots, 10 ml top agar (Golds medium 8; 16) supplemented with 10$ sorbose and 100 ug/ml GIIIB was added to both aliquots, and each reaction mixture was poured onto a plate of the spheroplast regeneration medium described above. For the determination of regeneration frequency 50 ul of the negative control (no DNA) is added to the same regeneration medium but without-61118. After 5-7 days incubation at 37°C, individual colonies become visible on this medium. 2ecovery of Vector DNA from Transformants. Colonies of GII18 resistant transformants from the previous step were inoculated onto malt extract plates supplemented with 100 ug/ml GlIIB. After II days of growth, the mycelia were scraped from these plates and DNA was isolated as described (19). Total DNA from GlIIB resistant 2. chrysospgrium transformants was used to transform 2. c_oIL__i using the method of Hanahan (21). High frequency transformation of 2. coL_i_ is critical since no transformants were recovered unless the trans- formation frequency was at or above 1x10°/ug DNA standard (CsCl-EtBr gradient purified pUC18). Transformed 2. coli were plated on LM medium (21) containing 50 ug/ml of kanamycin. RESULTS AND DISCUSSED! The Q” determinant of Tn903 (20), which encodes an aminoglycoside phosphotransferase is known to confer resistance to both kanamycin and Gina, and has been shown to be expressed in 2. g; as well as in several eukaryotic systems (22,23). Our initial results showed that growth of 2. hrysosmrium was completely inhibited on regeneration medium containing 50 ug/ml of OMB. A concentration of 100 ug/ml CRIB was thus chosen for selection in all trans- formation experiments. Transfor-tion of 2. W. Vector pRR12 transformed both strains of 2. chusosmrium (ME MIG and BKM-F) yielding about 20 61118 resistant transformants per ug DNA (Table 1). Negative controls with no DNA added or with salmon sperm DNA substituted for pRR12 DNA did not yield any transformants. The purity of the vector DNA appears to influence the transformation efficiency as the plasmids purified in a CsCl-ethidium bromide gradient gave approximately three-fold higher transformation efficiency as compared to that observed with plasmids isolated by the method of Holmes and Ouigley (17). Similar variation in transformation 722 261 Vol. 161. No. 2. 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table 1, Transformation of 2. chrysospgrium with vector pRR12a Strain Plasmid Transformation Transformation Frequency Efficiency (no./ug DNA) (no./regenerated spheroplast) MEAAG no DNA 0 O pRR12 6.6 3.3 x 10'3 pRR12b 22 1.1 x 10'2 BKM-F no DNA 0 O 11111112b 19.9 9.9 x 10'3 ‘Each strain was transformed with 10-20 ug vector DNA. A sample with no DNA added was used as a negative control. Transformants were counted as colonies growing on regeneration medium containing 100 ug/ml GA18. bPurified through a CsCl-ethidium bromide gradient (18). efficiency based on the purity of the vector DNA has been reported in other organisms also (211). The transformation efficiency, as determined by the number of transformants per regenerated spheroplast was comparable to that obtained for high frequency transformation of Neurosmra crassa (25), and Aspergillus nidulans (26). Recovery of Transforming Vector pRR12. He used two approaches to recover the transforming vector pRR12 from 2. chrysosmrium transformants. The first was to attempt to recover plasmids from individual transformants. The second was to make DNA preparations from a pool of individual transformant colonies on a plate. Both approaches were successful in recovering intact pRR12 by 2. 9922 transformation using genomic DNA of 2. chrysosmrium transformants. The plasmids 'recovered in 2. .1912 were identical to pRR12 (Fig. 2, panel A) in that all had the same restriction pattern as the original vector. Furthermore, identical Southern hybridization patterns were seen with both pRR12 and the plasmids recovered from individual GAB-resistant transformants when the 299" determinant of pRR12 was used as the probe (Fig. 2, lanes c and d). However, this plasmid appears to be present in too low a copy number in the total genomic DNA of fungal transformants to be detectable by Southern hybridization using the k_ag_" determinant as a probe. Maintenance of the transforming vector in low copy number is also suggested by the low frequency of recovery of pRR12 (.1- one transformant/10-3O ug genomic DNA) from fungal transformants. It is important to emphasize, however, that pRR12 was consistently recovered, although at a low frequency, from the trans- formants by 2. all transformation. The fungal transformants have been main- tained on selective medium (malt extract slants containing 200 ug/ml 61118) for 723 262 Vol. 161, No. 2. 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS A B up a b t: I! e f 9 h kbabcdeigh Figure 2. Recovery of pRR12 from 2. chrysosmrium transformants. One 113 of plasmid DNA was electrophoresed on a 0.71 agarose gel, blotted onto nitro- cellulose and hybridized with the Er determinant (1.7 kb PvuII fragment of pRR12). Panel A is the ethidium bromide stained photograph of the agarose gel and Panel B is the Southern blot of the same gel. Lanes a, c, e, contain uncut plasmid DNA and lanes b, d, f, and h contain PvuII digested plasmid DNA. Lane a and b contained pRR12; lanes c and d, and e and f contain separate plasmids recovered from the genomic DNA of hingal transformants via 2. c__oli transformation; plasmid shown in lane e was used to retransform wild type 2. chrysosgrium to 61118 resistance, and from the total DNA of one of those transformants the plasmid shown in lanes g and h was recovered. over a year and the vector recovered from such transformants was identical to pRR12. Plasmids recovered from the genomic DNA of transformants (Fig. 2, lanes e and f) were used to retransform 2. chrysosggrium to Gu18 resistance success- fully at frequencies similar to that observed with the original pRR12. Plasmids recovered from these second generation transformants (Fig. 2, lanes g and h) were also identical to pRR12. These results indicate that pRR12 can be passed intact through 2. chrysosporium repeatedly and recovered from CRIB resistant fungal transformants by 2. £222 transformation. Further studies are in progress to understand the nature of this novel transformation system mediated by vector pRR12. ACKNOWLEDGMENTS This research was supported, in part, by grant DE-FGOZ-8SERI3369 from the U.S. Department of Energy, Division of Basic Biological Sciences; NSF grant DMD-8H1271; and NIH grant 1-ROI-GM39032-01A1. He thank Novo Laboratories for the donation of Novozyme 23A. 7211 VbL.16L No.2,1989 263 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS d . can N) O5 U! 1: WM _a O 11. 12. 13. 1n. 15. 16. 17. 18. 19. 20. 21. 23. 2“. 25. 26. Crawford, R.L., and Crawford, D.L. (I984) Enz. Micrab. Technol. 6, ”33-480. Kirk, T.K., and Farrell, R.L. (1987) Ann. Rev. Microbial. A1, u65-505. Liwicki, R., Peterson, A., MacDonald, M.J., and Broda, P. (1985) J. Bacterial. 162, 641-6Au. 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