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IIIIII I IIIIE I'IIII‘W IiquII IIIIII: IIIRIII IIIIII I ‘1. ‘2' I I "III ' I II N v-‘IIII IIIII III}? III .II'I IIIIII I IIIIIIII IIII- IIIIII UIIII‘ ,‘I'K‘I'li III II” III‘ I’l’I‘zII‘IM”($371“IE2:I":'iI'IIIfiIIii?H ‘l, I} II II: IIIIII 'III‘I'I'I’ILI ”III 'IfIII IHIIII IIIIIIt :III IIIII 'II;IIIIIIIIJEIL‘L)IIIIWk 2‘ I I“ my IIIIIIIIIIII IiIII?!“ III 31%|}? ‘ “I I ' I III II I‘M}: 2 II :22}; .IIII 2'23." ."I III... 22 WII III III. ‘1. I I I J'j-{ESib This is to certify that the thesis entitled CONSTRUCTION OF NEW CLONING VECTORS FOR THE GENETIC MANIPULATION OF YEASTS presented by WEN HWEI HSU has been accepted towards fulfillment of the requirements for PH.D. degreein MICROBIOLOGY C’ MW Major profes/r Datey‘ZI/J>-g / / 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution \.____—-—A— _‘ -~..__r MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. 1.1.- A. l ‘ ”A CONSTRUCTION OF NEW CLONING VECTORS FOR THE GENETIC MANIPULATION OF YEASTS BY Wen Hwei Hsu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1983 ABSTRACT CONSTRUCTION OF NEW CLONING VECTORS FOR THE GENETIC MANIPULATION OF YEASTS by Wen Hwei Hsu Genetic cloning systems for yeasts are largely Therefore, studies were initiated to develop a broad host- range cloning vector for yeast. A yeast cloning vector, pHR40, which carries both'2p DNA and an autonomous replica— tion sequence (ars) from §. cerevisiae, and a kanr determi- nant from Escherichia coli transposon Tn601, which codes for an) aminoglycoside phosphotransferase that inactivates the antibiotics G418 and kanamycin. pHR40 transforms at high frequency a number of yeast genera besides §. cerevisiae into G418-resistance. This selection system eliminates the need for the construction of stable mutants to serve as the recipient strains. However, pHR40 does not appear to be the most desirable vector for gene transfer, because it is unstable in the host yeast. To study the factors which affect the stability of a cloning vector containing 532‘ determinant, several new, relatively small, yeast cloning vectors were constructed carrying 2n DNA, arg, and centromere (9323) genes, either singly or in r combination, from g. cerevisiae and a 523 determinant. All the vectors transform G418-sensitive s. cerevisiae to G418-resistance with a high frequency, and replicate autonomously in host yeast. The results suggested that the smaller the molecular size of the vector, the greater is its mitotic stability in §. cerevisiae. The presence of 9333 appears to enhance the mitotic stability of the vector. A new cloning vector (pHMR22) containing an ars from Candida utilis was constructed as a first step in developing a cloning system for Q. utilis. pHMR22 is small in size (6.6 kb), and has several unique restriction enzyme sites for gene cloning. Therefore, pHMRZZ should be a useful vector for cloning the desired genes in g. cerevisiae and perhaps also in g. utilis, and for the comparative study of ars in yeast. To Huiyu, Suting, Suchi and my parents ii ACKNOWLEDGMENTS I would like to express my appreciation to Dr. CMA. Reddy for his valued counsel during the course of this study. I am also grateful to Drs. P. Gerhardt, P.T. Magee, H.L. Sadoff, J.M. Tiedje, and M.T. Yokoyama for serving as my Research Guidance Committee. The critical suggestions and assistance of Ms. B.B. Magee, Dr. M. Clancy, Dr. J.B. Dodgson, Mr. RJL Kelley, and numerous others have been very useful and are gratefully acknowledged. I would like to thank Dr.l%C. Ma for his encouragement. Finally, I thank the Department of Microbiology and Public Health (Michigan State University), Food Industry Research and Development Institute (Republic of China), and National Science Council (Republic of China) for the financial assistance provided to me. iii TABLE OF CONTENTS Page List of Tables ....................................... vi List of Figures ...................................... vii General Intr0duction O...OOOCOOOOOOOOOOOOOOOOOOOO0.... Introduction ....................................... Transformation Methodology ......................... Spher0plast method ............................... 7 Alkali cations method ............................ 15 Selectable Genetic Markers for Transformation ...... 15 Cloning Vectors .................................... 20 Features of an ideal cloning vector .............. 20 Autonomously replicating vectors ................. 25 (I) 2n plasmid vectors ......................... 25 (i) properties of 2p DNA ..................... 25 (ii) Transformation with 2p plasmid vectors .. 29 (II) ars vectors ............................... 32 (i) pr0perties of ars ........................ 32 (ii) Transformation with ars vectors ......... 34 Integrating vectors .............................. 37 (I) Circular vectors ........................... 37 (II) Linear and gapped vectors ................. 41 Cosmids .......................................... 43 1 Literature Review. Gene Cloning in Yeasts ........... 6 6 7 Isolation and Expression of Cloned Genes in Yeast .. 44 Isolation techniques ............................. 44 Gene expression in yeast ......................... 53 (I) Expression of foreign genes ................ 53 (II) Expression of yeast genes ................. 56 Application of Gene Cloning in Yeast ............... 58 Conclusion and Outlook ............................. 62 Literature Cited ................................... 64 Chapter 1. Construction of a New Yeast Cloning Vector Containing Autonomous Replication Sequences from Candida utilis ............ 86 Abstract 0 O I C C O O O O O O I O O O O O O O O O O O O O O O O O O O O O O O O O 0 O O O O O 86 IntIOduction O O O O O O O O O O O O O O I O O O O O I O O O O O O O O O O O O O O O O O O 88 Mater ials and MethOdS O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O 9 1 iv Page Results ............................................ 96 Discussion ......................................... 116 Acknowledgments .................................... 122 Literature Cited ................................... 123 Chapter II. DevelOpment of a New Generalized ~ Transformation System for Yeasts ........ 127 Abstract ........................................... 127 Introduction ....................................... 128 Construction of a Broad Host-Range Cloning Vector Containing kanr, arsl and 2p DNA ................. 129 Generalized Transformation Procedure for Yeasts .... 130 Demonstration of pHR40 in Yeast Transformants ...... 138 Acknowledgments .................................... 143 References .............. ..... ...................... 144 Chapter III. Construction of New Cloning Vectors for Gene Transfer in Saccharomyces cereViSiae .0...OOOOOOOOOOOOOOOOOOOIOOI. 146 Abstract ........................................... 146 Introduction ....................................... 147 Materials and Methods .............................. 149 Results ............................................ 152 Discussion ......................................... 166 Literature Cited ................................... 169 Appendix. Alkaline Hydrogen peroxide pretreatment of Wheat Straw for Enhancing Cellulase Hydrolysis and Ethanol Production ......... 173 Summary ............................................ 173 Introduction ....................................... 174 Materials and Methods .............................. 177 Results ............................................ 180 Discussion ......................................... 194 Acknowledgments .................................... 197 References ......................................... 198 Table LIST OF TABLES Literature Review Table Table Chapter Table Table Chapter Table Table Chapter Table Table Appendix Table Table 1. 2. III 1. 2. l. 2. S. cerevisiae - E. coli shuttle vectors .. Some cloned yeast genes .................. Microbial strains and plasmids used and their sources ............................ Properties of the arg-YIpS plasmids isolated from Ura+ transformants ......... Sensitivity of various yeast strains to antibiotic G418 .......................... Transformation of various G418-sensitive yeast strains to G418 resistance with chimeric plasmids harboring kanr determinant .............................. Microbial strains and plasmids used and their sources ............................ Mitotic stability of different cloning vectors in S. cerevisiae ................. Effect of incubation temperature during NaOH and H 02-NaOH pretreatments on the susceptabiIity of wheat straw to cellulase ................................ Effect of NaOH and Hzoz-NaOH pretreatment on glucose and ethanol production from wheat straw and Sigma Cell ............... vi Page 22 46 92 102 133 135 151 161 192 193 Figures LIST OF FIGURES Literature Review Figure Figure U) N i" 0 Figure Figure 4. Chapter I. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Chapter II. Figure 1. Figure 2. Chapter III. Figure 1. Figure 2. Transformation of yeast : spheroplast method .................................. Transformation of yeast : alkali cations method .................................. Physical maps of the two forms of the 2p DNA .................................. Model for integration of a linear (A) or a circular (B) plasmid ............... Scheme for the isolation of Egg from S. utilis ............................... Analysis of the transforming plasmids of S. cerevisiae YNN27 in Ura transformants ........................... Restriction mapping of plasmid, pHMR22, containing §£§ of S. utilis ............. Subcloning analysis of arJ DNA from . utilis ............................... §r0b1ng of total yeast DNA with 2P-labeled pBR322 DNA (a) and SalI/ HindIII arJ fragment of pHMR22 DNA (b) .. Restriction maps of C. utilis DNA flanking cloned arJ DNA in pHMR22 ....... Construction of the broad host-range yeast cloning vector, pHR40 ............. gybridization of total yeast DNA to labeled pBR322 DNA .................. Construction of the yeast cloning vectors pHR40 and pHR7l ................. Construction of the yeast cloning vectors pHR2 and pHR31 .................. vii Page 16 27 39 97 99 104 107 111 114 131 139 153 156 Figure Figure Figure Appendix Figure Figure Page Restriction analysis of the pHR2 DNA containing 9323 gene of S. cerevisiae ... 158 gybridization of total yeast DNA to p-labeled pBR322 DNA ................... 163 H 02-NaOH pretreatment of wheat straw: e fect of NaOH and H202 concentration on (a) delignification as shown by increase in absorbance at 280nm, (b) loss of dry weight, and (c) saccharification ........................ 181 H 02-NaOH pretreatment of wheat straw: effect of pretreatment time on (a) delignification as shown by increase in absorbance at 280nm, (b) loss of dry weight, and (c) saccharification ........ 187 viii General Introduction Recombinant DNA methodology involves cleavage of a given host DNA by restriction enzymes and insertion of one or more host DNA fragments into a cloning vector, which is then used for transformation of a suitable recipient cell. Thus, genetic information from one organism may be transferred to and expressed in a related organism, or often in an unrelated organism as well. This powerful new technology allows one to understand the molecular mechanisms controlling the functions of the genes inside the cell and to alter the genetic make-upiof an organism precisely according to plan so that one can create an organism tailored to produce a desired product inexpen- sively and efficiently. Genetic engineering techniques became available for the study of yeasts with the estab- cerevisiae in 1978 (95). A great deal of progress has been made since then in developing many different type of yeast cloning vectors (13,14,21,27,119,149,188). However, at the present time, gene cloning systems are essentially restricted to Saccharomyces and Schizosaccharomyces pombe (9,13,106). Development of gene cloning techniques for other yeast genera has been hampered by the fact that suitable cloning vectors and transformation systems are not 2 available for these organisms. Many yeasts, such as Candida utilis are important from the stand point of biotechnology. They can be used for the production of single cell protein, chemicals, enzymes and other products" To apply recombinant DNA methodology to these industrially important yeasts, a high frequency transformation system is prerequite. Such a system will allow the development and utilization of many different yeasts as host cells for foreign genes and will provide a means for the further analysis of structure and regulation of various yeast genes. Gene cloning will be particularly useful for genes such as xylose isomerase, which are industrially important but are absent in S. cerevisiae. High frequency transformation in yeast has been accomplished primarily with two kinds of plasmids: 1) those containing fragments of 2 u DNA, which clearly are capable of self-replication in yeast (13,14,21,27); and 2) those containing a certain yeast chromosomal sequence called autonomous replication sequence (SEE): which presumably contains replication origin 1km: DNA synthesis (21,111,119,188). Efficient replication and maintenance of the 2 u chimeric vector in host yeast requires, in addition to the replication origin, the DNA regions ggpl, £322 and ggp3 which almost span the entire 2 u DNA and are implicated in copy number control. Two of these (£221 and £322) code for proteins that are active in trans (23). 11 third locus (r__e_p3) is required in cis (G. Tschumper, 3. personal communication). This system maintains the 2 u plasmid copy number at about 50-100 copies per diploid cella‘Vectors containing the whole 211DNA.are relatively large and have too many restriction endonuclease sites to serve as ideal cloning vectors. Therefore, a number of smaller and more useful 2 u plasmid vectors, which contain replication origin of the 2 u DNA but do not have all three £32 sequences, have been constructed (l3,14,21,27L. These plasmids have mostly been used to transform yeast strains containing endogenous 2 u DNA, which supplies the £32 gene functions. These type of plasmids, however, may not be suitable as cloning vectors for yeasts other than Saccharomyces and Schizosaccharomyce because 2 u DNA or sequences complimentary to 2 11 DNA have not been demonstrated in other yeast genera (86,219). Autonomous replication sequences,ixicontrast_to:2u DNA, are reported to be widely distributed and have a broad host range» For example, the Eggs from several phyloge- netically distant eukaryotes (121,200,232) as well as prokaryotes (80) were known to confer on yeast integrating plasmid (e.g. YIpS) the ability to replicate autonomously in S. cerevisiae. A question of considerable interest is whether a plasmid containing ars would serve as broad host range cloning vector for a wide variety of yeasts other than Saccharomyces. Most selection systems used in yeast recombinant DNA methodology currently employ a suitable cloning vector, 4 which carries a gene responsible for a specific step in the biosynthesis of a nutrient such as an amino acid or nucleotide, and a cmuresponding auxotrophic recipient strain (12,95,100,138,200). Selection based on the use of auxotrophic mutants obviously has the disadvantage of requiring the construction of stable auxotrophs which is not always easy. With the exception of S. cerevisiae and S.‘pgm§g, stable auxotrophs suitable for genetic cloning are currently not available for most other yeasts. An antibiotic-sensitive yeast strain could be used as a recipient strain 1J1 place CHE nutritional auxotrophs, provided the yeast cloning vector carries the genetic determinant for that particular antibioticu A number of antibiotic resistance determinants of bacterial origin have been known to be expressed in yeast (22,36,107,182), but most cells are relatively resistant to these antibiotics (45,106). One exception is G418, an aminoglycoside antibiotic, which was reported to inhibit the growth of Saccharomyces. Jimenez and Davies (114) recently described a transformation system for S. cerevisiae in which a I cloning vector carrying kan determinant from Escherichia 99;; transposon Tn601 and a G418-sensitive recipient yeast strain were employed. Since selection based on drug resistance would eliminate the requirement for the con- struction of a stable yeast mutant of a specific genotype to serve as the recipient, it appeared that this type of selection system may be valuable for the genetic manipula- 5 tion of wild type strains of S. cerevisiae and perhaps other industrially important yeasts. The main objectives of this study were as follows: 1. To isolate and characterize the ars sequences from S. utilis; 2. To determine whether a plasmid containing ars and 532‘ would serve as a broad host-range cloning vector; 3. To analyze the factors controlling the mitotic stability of vectors containing Sggr and BEE- The results show that the cloned a__r__§ of _C_. g_t__i__l__i_§ has no detectable homology to the S. cerevisiae genome but is fully functional in this yeast, and that the ESEF/G418 selection system coupled with LiCl transformation procedure is useful for the transformation of a number of yeast genera, besides Saccharomyces. 'Theinitotic stability of kanr-ars plasmids apparently depends on the molecular size of the vector. The smaller the molecular size of the vector, the greater is its mitotic stability in S. cerevisiae. The presence of a centromere element (9233) appears to enhance the mitotic stability of a vector. LI TERATURE REVI EW Gene Cloning in Yeasts Introduction Yeast, especially Saccharomyces species, have become important tools for genetic research in the past few years. This is partly due to the fact that the organization of the yeast genome is relatively less complex than that in most other eukaryotes.(68,17l,l73L. Further, genes from other eukaryotic organisms which are not selectable or compatible with bacterial systems can be cloned in yeasts. Furthermore, many yeasts can be propagated either in the haploid or diploid state with relative ease. The popularity of yeast as genetic tools also stems from the fact that many of them can be sporulated and that each spore can be germinated and studied individually. Studies on the genetics of yeasts have particularly mushroomed after the transformation system(s) which permitted the isolation of virtually every gene became widely available. Yeast cloning technology provides a powerful tool for genetic studies because the effect of changes induced i2 31559 within the cloned gene can be monitored ig‘yiyg by transforming the recombinant plasmid back into a suitable recipient yeast cell (84,143,189,209). This has thus paved the way for extensive studies on the mechanism of gene expression in yeast. In this paper, I have reviewed the procedures for yeast transformation and described the properties of different types of cloning vectors currently available for yeast, especially S. cerevisiae. Considerable emphasis is placed on reviewing the isolation and expression of genes of yeast origin as well as foreign genes in yeast. Finally, some possible applications of yeast cloning technology and a few comments about future challenges in this field are presented. The current advances in yeast recombinant DNA technology are particularly emphasized. Readers who are interested in the general area of gene cloning in yeast are referred to several excellent recent reviews (13,96,106,l63). Transformation Methodology Spheroplast method Hinnen et a1. (95) were the first to develop a Spheroplast method for the transformation of S. cerevisiae and several variations of this basic procedure have been reported subsequently (12,110,112L. The basic steps (Fig. 1) involved in this procedure may be summarized as follows: cells in the logarithmic phase of growth are treated with a mixture of glucanases to produce yeast spheroplasts. The spheroplasts arelextensively washed with 1M sorbitol and then treated with calcium chloride and polyethylene glycol (PEG; 4000 or 6000 M.W.) to promote the uptake of the transforming DNA. The transformed spheroplasts are Fig. 1. Transformation of yeast: spheroplast method. Yeast cells in log phase Treatment with 2-mercaptoethanol or dithiothreitol Digestion of cell walls with Glusulase or Zymolase Washing spheroplasts with 1M sorbitol or 0.6M KCl Treatment of spheroplasts with calcium chloride Competent cells Addition of transforming DNA Treatment with PEG Incubation in complete medium and embedding in selective medium containing 2-3% agar Transformed yeast colonies 10 embedded in a selective medium containing a high percentage of agar, usually 2-3%, to facilitate the regeneration of the cell wall (225L. In the final step, the spheroplasts are plated on a selective medium which would allow the growth of the transformed clones only. Yeast cell walls are composed of a mixture of polysaccharides, such as glucans, mannans and chitin, and other components such as amino sugars, proteins, lipids and phosphates (3,45”. The cell wall must be digested, at least partially, with a mixture of hydrolytic enzymes to generate spheroplasts and thus to promote the effective uptake of transforming DNA (13,95). The effectiveness of various hydrolases is thought to be influenced by interactions betweentflmepolysaccharide and the other polymers (such as proteins) within the cell walls (7). Reducing agents such as, 2-mercaptoethanol (54,192), and dithiothreitol (192) are often used in transformation procedures because they reduce disulfide bridges in the cell wall proteins (67) and thus apparently renders the cell wall polysaccharides more susceptible to digestion.by glucanases. However, it is still not known exactly how the reducing agents make the cell walls more vulnerable to hydrolysis. The pretreatment of yeast cells with reducing agent prior to the addition of hydrolytic enzymes is not always essential and its requirement probably depends on the yeast strain used and the physiological state of the cells to be treated (13). The 11 properties of lytic enzymes used for the preparation of yeast spheroplasts had been extensively reviewed by Kuo and Yamamoto (130) and by Hamlyn et a1. (87). Most of the enzyme preparations used for making spheroplasts are rather crude mixtures of hydrolytic enzymes such as those in snail gut extracts and are capable of hydrolysing polysaccharide components such as chitin and B-glucans, which are known to play a major role in the cell wall mechanics of budding yeasts such as S. cerevisiae (3,63). Preparations such as Glusulase (87,130) and Zymolase (130), which contain high levels of B-D-glucanase and chitinase activities, generally give the best spheroplast yield from S. cerevisiae (87). The fission yeast eehiaeseseheremeee 22292 differs chemically from budding yeasts in that a-mannan and chitin are absent from its cell wall, and in that about one—third of the glucan contains (l+-3)-a-1inked glucose residues (7,28). This apparently accounts for the resistance of its cell wall to the extensive hydrolysis by (1+3) and (1+6) B- glucanases (58,118,217). ‘Kopecka (126) using a combination of snail and Trichoderma viride enzymes, and Schwenke and Nagy (191) using a combination of snail enzymes and a (1+3) cwglucanase and a (l+3)-B-glucanase, have reported quantitative conversion of exponentially growing cells of S. 99999 to spheroplasts. Dickinson et a1. (60) described the use of Novozym 234, which contains high levels of a-D- glucanase and B-D-glucanase activities, to prepare spheroplasts from S. pngg. Both exponential growing and 12 stationary phase cells can be quantitatively converted to spheroplasts on a large scale by using this procedure. Beach and Nurse (9) have also successfully used Novozym 234 to develop an efficient transformation procedure for S. @- Variations in susceptibility to attack by lytic enzymes has been observed between different yeast strains of the same species and between species (130). Even within a single strain, susceptibility is highly dependent on the physiological state of the yeast (130). This is undoubted- ly due to the relative proportion of the different glucan components in the walls and, perhaps more importantly, their susceptibilities to hydrolysis by the particular glucanases in the digestion mixture. Generally, cell walls of exponentially growing yeast cells are more susceptible for digestion (56,64,184,185) and give a higher percentage of regeneration of spheroplasts than stationary phase cells. Therefore, exponential phase yeast cells, rather than stationary phase cells, are used for yeast transforma- tion. The extent of cell wall digestion must be carefully standardized to obtain an optimal level of yeast transfor- mation because extensive cell wall removal will adversely affect the regeneration of spheroplasts and consequently lead to poor transformation efficiency (96). The extent of cell wall digestion can be monitored by lysing the spheroplasts with sodium dodecyl sulfate and‘viewing the 13 samples with a light microscope (192). The extent of cell wall digestion can also be monitored by adding four parts of water to one part of spheroplast solution and measuring the decrease in optical density at 800 nm (122,123,124) in comparison with that obtained when 1M sorbitol, instead of water, was added to the spheroplast suspension. Use of cells that are only partially converted into spheroplasts may be preferable because this will reduce spheroplast fusion occuring during transformation. Glusulase (95) and Helicase (13) are widely used for the transformation of S. cerevisiae, and S. carlsbergensis, since with these preparations overdigestion of the cell wall rarely, if ever, occurs. Although Helicase is a cruder preparation than glusulase, the tranformation efficiencies obtained with these preparations were very similar. In general, Zymolase treatment has to be timed more accurately because long exposure can extensively digest the cell wall and result in poor regeneration frequencies of spheroplasts (96). Since spheroplasts are osmotically fragile, an osmotic stabilizer such as sorbitol must be added to the suspending medium. Although sorbitol at concentrations ranging from 0.9 to 1.2 M (106,130) has been recommended, 1.0 M sorbitol should be the best starting point in developing transforma- tion for a new yeast species (4). In cases where sorbitol is not desired as an osmotic stabilizer, because the yeast species in question can use it as a carbon source, one can 14 use KCl (4,60,106,130L. The recommended concentration for S. cerevisiae is 0.6 M KCl (4,106) because this is close to the osmolality which elicits incipient plasmolysis (5). The metabolic activities of the spheroplasts are influenced by the concentration and nature (ME the stabilizer (130,142,190). In some cases, potassium chloride leads to even better spheroplast regeneration frequencies than those observed with sorbitol (106). DNA uptake by the spheroplasts is promoted by treatment with calcium chloride and by the addition of 20— 40% PEG (13). Addition of PEG results in the fusion of some of the spheroplasts (92,225). Apparently, the function of the PEG is to induce better contact between cells (213,214). This in turn may result in more cell fusion during incubation in the regeneration medium (92,214). To facilitate regeneration (M5 the cell wall, spheroplasts are embedded in a solid matrix which contains 2-3% agar, is osmotically stabilized and contains certain specific nutrients required for the growth of the specific recipient yeast (12,95,192). Colonies develop in the agar in 2-7 d depending on the particular vector and recipient yeast strain used. Schizosaccharomyces spheroplasts can regenerate the cell wall even in liquid medium (154) and on the surface of agar (9). The transformation efficiency (transformants/pg DNA) is influenced by several variables including the strain 15 (106,115), the extent of spheroplast formation and regeneration (106), the plasmid type (13) and the selection system (106). Genetic data from crosses between high- transformation and low-transformation strains suggested that the transformation frequency is 23 polygenic inheritance and high-frequency transformation is inherited in a recessive fashion in S. cerevisiae (115). Alkali cations method Ito et al. (113) recently described a new transforma- tion procedure for S. cerevisiae in which alkali cations such as Li+ and Cs+, instead of glusulase, were used for preparing competent yeast cells (Fig.2D. This procedure does not require an osmotic stabilizer and regeneration agar. However, the transformation efficiency is 10-100 fold less than that obtained with conventional spheroplast procedure. Selectable Genetic Markers for Transformation Most selection systems used in yeast recombinant DNA methodology currently employ a suitable cloning vector, which carries a gene responsible for a specific step in the biosynthesis of a nutrient such as an amino acid, (12,95,110) or nucleotide (138,200), and a corresponding auxotrophic recipient strain. To eliminate background reversion of the selected marker in the transformation system, stable yeast mutants with double mutations such 16 Fig. 2. Transformation of yeast: alkali cations method. 17 Yeast cells in log phase Treatment with Cs+ or Li+ Competent cells 1 Addition of transforming DNA Treatment with PEG 1 Heat shock (42°C) Washing with water 1 Incubation in complete medium and spreading on the surface of selective agar medium l Transformed yeast colonies 18 (e.g. 1332 locus) or deletion mutations (e.g. 3533 or SL133 locus) are often used (188,200). These mutations have a reversion frequencies lower than 10"8 (96) and therefore background reversion is not a problem even when the integrating vectors are used. Selection based on the use of auxotrophic mutants obviously has the disadvantage of requiring the construc- tion of stable auxotrophs, which is not always easy. With the exception of S. ggggyigigg and S. 29393, stable auxotrophs suitable for genetic cloning are currently not available for most other yeasts. An antibiotic-sensitive yeast strain could be used as a recipient strain in place of nutritional auxotrophs, provided the yeast cloning vector carries the genetic determinant for that particular antibiotic. However,an:the present time drug-resistant genes of yeast origin which are dominant are not avialable as selective markers for transformation of wild type strains. Antibiotic resistant mutations which lead to alterations in the primary structure of ribosomal proteins are generally recessive. One such gene, encoding for trichodermin resistance in S. cerevisiae, has been cloned and shown to code for ribosomal protein L3 (74). A direct transformation selection for plasmids carrying the trichodermin-resistant gene was not possible. With the knowledge that the genes of drug resistance, such as those arising from R-factors of Escherichia coli, are capable of expression in the most varied species of l9 bacteria (65),tflmeexpression of some of these bacterial antibiotic genes in yeast.has been studied. .Although the ampicillin determinant has been shown to be expressed in yeast (22,36,107,182), it is not useful as a selective marker for transformation, because S. cerevisiae is not sensitive to ampicillin (106). .Jimenez and Davies (114) recently described a transformation system for S. cerevisiae in which a cloning vector carrying S. ggli transposon Tn601/Tn903 (82,161) which contains the Eggr determinant (code for a phosphotransferase that inactivates the aminoglycoside antibiotics G418 and kanamycin) and a recipient yeast strain sensitive to G418 were employed. The yeast transformants obtained were resistant to high levels of antibiotic G418. Growth and metabolism of yeasts on nonfermentable carbon sources (glycerol, alcohol, etc.) depend on the functioning of the mitochondria, which are inhibited by chloramphenicol. Yeast cells containing a plasmid bearing a chloramphenicol resistance gene (ggmr) from on S. 9911 plasmid can grow in a medium with a nonfermentable carbon source in the presence of small quantities “LS mg/ml)iof antibiotic (45). However, since yeast cells are relatively resistant to chloramphenicol this antibiotic determinant is not very useful as a selective marker in transformation. .A deletion of 120 bp immediately before the beginning of the structural part of the ggmr gene enhances expression of the gene and leads to a 50-fold increase in acetyltransferase 20 activity in yeast cells (44). Transformants containing this plasmid may be directly selectable on medium containing chloramphemicol but this fact has never been established. It is possible that with further research chloramphenicol resistance may prove to be a useful selectable marker, similar to G418 resistance. Nevertheless, based on the data available to date, it appears that antibiotic resistance determinants have the potential to serve as useful selectable markers for the transformation of agriculturally, medically or industrially important yeasts for which stable auxotrophic or other mutants are not available. Cloning Vectors Features of an ideal cloning vector General purpose cloning vectors for yeast are genetically and structurally well-characterized and can be readily purified in large quantities. These plasmids are almost exclusively chimeric plasmids containing S. 99$} replicon and DNA sequences which include genes useful for selection in S. 99;}; or in yeast transformation. In contrast to integrating vectors (64L, YIp plasmid), the cloning vectors which are capable of autonomous replication in recipient yeast cells additionally carry DNA fragments containing a replication origin for 2 u DNA (eqp, YEp plasmid) or autonomous replication sequence (SEE? e4L, YRp plasmid). The properties of S. cerevisiae cloning vectors 21 described to date are listed in Table 1. An ideal plasmid cloning vector for yeast should possess the following features: (a) should be relatively small so that high amounts of DNA can be cloned into it; (b) should contain a bacterial replicon such as the pBR322 origin which permits amplification of the plasmid in S. 9911 cells; (c) should carry selectable genetic markers for both yeast and S. gg_l__i_ transformation; (d) should contain unique restriction sites for as many of the commonly used restriction endonucleases as pmssible; (e) should possess one or more promoters that can actively transcribe cloned genes, (f) should have properties that permit the detection or selection of hybrid molecules easily, and (9) should contain an yeast replication origin and be stably maintained in the host yeast during cell propagation. The type of yeast vector one chooses is clearly dictated by the specific cloning problem. If a high level of expression of the cloned gene is desired, a cloning vector with high copy number in the recipient yeast cell is advisable» iHowever, with high copy number the potential deleterious effects from high gene dosage also have to be considered and might in some cases favor the use of a low copy number cloning vector such as the cloning vector containing a centromere gene and the replication origin from S. cerevisiae (7,110). 22 Table 1. S. cerevisae - S, coli shuttle vectors Stabilizinga Selective Cloning References Vectors Size elements markers sites or sources (kb) YIpl 9.8 ori Amp”, h133 EcoRI,Sa11 XhoI 21 YIpS 5.5 ori Amp”, Tet” EcoRI, BamHI 21,188, ura3 SalI, HindIII 201 SmaI YIp25 11.9 ori Tet”, hisu BamHI, HindIII 21 YIp26 7.8 ori Amp”, leu2 BamHI, SalI 21 ura3 SmaI Y1p27 7.8 ori Amp” leu2 BamHI, SalI 21 ura3 SmaI YIp28 7.8 ori Amp” leu2 BamHI, SalI 21 ura3 SmaI YIp29 7.8 ori Amp” 1222 BamHI, SalI 21 ura3 Smal YIp30 5.5 ori Amp” ura3 EcoRI, BamHI 21 Sell, SmaI YIp31 5.5 ori Amp”, ura3 EcoRI, BamHI 21 SalI, SmaI YIp32 6.7 ori Amp”, leu2 BamHI, SalI 21 PstI, HindIII YIp33 6.7 ori Amp”, leu2 BamHI, Sell 21 PstI, HindIII er7 5.7 arsl, ori Amp”, Tet” BamHI, SalI 21,199 trpl YRp12 7.0 ars1, ori Ampr, Tetr BamHI, SalI 188 trp1, ura3 HindIII chsuu 10.3 arsl, ori Amp”, Tet” BamHI, SalI 119 trpi pLC1 8.7 arsZ, ori Tetr, argfl BamHI, EcoRI 111 pYe(cen3) 9.2 ars1, cen3 leu2 BamHI, HindIII M1 91 ori SalI Table 1 (cont'd) 23 Stabilizinga Selective Cloning References Vectors Size elements markers sites or sources (kb) YEp6 7.9 2p, ori Amp”, his3 EcoRI, Xhol 21 Sell YEp13 10.7 2p, ori Amp”, Tet” BamHI, PvuII 27 leu2 SstI YEp16 10.7 2p, ori Amp”, Tet” BamHI, PvuII J. Hicks leu2 SstI, SalI YEp20 10.u 2p, ori Amp”, leu2 BamHI, Sell 21 PstI YEp21 8.8 2p, ori Amp”, leu2 BamHI, SalI 21 YEpZN 7.6 Zn, ori Ampr, ura3 BamHI, SalI 21 SmaI pJDBZO? 6.9 2u, ori Amp”, Tet” BamHI, SalI 13,1u leu2 PstI, HindIII pJDBllO 6.7 Zn, ori Ampr, ura3 BamHI, SalI 13,1“ EcoRI pJDB210 7.9 2p, ori Amp”, leu2 BamI, SalI 13,1u ura3 pJDBle 7.9 2u,,ggi Amp”, 1232 BamHI, SalI 13,1u ura3 pDBzuB 10.1 2n. ori Amp”, Tet” BamHI, HindIII 9 leu2 PstI pFL 2 7.6 2p, ori Amp”, Tet” BamHI 37 ura3 pEYlurl 9.0 Zn, ori Amp”, Tet” BamHI, SalI 1A1 ura1 prT1u- 13.9 2n, ori 0am”, Kan” BamHI, SalI J. Marmur kan5 Tetr pDBZ62 9.8 Zn, ori leu2 BamHI D. Beach & P. Nurse ZN Table 1 (cont'd) Stabilizinga Selective Cloning References Vectors Size elements markers sites or sources (kb) chlb 10.0 2p, ori Amp”, his3 BamHI, EcoRI 100 pBT1-1b 11.1 2n, ori Amp”, Tet” BamHI 1A9 leu2 pBT1-10b 7.7 ars1, ori Tet” trp1 BamHI 1H9 a. DNA sequences capable of conferring mitotic stability on autonomously replicating vector in yeast (e.g., 2 u, §§§_or ggg_3) or S. coli (221): b. Cosmid. 25 Autonomously replicating vectors (I) 2u_p1asmid vector (1) Properties of Zn DNA. The yeast 2u plasmid, sometimes referred to as S. cerevisiae plasmid (Scpl), is a circular, double-stranded-DNA molecule of about 6.3 kb present in most S. cerevisiae strains at 50-100 copies per a diploid cell (43). In yeast Species other than Saccharomyces and S. pgggg 2n DNA has not been found (86,219). The intracellular location of the plasmid is not clear, although many indirect arguments suggest that its actual location within the cell is in the nucleOplasm. The fact that autonomously replicating hybrid plasmid, consisting of a 21.1 DNA and a yeast gene, can recombine with the homologous chromosomal DNA (12,62,78), that it is packaged into nucleosomes containing the normal composition of core histones (136,155), that it is extensively transcribed, presumably by RNA polymerase II (24), and that its replication is under the control of nuclear DNA (137,174), all support the idea that 2u DNA resides in the nucleoplasm. The evidence against a nuclear location of 2p DNA is that nuclei isolated by conventional methods contain less than 5-10% of the total cellular plasmids (43). However, it has recently been reported that using different lysis procedures, 90% of Zn DNAs are associated with the nuclear matrix (216). Thus, it seems likely that the plasmid is associated with the nucleus, but the nature of this association is not known. 26 The function of the 2p plasmid in yeast is not known, but its structure is well defined (23,25,26). The complete nucleotide sequence of this plasmid has been determined by Hartley and Donelson (88). The restriction map of 211 DNA is depicted in Fig. 3. 'The plasmid consists of 2 unique segments of DNA (2774 bp and 2346 bp) separated by two copies of a 599bp sequence which are precise inverted repeats of each other. Reciprocal intramolecular recombination between the two inverted repeated sequences produces two forms of plasmid, form A and form B, that differ in the orientation of one unique region with respect to the other (23,25). .At least two physical variants of the 2 u DNA (Scp. 2 and Scp 3) have been reported as natural isolates from various strains of S. cerevisiae (30,135). Restriction analysis Scp 2 showed a 125 bp deletion, which removes HpaI restriction site in the large unique region (2774bp segment; Fig. 3). Scp 3 variant contain a 220 bp deletion, which removes both HpaI and AvaI restriction site, in the large unique region. Since the deletions in Scp 2 and Scp 3 are located at a site where a series of tandem repeats are present in 211 DNA, it seems likely that these variants arose by equal or unequal intermolecular recombination at these repeats (23). The sizes of deletions in Scp 2 and Scp 3 are consistent with the removal of 2 and 4 copies of these repeat sequences, respectively. The replication of Zn DNA is responsive to the 27 Fig. 3. Physical maps of the two forms of the 2p DNA. The approximate location of the origin of replication of the 2p circle is indicated by the heavy line in the schematic diagram of Form A. The regions Sgpl and £322 encode proteins that promote high copy levels of 2p circle plasmid. (Adapted from reference 23). 28 ' ‘ 2000 mm: Pvul ID 0 D 5“! Hind- . . . Econ! xm 5000 9'“ 3000 PU Ibo! r . 2 (Hlncl) ‘2 Em! M Ava! M m HM. §III 5mg ulna 3000 M l O 0 0 1000 I Put Xbcl line! 0 5000 Hind. M1 M1 Econ! 29 cellular control mechanism that limits chromosomal DNA replication to one round per cell division (137,1751. The 2p plasmid was shown by electron microscopy to have an active DNA replication origin (32,125). Using high frequency transformation of yeast as a criterion for origin function, Broach and coworkers (25,26) have identified a single 350 bp region of the 2p plasmid which contains the replication origin. The region lies predominantly within one inverted repeat region but extending approximately 100 bp into the continuous large unique region (26). (ii)Transformation with 2u plasmid vectors. Yeast—S. 99$; chimeric plasmids, such as YEp containing part or all of the 2p DNA can transform yeast cells with frequencies as high as 103-105 transformants/pg of DNA (lZfiULllO). Southern hybridization analysis clearly showed the presence of copies of the transforming plamid and genetic analysis revealed a 4+: 0' segregation pattern indicative of plasmid-mediated inheritance (92). These results reflected that the 2u' plasmid vector can autonomously replicate in host yeasts. The transformation efficiency and mitotic stability of the 211 DNA chimeric vector varies depending on which part of the Zn sequence is carried by the vector and the presence of endogeneous 2u DNA in the recipient yeast strain (26,92). The replication origin of 211 DNA is required for the replication of the yeast -S. 99;; chimeric plasmid in recipient yeast. Plasmids containing the entire 30 Zn sequence transform both Cir+ and Ciro (2n DNA-free) strains with high efficiency and the transformed phenotype is relatively stable (18,26,61). The 3.9 kb and 2.2 kb EcoRI fragments, from 211 DNA forms A and B, respectively, contain the replication origin of 2 11 DNA (Fig. 3). Each of these fragments confers on the hybrid plasmid high efficiency of transformation and stable maintenance in a Cir+ strain of S. cerevisiae. In contrast, plasmids carrying the 2.4 kb or the 4.1 kb EcoRI fragments (derived from 2 u DNA form A and B, respectively) which do not contain the replication origin of Zn DNA, have a relatively high transformation efficiency but are not stably maintained (26). However, if the recipient yeast is a Ciro strain, the hybrid plasmid carrying the 2.2 kb or the 3.9 kb EcoRI fragment is not stably maintained in recipient yeast and those carrying the 2.4 kb or 4.1 kb EcoRI fragment fail to yield any transformants. These results indicated that efficient replication and maintenance of 2 u chimeric vector in host yeast requires, in addition to the replication origin, the DNA regions £321, £322 and £323 which almost span the entire 2 u DNA and are implicated in copy number control. Two of these (£321 and £322) code for proteins that are active in trans (23) and a third locus (£323) that is required in cis (G. Tschumper, personal communication). Under normal conditions, replication of 2 u circles is strictly under cell cycle controls that limit the replication of Zn DNA to one round per cell division 31 (23). Apparently £32 proteins act to override cell-cycle control of 2p DNA replication and to induce multiple rounds of replication during a single cell-division cycle. Yeast-S. 32;; hybrid plasmids containing the whole 2p DNA can transform both Ciro and Cir+ strains of S. cerevisiae. However, many of these plasmids are relatively large and have too many restriction enzyme recognition sites to serve as convenient cloning vectors. Therefore, a number of smaller and more useful 2n plasmid vectors (Table l) which contain only the 2.2 kb EcoRI fragment or a similar fragment of 2p DNA, are capable of high frequency transformation of yeast and have a high copy number in host yeast have been constructed. These plasmids which contain replication origin of the 2 u DNA but do not have all three £32 sequences have mostly been used to transform yeast strains containing endogeneous 211 DNA which supplies the £32 gene functions. These types of plasmids, however, are associated with two types of instability in Cir+ cells. First, they frequently recombine with endogeneous 2n DNA and subsequently rearrange their DNA sequence (62,149). Secondly, under nonselective conditions, the endogenous 2p DNA is, in most cases, retained at the expense of the transforming plasmid because the latter uses the same replication and transmission system as endogenous 2u DNA (106). Beggs (13) reported that the mitotic stability of the Zn plasmid vector is associated with its copy number. 32 Plasmid pJDB207 has a copy number of about 50 in the transformed yeast whereas the copy number of pJDBllO is 5- to 6-fold lower than that of pJDB207. After propagation of the transformants for 20 generations on nonselective medium, 85% of the cells retained the pJDBZO7 while only 10-50% of cells contained pJDBllO. This may be due to the fact that recombinant plasmids maintained at a high copy number per cell are segregated out less frequently than those with a low copy number. Endogenous 211 DNA has a copy number of 50-100 and may be able to outcompete cloning vectors containing 2p DNA sequences in a population of transformed cells under nonselective conditnmua Therefore, Ciro strains of S. cerevisiae are more desirable as recipients in gene cloning experiments if a useful 2u plasmid vector which contains the replication origin and re2 1 and re2 2 sequences is available. (II) ars vectors (1) Properties of 3£3. Yeast transformation studies with recombinant DNA plasmids have identified a class of DNA sequences which promote high-frequency transformation and extrachromosomal maintenance of plasmid DNAs (10,34,200). These sequences are called autonomous replication sequences (3£3s) and their properties are believed to be due to their ability to serve as initiation sites for DNA replication (32). 3£Ss capable of high- frequency transformations have been reported to occur once in 30-40 kb in the S. 33£3313133 (199) genome. The 33 frequency of one replication origin per 36 kb was independently confirmed by the average spacing of initia— tion sites in S. 23£3113133 as detected by electron microscopy in small molecules of yeast DNA (156,157). 'The coincident frequency between the 3£3 and replication origin in S. cerevisiae strongly suggest that the 3£3 sequences isolated by high-frequency transformation contain the origins of replication of S. 33£3yi§i33. Recently, Celniker and Campbell (32) developed an 32 gi££2 system for the replication of both 211 circle and 3£§_-containing plasmids. They found that only plasmids containing a functional yeast 3£3 initiate replication at a specific site within the 3£3 region 32 yi££2. These data supported the view that 3£3 sequences contain specific origins of chromosomal replication in yeast. The DNA sequences of 3£31, 3£32 and 3£33 of S cerevisiae have been studied in detail (198,221,222). Extensive homologies have not been observed among these three 3£Ss and Zn DNA (198,222). A canonical shared sequence, TAAPyAPyAAPu, is present in 3£31, 3£33 and Zn DNA but absent in 3£32. Lack of sequence homology between the 3£3s of S. 33£3313333 was also indicated by their respective restriction enzyme maps. The essential region of the S. cerevisiae arsl DNA resides at or near a PstI site (221,222), whereas a similar site is lacking in 3£32 (198). These data indicate that the DNA sequences corresponding to 3£3 function are very diverse. This 34 observation is in striking contrast to the 2£S sequences of different bacterial species which were shown to have remarkable sequence homologies (236). Non-yeast DNA can allow autonomous replication of an integrating plasmid in.S. cerevisiae. Stinchcomb et al. (200) found that DNA fragments from a wide variety of eukaryotes (Neurospora crassa, Dictyostelium discoideumL Caenorhabditis elegans, D£oso2hila Eelanogaster and S33 23y3) are capable of conferring on yeast integrating plasmid (YIp) the ability to replicate autonomously in S. cerevisiae. The replication origins from both the DNA of Tetrahymena thermophila (121) and the mitochondrial DNA of 35322223 laevis (232) also promote high-frequency transformation and extra-chromosomal replication of the YIp plasmid. In addition to the DNA sequences from eukaryotes, a Staphylococcus aureus plasmid was found to act as an ars in S. cerevisiae (80). This evidence suggests that the ars sequences possess broad host-range specificity. UJJTransformation gith ars vectors. Plasmids con- taining 3£3 sequences, such as YRp, have been shown to be capable of high frequency transformation of yeast (103-104 transformants/ pg DNA) and were shown to be unstable during both mitotic and meiotic divisions of yeast transformants (110,119,211L. In general, when yeast transformants are propagated in nonselective medium for 20 generations, 95- 99% of the cells lose their selective phenotype and this in turn is associated with the loss of the entire hybrid 35 transforming plasmid. Kingsman et al. (119) grew the 3£3- vector transformants in selective medium and found that only 30-50% of the cells in transformed clones retain the selective phenotype. The mitotic stability of the 3£3-vector appears to be affected by its size. Zakian and Scott (234) constructed a novel 1.45 kb yeast plasmid 3£21 RI circle which contains 3£3 plus ££21 and consists solely of yeast chromosomal DNA. The ££2l RI circle occurs at 100 to 200 copies/cell (57,234) and was shown to be stable during both the mitotic and the meiotic cell cycles. In contrast, YRp 7 plasmid which contains the 1.45 kb ££2l RI circle plus pBR322, has a relatively low copy number (20-30/ce11) and is unstable. It is unlikely, however, that the stability of the 3£2l RI circle can be attributed solely to its high copy number. For example, plasmid pXEY26 which is relatively unstable, similar to YRp 7 (232), is found at 200 copies per cell in some yeast strains (233). The mitotic instability of pXEY26, inspite of its high cepy number suggests that 3£3- containing hybrid plasmids segregate nonrandomly. It is unlikely that the pBR322 sequence is reSponsible for the instability of YRp7. Stinchcomb et al. (198) constructed another vector, consisting only of yeast sequences, by circularizing an 8.0 kb XhoI/SalI fragment of S. cerevisiae chromosomal DNA carrying 3£2l and 3£31 (Sc4128), this fragment, when transformed into yeast, was also shown to be mitotically unstable. Therefore, the reasons for the 36 mitotic instability of YRp 7 are not clear. The stability of 2£21 RI circle is on theiother hand is most likely due to its small size which may permit it to escape the compartmentalization that causes asymmetric segregation of 3£3 vectors. The stability of 3£3 vectors in transformed yeast can be enhanced by the addition of a cloned yeast DNA fragment containing a functional centromere (33,40,4l,72,73). When the transformants are propagated in nonselective medium, 60-100% of the cells retain the centromere (332) - containing 3£3 vectors. The range of stability values obtained for the different 332-containing plasmids is not correlated with the size of the yeast DNA insert. For example, Fitzgerald-Hayes et a1.(73) reported a similar range of mitotic stabilities for plasmids containing fragments of DNA subcloned from the yeast insert in pYe (23214) 2 (5.2 kb insert), pYe (33211) 12 (1.6 kb insert) and pYe (33211) 5 (0.9 kb insert). Thus, the factors which determine the stability of the 3£3 - 332 vectors are still unclear. Hybrid plasmids containing centromeric DNA sequences in combination with a yeast 3£3 exhibit typical Mendelian segregation (2+ : 2‘) through meiosis (33,40,41,72,1\11). No specific class of chromosomal replicators appears to be required for proper functioning of the 332 - containing mini-chromosomes. Hsiao and Carbon (111) found that the replicator function in a plasmid containing 3323 can be 37 supplied equally well by 3£31, 3332 or even 211 DNA. The 3£3 - 332 vectors allow the stable introduction of a single gene into a yeast cell and, therefore, may be an useful and reliable gene cloning vectors, especially where a low copy number is not a problem and high genetic stability is required. Integrating vectors (I) Circular vectors A yeast integrating vector essentially consistscnfa bacterial cloning vector and a suitable yeast gene which can be used in the selection of yeast transformants and which provides homology to promote integration of the plasmid DNA into the chromosomal DNA of the recipient yeast cell (Table 1). Yeast transformation occurs as a result of the integration of the entire plasmid into the yeast genome. Such integrations can be confirmed by tetrad analysis in which the transformed gene shows Mendelian segregation patterns of 2+:2'. Since the transformation process with an integrating vector includes the step of DNA integration (48,92,95,l93,235), the efficiency 10f transformation is relatively low (1-10 yeast transformants per microgram of DNA; 98) and the transformed phenotype is relatively stable. After 20 generations of growth under nonselective conditions approximately 99% of the cells retain the selective marker (13). In the first transforma- tion experiments described by Hinnen et al. (95), they 38 found that the SSS plasmid pYe 33210 occasionally integrated at a genomic site other than at the homologous 3332 region. Subsequently it was found that a subcloned fragment of pYe 3310 contains a repeated sequence (120). This subcloned plasmid integrated at the dispersed copies of the repeated sequence as well as at the 2322 locus (95). Thus, all integrations appear to result frbm recombination between homologous sequences. In about one - third of the transformants, yeast sequences on the plasmid are substituted for the chromosomal sequences without integra- tion of theHuooHom . 3.5m: omv HHomu: 5H3 poucngmmsm =5ng o>HuooHom E 5385 n I .mucwEHuomxo mucummwm woufi Ho «.9395 am now @3835 E manS muHHHnmum .hmzzw omHmH>ouoo .m 35 Benownmcmuu mcHEmmHm so @958qu 9.83 35.5 nomad wuHHHnmum 03035 :5 om H A 0.". m.~ comm m.H «Hg mm H A oé m.~ ooom o.H mmmzmm H H A ode m.m oOHH mé $sz mm H A oé m.m oomH Hé Hams—E :5ng =5ng :5ng :5ng 22a ms mom 3v: mpHEMHm o>Huoonm w>Huoonmcoc o>HuooHom w>HuooHomcoc mucflfiemcmav oNHm nco wcoHumuocmmlom noumm nco Esau—Ounce» mo mucoHonuw uuomcH +95 @5533 .2chqu 35 063 833360 5323355 wmu§u0mm§uu +3: ERG ©3303 mpHammHm mmglmm on» mo 833905 .m 033. 103 20 generations in nonselective medium, 99% of the transformants lost the Ura+ phenotype. Frequency of a1§_1oci. To obtain a rough estimate of the frequency of Egg in g. utilis genome, g. 99;; HBlOl was transformed with the pool of YIp5 g. utilis hybrid DNAs. Twelve clones, each containing inserted YIp5 (ampicillin- resistant and tetracycline-sensitive), were isolated, and the plasmid DNA isolated from each individual clone was used to transform g. cerevisiae YNN27. One of these 12 hybrid plasmids allowed high-frequency transformation of YNN27. The average size of the MboI inserts in 12 hybrid plasmids is about 2.5 kb. This frequency of one ars per 30 kb is consistent with the demonstrated frequency of one ars per 30-40 kb in g. cerevisiae genome (2,8). Restriction mapping of ars plasmid pHMR22. pHMR22 was first digested with various enzymes and the fragment patterns compared to those of YIp5. Single enzyme diges- tion indicated that the ars does not have a site for PstI, SalI, HindIII, EcoRI and PvuII (Fig. 3A). Also, the ars plasmid still contains a cleavage site for BamHI after BamHI/MboI ligation (Fig. 3A, Lane C). Double enzyme digestion indicated that the BamHI site is near or at one of the ends of the ars close to the SalI site of the YIp5 sequences (Fig. BB). The restriction map of plasmid pHMR22 containing the ars of g. utilis is shown in Fig. 4. Subcloning of ars in pHMR22. To determine whether the BamHI site is located in that part of the ars essential for Fig. 3. 104 Restriction mapping of plasmid, pHMR22, containing 355 of Q. utilis. a. Single digestion of pHMR22 and YIp5, respectively, with EcoRI (lane A and B), BamHI (lane C and D), PstI (lane E and F), PvuII (lane G and H), HindIII (lane I and J) and SalI (lane K and L). The DNAs, after restriction, are electropho- resed on 0.7% agarose gel. Lane M con- tains size markers ofALDNA-HindIII fragments. b. I. Double digestion of YIp5 and pHMR22, respectively, with HindIII/BamHI (lane B and C) and SalI/BamHI (lane D and E). II. Double digestion of pHMR22 with HindIII/SalI (lane G). Lanes A and F contain size markers of A DNA-HindIII fragments. 105 a.ABCDEFGHIJKLM b. FG III [III 106 function, we constructed a new plasmid (pHMR23) containing a 1.35 kb HindIII/BamHI fragment of pHMR22 and a 5.25 kb HindIII/BamHI fragment of YIp5 (Fig. 4A) and examined the autonomous replication of this plasmid in g. cerevisiae YNN27. If the BamHI site is not located in the essential portion of the ars, the 1.35 kb HindIII/BamHI fragment of pHMR22 should confer on YIp5 the ability to replicate autonomously in §. cerevisiae. pHMR22 and YIp5 were cleaved with HindIII/BamHI and the resulting fragments were separated by agarose gel electrophoresis. The small DNA fragment uhas kb) of pHMR22 and the large DNA fragment (5.25 kb) of YIp5 were isolated from the agarose gel by electrophoretic elution and then ligated with T4 DNA ligase. The pool of hybrid DNA was transformed into E. coli HBlOl and ampicillin-resistant and tetracycline- sensitive clones were selected. We isolated three plasmids containing the desired DNA fragment from pHMR22 and YIp5. All of these three plasmids were capable of transforming g. cerevisiae YNN27 to Ura+ with high efficiency similar to pHMR22 (data not shown). 'These results show that the BamHI site is not located in the essential portion of the Egg. The ars in pHMR22 was subcloned further to localize the chromosomal replicator (Fig. 4B). pHMR22 was digested with HindIII/SalI and separated on an agarose gel. The Egg fragments were isolated from agarose gel and subjected to Sau3A digestion. To prevent the self-ligation of Sau3A fragments, the pool of Sau3A fragments was treated with Fig. 4. 107 Subcloning analysis of ars DNA from Q. utilis. Plasmid pHMR22 is YIp5 plus a 1.05 Kb MboI restricted fragment of Q. utilis which includes 352. A new plasmid, pHMR23, was constructed using 1a35 kb HindIII/BamHI fragment of pHMR22 and aELZS kb HindIII/BamHI fragment of YIp5 (Fig. 4a). Subcloning of the Egg in pHMR22 after Sau3A digestion is shown in Fig. 4b. Restriction enzyme sites are as follows: EcoRI (+), HindIII (X), BamHI ( A), Pstl (T), SalI (O), PvuII (a), and Sau3A (0). Not all Sau3A sites are shown. 108 pHMR22 (6.6 Kb) . I (33 y 1) Hind Ill/BamHI 2) Fragment isolation 1 Hi 1) Hind III/BamHI 2) Fragment isolation ‘A pr—w T 4DNA ligase pHMR23 (6.6 Kb) 109 pHMR22 (6.6 Kb) 1) Hind III/SalI 2) Fragment isolation x-CCZZA-O BamHI 1) Sau3A 2) Alkaline phosphatase T4 DNA ligase llO calf alkaline phosphatase to remove the terminal 5'- phosphates from the DNA (6). After the ligation of the Sau3A fragments with BamHI-restricted YIp5 plasmid DNA, the pool of hybrid plasmids was transformed to E. coli HBlOl. About 500 of the tetracycline-sensitive and ampicillin- resistant clones of transformed g. 9911 were pooled together for total plasmid isolation. A pool of resulting hybrid plasmids (10 ng) was used to transform g. cerevisiae YNN27. No Ura+ transformant harboring plasmid was isolated. These results indicated that the Sau3A site is located in the essential portion of cloned ars. Yeast sequences homologous to the cloned ars,of4pHMR22. The results of hybridization of the 32P-labeled ars fragment with total yeast DNA under low stringency condi- tions are shown in Fig. 5. We also conducted an experiment under high stringency hybridization conditions and found that the hybridization spectrum was the same as that shown in Fig. 5 (data not shown). We then asked the question whether DNA complementary to the ars was present elsewhere tannophilus were isolated and subjected to restriction enzyme digestion. After separation of DNAs by agarose gel electrophoresis, the DNAs were transferred to nitrocellulose paper and hybridized with 32P-labeled HindIII/SalI ars DNA or 32P-labeled pBR322 DNA. As shown iniFig. 5a, pBR322 did not hybridize to yeast DNA even at Fig. 5. 111 Probing of total yeast DNA with 32P- labeled pBR322 DNA (a) and SalI/HindIII 3_r__s_ fragment of pHMR22 DNA (b). About 5 ug of each DNA preparation was digested with restriction enzymes and electropho— resed in 0.7% agarose gel. 9. utilis DNA was digested with EcoRI (lane A). BamHI (lane B), HindIII (lane C), EcoRI/BamHI (lane D), HindIII/BamHI (lane E) and EcoRI/BamHI/HindIII (lane F). g. cerevisiae (lane G), C. albicans (lane H),and g. tannophilus (lane I) DNAs were restricted with EcoRI. Lane J was blank and lane K contained 1 ug of BamHI restricted pHMR22. Blot hybridization was carried out under low stringent conditions as described in Materials and Methods. 112 v. H. ..IOu..moom< .... . v. ... :cumaom.‘ 113 very low stringency hybridization conditions. This result precluded the possibility of the pBR322 sequences which flank the ars cross-hybridizing with yeast chromosomal DNAs. Tracks G, H and I of Fig. 5B are EcoRI cleaved DNAs of _S_. cerevisiae, _C_2. albicans, and g. tannophilus; none of these showed homology to the cloned ars of Q. utilis even under conditions that favor the formation of mismatched hybrid. Tracks A to F are the total DNA of Q. utilis cleaved with different restriction enzymes. Surprisingly, there were two bands seen in lane C, i.e.‘when total DNA was cleaved with HindIII, and only one band was present in lanes A, B, D, E and F (Fig. 5B) where the DNA was treated with other restriction enzymes individually or in combina- tion. These results indicated that the cloned ars hybridized with two different fragments of g. utilis genome DNA (see Fig. 6 and Discussion below). Fig. 6. 114 Restriction maps of Q. utilis DNA flanking cloned ars DNA in pHMR22. (a) and NH show two different fragments of g. utilis DNA containing _a__r_§_. Restric- tion maps are deduced from the hybridization experiments (Fig. 5). The distance between restriction enzyme sites shown is relative. Restriction enzyme sites are as follows: EcoRI (+), HindIII (X), BamHI (A), and Sau3A an. Not all Sau3A sites are shown. 115 mum mum db So 23 116 DISCUSSION The results of this study show that several DNA fragments of g. utilis have been cloned into YIp5 and that these DNA fragments confer on YIp5 the ability to replicate autonomously in S. cerevisiae. All of these hybrid plasmids transformed g. cerevisiae (Table 2) and E. coli at high frequency and could be reisolated from transformants without any detectable change in plasmid structure or function. These results suggested that the cloned DNA fragments fit description of ars previously isolated from other eukaryo—tic cells (1,2,21,38,39). To the best of our knowledge this is the first report describing the isolation of an 2L5. from 9- 2211s- Ags capable of high-frequency transformations have been reported to occur once in 30-40 kb in the S. cerevisiae chromosome (2,8) and once in 15 kb in the Drosophida melanogaster chromosome (38). The frequency of one replication origin per 36 kb was independently confirmed by the average spacing of initiation site in _S_. cerevisiae as detected by electron microscopy in small molecules of yeast chromosomal DNA (27). Our results, suggesting the presence of one ars in 30 kb, are consistent with the demonstrated frequency'of‘args in §.icerevisiae genome, but the number of clones analyzed by us is too small to consider the data conclusive. The observations of high-frequency transformation, extrachromosomal copies of the ars-YIp5 plasmid, and coincident frequency between the 117 ars in S. EEilié and the replication origin in S. cerevisiae strongly suggest that the BEE sequences we isolated by high-frequency transformation include the origins of replication of S. BEiliE genome. This conclusion is supported by the recent finding of Celniker and Campbell (5) that Sggl and a£§2 in S. cerevisiae are specific origins of chromosomal replication. Initiation of DNA replication occurs at multiple internal sites in the chromosomes, and these sites may be activated at different times during the replication phase of the cell cycle (28). If the origins share DNA sequence homology, then the SEE should hybridize to several yeast chromosomal DNA fragments. We have shown that the cloned 2.1-1.5. from S. _ut__i__l__i__s_ only hybridizes to two fragments of S. Bfiilié genome even under low stringency hybridization conditions. This suggests that the cloned 2£§ is unique or shares limited homology with other replicator sequences. The nucleotide sequence responsible for the replication of several S. cerevisiae ars has been studied in detail (37,41,42). Extensive homologies have not been observed among 2£§1 (42), 3552, 2£§3 and 2 u DNA (17,42). A canonical shared sequence, TAAAPyAPyAAPu, is present in Sggl, 3553 and 2 u DNA but absent in EEEZ- The signifi4 cance of this canonical sequence is, therefore, unclear. This is in striking contrast to the comparison between ggi sequences of different bacterial species where remarkable sequence homologies are evident (44). Apparently, the DNA 118 sequences corresponding to §£§ function are very diverse. This conclusion is supported by the findings of this study that little homology exists between the present cloned EEE and other replicator(s) in S. EEilié genome. Sequence diversity may reflect many different classes of replicators in one yeast which are temporally and coordinately activated by respective signals during S phase. Although definitive information is not available at this time, results of several previous investigators suggested that, in S. gggeyigigg, all DNA initiation sites are not activated simultaneously'(28,29,30) and some of the EEE are reiterated in the genome (7,8). Non—yeast DNA can allow autonomous replication of a yeast integration plasmid in S, cerevisiae (22,38,43). Sequence homology between one of these DNAs (the EEE of Tetrahymena found in rDNA) and S. cerevisiae arsl, 飧2r and the replication origin of 2 u circle have been investigated (22). These results showed that no extensive homology exists among these DNA replicators. Extensive homology between the cloned Egg of S. utilis and sequences in the S. cerevisiae genome was not detected by hybridiza- tion experiments in our study (Fig. SB, Track G), even though the E£§ exhibits high-frequency transformation in the latter yeast. Lack of sequence homology between the cloned 飧 of S. Bfiilifi and the E£§ of S. cerevisiae was also indicated by their respective restriction enzyme maps. The essential region of the S. cerevisiae arsl DNA resides 119 at or near a PstI site (37,41,42), whereas cloned Egg of pHMR22 does not have this site (Fig. 4). The only similarity between 2£§1 and the cloned SEE described in this paper is that the functional DNA sequences in both these Eggs contain a Sau3A site. In contrast, 2£§2 (42) does not.havera Sau3A site in the region required for its function. However, neither E£§2 nor the E£§ cloned in pHMR22 have a PstI site. All of these data indicate that there is too little homology between DNA sequences corre- sponding to the 飧 of S. Efiillfi and S. cerevisiae to be detected by standard hybridization procedures. These results extend the recent findings of Kiss et al. (22) that S. cerevisiae "initiation proteins" can recognize sequences corresponding to heterozygous replication origins. The BamHI site almost always disappears after a MboI/BamHI ligation (25L. However, we did find a BamHI site in pHMR22 which was constructed by MboI/BamHI liga— tion. The restriction map of _C_. _u_t_i_l__i__s_ genome DNA in the area of the Egg (Fig. 6) based on hybridization experiments (Fig. 5) revealed that there is no BamHI site in the E£§ before cloning. 'Thus, we believe that the BamHI site in the cloned SEE was created from a MboI/BamHI ligation and should be at the junction of YIp5 and cloned DNA (Fig. 4). Availability of unique cloning site(s) is one of the requirements for an ideal cloning vector. In addition, the smaller the cloning vector the greater is the amount of foreign DNA that can be cloned into it, at least on 120 theoretical grounds. The constructed plasmid, pHMR22, offers several advantages. It is small in size “L6 kb) and therefore a significant amount of DNA can be cloned into it. It has unique restriction sites for BamHI, SalI, HindIII, EcoRI and PvuII restriction endonucleases and these sites are not located in the region of the §£§ which is essential for its function. These features make pHMR22 a useful vector for cloning desired genes in S. cerevisiae and perhaps in S. utilis when a selectable marker for this yeast becomes available. We observed that the chromosomal DNA of 9. 251115 digested with HindIII gives two bands which hybridized with the cloned S£§_(Fig. 5B, track C) even though there is no HindIII site in the BEE (Fig. 6). Thisresult is consistent with the suggestion that the organism is diploid, and with HindIII site polymorphism on the two homologues bearing the EEE- However, if the BEE is randomly repeated in the genome, either tandemly with a site for HindIII between the two copies, or separately as a part of a larger homologous sequence, the same result might be obtained. In principle, probing with a number of separate clones might give evidence for a ploidy higher than one if all gave two or more bands. In conclusion, we have for the first time constructed a new cloning vector (pHMR22) containing an E£§ from S. utilis. The cloned 2£§ is fully functional in S. cerevisiae although there is no demonstrable homology 121 between the ars DNA and S. cerevisiae genome DNA. The fact that pHMR22 is relatively small in size “L6 kb) and that its §£§_has unique sites for several restriction enzymes frequently used in genetic cloning work makes it a useful vector for cloning the desired genes both in S. cerevisiae and S. BEilié and for the comparative study of 2£§ in yeasts. 122 ACKNOWLEDGMENTS This research was supported in part by a grant from the Michigan Agricultural Experiment Station. We acknowledge Drs. Jerry Dodgson and Mary Clancy for helping with the techniques, R. W. 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Origin of replication from Xenopus laevis mitochondrial DNA promotes high-frequency transformation of yeast. Proc. Natl. Acad. Sci. USA 1S:3128-3132. Zyskind, J. W., N. E. Harding, Y. Takeda, J. M. Cleary, and D. W. Smith. 1982. The DNA replication origin region of the Enterobacteriaceae. ICN-UCLA Symp. Mol. Cell. Biol. nglB-ZS. CHAPTER I I Deve10pment of a New Generalized Transformation System for Yeasts. Abstract We have described here for the first time a generalized lithium chloride transformation procedure and kggr-G418 selection system suitable for the transformation of a number of yeasts belonging to several different genera. This system is useful for the transformation of any G418-antibiotic sensitive yeast and consequently eliminates the need for the construction of stable mutants of known geno -type (e.gu, auxotrophs) to serve as the recipient strains. We constructed a broad host-range yeast cloning vector, pHR40, which carries both a 2 n DNA replication origin and an autonomous replication sequence (ars) from Saccharomyces cerevisiae, and a kanr determinant from Escherichia coli transposon Tn601 (Tn903), which codes for an aminoglycoside phosphotransferase that inactivates antibiotics G418 or kanamycin. pHR40 transforms a number of G418-sensitive yeast strains, belonging to several different genera, to G418 resistance with a high frequency. 127 128 INTRODUCT ION At the present time genetic cloning systems for yeasts are restricted only to Saccharomyces cerevisiael'2'3'4'5, S. carlsbergensis6 and Schizosacharomyces p9mbe7, because suitable cloning vectors and selection systems are not available for the other yeasts. Most genetic cloning systems in yeasts currently employ a cloning vector which carries a gene responsible for a specific step in the 8 and a biosynthesis of an amino acidl'2'5'7 or nucleotide corresponding auxotrophic recipient strain. However, stable auxotr0phs with the desired genotype are generally not available for yeasts other than Saccharomyces and Schizosaccharomyces. An antibioticsensitive yeast strain could be used as a recipient strain in place of nutritional auxotrophs, provided the yeast cloning vector carries an dominant genetic determinant for an enzyme which inactivates that particular antibiotic. A number of antibiotic resistance determinants of bacterial origin have been known to be expressed in yeast, but most yeast cells are relatively resistant to these 9,10. antibiotics On exception is G418, an aminoglycoside antibiotic, which is reported to inhibit the growth of Saccharomycesll. Jimenez and Davies recently described a transformation system for S. cerevisiae in which a cloning vector carrying kanr determinant from Escherichia coli transposon Tn601 and a recipient yeast strain sensitive to G418 were employedlz. The yeast transformants obtained 129 were resistant to high levels of G418. Resistance to G418 in these transformants was shown to be due to the presence of an aminoglycoside phosphotransferase encoded by the 533‘ determinant. These results suggested the possibility that the kggr/G418 combination might serve as a generalized selection system for yeasts other than S. cerevisiae, especially since this selection should work with any G418- sensitive yeast strain as the recipient, there being no need to construct an appropriate genotype. High frequency transformation in yeast has been demonstrated with cloning vectors containing 2 p DNA2'4'6 or certain yeast chromosomal sequences called autonomous replication sequences (§£§)3'5. Both types of DNA were shown to contain a yeast replication origin13. Since ££§ have been shown to have a relatively broad host 8,14,15' specificity we felt that a plasmid containing ars, 2 u DNA sequences and the kanr determinant from Tn601 might serve as a generalized cloning vector for transforming G418-sensitive yeasts. The results presented here show that this is indeed the case. The cloning vector constructed, pHR40, has a broad host-range and transforms a wide variety of different G418-sensitive yeast strains to G418 resistance at a high frequency. Construction of a broad host-range cloning vector contain- ing kanr,and 2 p DNA Plasmid YRp12 carries.aIL45 kb EcoRI fragment from the S. cerevisiae chromosome which contains the £521 and 130 the EEElG- The portion of §£§1 which is essential for replica—tion is located within a PstI/EcoRI fragment close to the tetracycline resistance determinant of YRp1217'18. Thus, to construct a yeast-S. ggli "shuttle vector" carrying the 2 p DNA sequence, Sggl, 532‘, and the replica- tion origin of S. coli, plasmids YRp12 and pTYl4-kgn5 (a plasmid carrying the 532‘ determinant from Tn601 and 2 )1 DNA sequences; a gift from Dr..Julius Marmur, Department of Biochemistry, Albert Einstein University, New York) were digested with PstI and the resulting fragments were ligated with T4 DNA ligase (Fig. 1). Total hybrid DNA was used to transform S. 9911 HBlOllg. The transformants resistant to G418 and tetracycline and sensitive to chloramphenicol and ampicillin were selected. A 13.5 kb hybrid plasmid, pHR40, isolated from one of the transformants, has a unique site for the restriction endonuclease BamHI, and transformed S. 9911 HBlOl to tetrancycline resistance or kanamycin resistance with equal efficiency (105 transformants per ug DNA) . Generalized transformation procedure foryyeasts Eleven yeast strains belonging to several different genera were tested for their sensitivity to G418 and were shown to be highly sensitive to this antibiotic at a con- centration of 100 ug/ml (Table 1). This concentration is considerably lower than that used previously for S. cerevisiaelz. This result indicated that the kggr-G418 Fig. l. 131 Construction of the broad host-range yeast cloning vector, pHR40. Plasmids YRp12 and pTYl4-SSSS were digested with PstI and then ligated with T4 DNA ligase. The pool of hybrid plasmids was used to transform S. 9911 HBlOl. Transformants which were resistant to tetracycline and kanamycin and sensitive to ampicillin and chloramphenicol were selected. The structure of the resulting plasmid, pHR40, was deduced by restriction endonuclease digestion followed by agarose gel electrophoresis (unpublished data). Symbols:-w-~h, yeast 2 )1 DNA; —|_ , EcoRI;—u-—, HindIII;—A— , BamHI; , PstI; + , SalI;-D—, PvuII. Not I HindIII and SalI sites in pHR40 are shown. 132 133 Table 1. Sensitivity of various yeast strains to antibiotic G418a Concentration of G418 Recamiendedb (1g/ml) G418 concentration Yeast strains 0 25 50 100 150 200 300 (pg/ml) Candida utilis NRRL Y-900 + — - — - - - 60 S. utilis NRRL Y-1084 + + - — - - - 60 S. lusitaniae NRRL Y-5395 + + + + + - - 200 Torulopsis molischiana NRRL Y-2237 + + - - — -- - 60 _T. wickerhamii NRRL Y—2564 + + + - — — - 100 Schwannianyces castellii NRRL Y—2477 + + + - - - - 100 Kluyveranyces Eicerisporus NRRL Y—8277 + + + — - - - 100 Pachysolen tannophilus NRRL Y-2460 + + - - - - - 60 Wridim toruloides A'ICC26194 + + - - — - - 60 Schizosaccharomyces E1113 NRRL Y-164 + + - - — - - 60 S. cerevisiae YNN27 + + + + + + — 350 134 aCell were grown at 30°C unti% stationary phase ( 5 x 108 cells ml' ) in YPD medium 0 and were harvested by centrifugation. One ml of cell suspension was mixed with 20 m1 of melted YPD agar (47-50°C), containing various concen- trations of G418 as indicated and poured immediately into a sterile Petri dish. The plates were scored for the appearance of G418-resistant colonies after 3 d incubation gt 30°C. +, growth; -, no growth. Desired concentration of antibiotic G418 in the medium for obtaining optimal results in the transformation experiment (Table 2). Table 2. 135 determinanta. Transformation of various G418-sensitive yeast strains to G418 resistance with chimeric plasmids harboring kanr No. of G418-resistant coloniesb Yeast strains pTYl4- pHR40 -kan5 no DNA 9. utilis NRRL Y-900 5000 (1667) 375 (13) 0 S. utilis NRRL Y-1084 300 (100) 22 (8) 0 S. lusitaniae NRRL Y-5394 89 (30) 14 (5) 30 S. molischiana NRRL Y-2237 3000 (1000) 806 (269) 7 S. wickerhamii NRRL Y-2564 1300 (433) 225 (75) 0 O S. castellii NRRL Y-2477 1300 (433) 300 (100) 20 S. cicerisporus NRRL Y-8277 4800 (1600) 94 (31) 8 S. tannophilus NRRL Y-2460 800 (267) 7 (3) 0 S. Toruloides ATCC26194 550 (183) 300 (100) 46 S. pombe NRRL-164 3000 (1000) 600 (200) 0 S. cergvisiae YNN27 3600 (1200) 2531 (844) 60 aCell to be transformed werg grown in YPD medium to late exponential phase (1-2 x 10 cells per ml), collected by centrifugation, washed with TE buffer (10 mM Tris plus 1mM EDTA, pH 7AM and resuspended in TEL buffer (TE plus 1 M LiCl). After incubation at 30°C for 1 hr, the cells were centrifuged and resgspended in the appropriate volume of TEL to give 10 cells per m1. Plasmid DNA (3mg) was added to 200u1 of the cell suspension and after 136 incubating the mixture at 30°C for 30 Hum“ 1.5 ml of 40% polyethylene glycol 4000 (BDH Chemicals Ltd., Poole, England) was added and the mixture reincubated for 1 hr at 30°C. 'The cells in the incubation mixture were then heat shocked at 42°C for 5 min, washed twice with sterile distilled water, and reincubated in 200 pl YPD at 30°C for 2 hr. Cells were plated directly on the surface of YPD plate with or without G418. The concentrations of G418 used for each organism is shown in Table l. G418- resistant transformants on the plates were counted after -4 d of incubation. The number of transformants given was for 3 ug of DNA of the respective plasmid. Data shown in parentheses represent transformation efficiencies (transformants per mg plasmid DNA). 137 selection system might be useful for the transformation of yeasts other than S. cerevisiae. Plasmids pHR40 and pTYl4- 5225 were used to transform yeast cells to G418 resistance. Since the glusulase method20 gave either a low transforma- tion efficiency or no transformation at all for most of the yeast strains (S. Egggyigigg was the exception), we evaluated the suitability of the lithium chloride procedure (K. Murata, personal communication) for yeast transforma- tion. Our results showed that the use oftLJ.M LiCl in the procedure recommended by Murata works only for S. cerevisiae and S. 29393 but not for the transformation of other yeast strains tested (data not shown). Hence, we used 1.0 M LiCl in all the transformation experiments (Table 2). All yeasts, except 9929122 lEEiEEEiEEI transformed with pHR40 gave a much higher number of G418- resistant transformants than that either obtained with pTYl4-_lg_a__n$ DNA or the number of spontaneous mutants observed in the "no DNA" control experiments. These results indicated that the SSSr/G4l8 selection system coupled with the LiCl transformation procedure is useful for the transformation of a wide-range of G418-sensitive yeasts. The higher frequency of transformation obtained with pHR40, which contains both S£§_and 2n DNA, as compared to pTY14-SSSS, which contains only 2p DNA, presumably reflects the relatively narrow host-range for Zn DNA whcih has so 138 far been found only in SaccharomyggS and 2122,23,24. Schizosaccharomyces The lower transformation efficiency observed with pTYl4-SSSS perhaps is also an indication of the instability of this plasmid in organisms phylogenetically distant from Saccharomyces. In contrast to 2p DNA, SSSS have been shown to have a broader host specificity. For example, non-yeast DNA containing the Eggs from a wide variety of eukaryotes as well as the SSS from S. Bfiillé were shown to confer on the yeast integra- tion plasmid (YIp5) the ability to replicate autonomously in S. cerevisiae8'25. The broad host specificity of the 2£§ is further supported by the finding that pHR40 containing the arsl of S. cerevisiae is able to transform a wide variety of yeast strains with high efficiency. Demonstration of pHR40 in yeast transformants Total DNA was extracted from untransformed S. utilis, Torulopsis molischiana and Pachysolen tannophilus and the same yeasts transformed with pHR40. All the DNAs were fractionated individually by agarose gel electrophoresis. blotted to nitrocellulose paper and hybridized with 32p— labeled pBR322 probelg. The results (Fig. 2) show that the labeled pBR322 does not hybridize with untransformed yeast DNA (lane G, H, and I), but hybridized only with the DNA isolated from transformed yeasts (lanes A to F). Since the pHR40 contains part of the pBR322 sequence, these results indicate that the resistance of the transformed yeast to G418 is due to the continued presence of the transforming 139 Fig. 2. Hybridization of total yeast DNA to 32P-labeled pBR322 DNA. Untransformed yeast strains were grown in YPD medium and G418-resistant trans- formant strains were grown in YPD medium containing G418 at a concentration recommended for each organism (see Table 1). DNA was extracted from yeast as described by Beach and Nurse26. Total DNA preparations (10 ug each) from two transformants each of C. inulis (lanes A and B). _T_. molischiana (lanes C and D), and S. tannophilus (lane E and F), and from untransformed (2. 231115 (lane (H, T. molischiana (lane _H) and S. tannophilus (lane 1) were fractionated by electrophoresis on 0. 7% agarose gels, stained with ethidium bromide, photographed and blotted onto nitrocellulose BA85 (Schleichelr 9andfchuell) as described by Maniatis et a1.1 2P dATP was incorporated into pBR322 DNA by nick translation with S. coli polymerase l. Blotted filter paper was prehybridized for a minimum of 4 hr at 42°C with hybridization buffer containing 50% (vol/vol) formamide, l M NaCl, 10 mM Tris.HCI at pH 8.0, 1 mM EDTA, 0.05 M Na4P207 at pH 6.8, 5X Denhardt' 5 solution, and 10 pg of poly(rA), and9 8 uazof heat-denatured salmon sperm DNA per P-labeled pBR322 probe was then added to the hybridization solution and hybridized for 40 hr at 42° C. The hybridized filters were washed twice at ambient temperature for 5 min in a wash buffer (10 mM Tris. HCl, pH 7. 5/1 mM EDTA/O. 1% sodium dodecyl sulfate/0. 1% Na4 P2 0/ 50 mM.NaC1) followed bye four 15 min washes wiZh the same buffer at 65 C. Autoradiography was performed at -70° with Kodak XAR film and intensifying screen 140 ABCDEFGHI 141 plasmid pHR40. To determine whether the plasmid pHR40 can be reisolated from yeast transformants, we transformed S. 99;; HBlOl with the total DNA isolated from S. utilis, _T_. molischiana and S. tannophilus transformants. Transformed cells were plated on L agar, L agar containing tetracycline (10 pg ml’l) and L agar containing kanamycin (50 pg ml') 1'19. No antibiotic-resistant transformants were obtained. There are at least two possible explanations for our failure to isolate plasmid pHR40 from G418-resistant transformants. First, the copy number of the transforming plasmid in host yeasts may be too low to be isolated. The average c0py number of pHR40 in host cells may have decreased dramatically due to spontaneously occurring G418- resistant mutants among the transformants. 11 second possibility is that a recombination has occurred between the transforming plasmid and the yeast genome and as a result the recombinant plasmid lost its ability to transform S. 9911. These possibillities are currently being investigated. In conclusion, we have described here for the first time algeneralized transformation system which utilizes Saar-G418 selection and the LiCl transformation procedure. This system is useful for the transformation of any G418- sensitive yeast and consequently eliminates the need for the construction of stable mutants of known genotype (84L, auxotrophs) to serve as the recipient strains. The data 142 indicate that pHR40 transforms a number of different yeast genera. However, in spite of its broad host-range, pHR40 does not appear to be the most desirable vector for gene transfer because it is unstable in the host yeast. We do not know whether deletion of some part of the pHR40 (e.g., 2 u DNA) could be carried out without affecting the transformation efficiency. If this can be done, the determination of stability and copy number of the resulting plasmid would be of considerable interest. 143 Acknowledgements We acknowledge PhT. Magee for his invaluable counsel, R.W. Davis for supplying YRp12, J. Marmur for supplying pTYl4-Sgg5, the Northern Regional Research Laboratory, Peoria, Illinois, U.S.A. for supplying the yeast cultures, and Schering Corporation (60 Orange St., Bloomfield, N.J.) for donating the antibiotic G-418 sulfate. 10. 11. 12. 13. 14. 15. 144 REFERENCES Hinnen, A., Hicks, J.B. & Fink, G.R. Proc. Natl. Acad. Scil. USA 75, 1929-1933 (1978). Beggs, J.D. Beggs, J.D. Nature 275, 104-108 (1978). Beach, D., Piper, M. & Shall, S. Nature 284, 185-187 (1980). Broach, J.R., Stralhern, J.N. & Hicks, J.B. Gene 8, 121-133 (1979). Hsiao, C. & Carbon, J. Proc. Natl. Acad. Sci. USA 76, 3829-3833 (1979). Broach, J.R. & Hicks, J.B. Cell 21, 501-508 (1980). Beach, D. & Nurse, P. Nature 290, 140-142 (1981). Stinchcomb, D.T., Thomas, M., Kelly, J., Selker, E., 8 Davis, R. W. Proc. Natl. Acad. Sci. USA 77, 4559-4563 (1980). Cohen, J.D., Eccleshall, T.R., Needleman, R.B., Federeff, H., Buchferer, B.A. & Marmur, J. Proc. Natl. Acad. Sci. USA 77, 1078-1082 (1980). Hollenberg, C.P., Kustermann-Kuhn, B., Mackedonski, V. & Erhart, E. Alfred Bengon Symp. 16, 109-120 (1980). Daniels, P.J.L., Yehaskel, A.S. & Morton, J.B. 13th Interscience Conf. Anti-MicrobialtAg. Chemother., Washington, Abstr. 137 (1973). Jimenez, A. & Davies, J. Nature 287, 869-871 (1980). Celniker, S.E. & Campbell, J.L. 931“]; 31, 201-213 (1982). Kiss, G.B., Amin, A.A. & Pearlman, R.B. ICN-UCLA Symp. Molec. Cell Biol. 22, 607-614 (1982). Zakian, V.A. Proc. Natl. Acad. Sci. USA 78, 3128-3132 (1981). 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 145 Struhl, K., Stinchcomb, D.T., Scherer, S. & Davis, R.W. Proc. Natl. Acad. Sci. USA 76, 1035-1039 (1979). Tschumper, G. & Carbon, J. Gene 10, 157-166 (1980). Stinchcomb, D.T., Stuhl, K. & Davis, R.W. Nature 282, 39-43 (1979). Maniatis, T., Fristsch, E.F. & Sambrook, J. Molecular Cloning: A Laboratory_Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Sherman, F., Fink, G.R. & Hicks, J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1981). Grerineau, M., Grandchamp, C., Paoletti, C. 8: Slonimski, P. Biochem. Biophys. Res. Commun. 42, 550-557 (1971). Toh-E. A., Tada, S. & Oshima, Y. J. Bacteriol. 151, 1380-1390 (1982). Gunge, N., Tasuko, A., Ozawa, F. & Sakaguchi, F. J. Bacteriol. 145, 382-390 (1981). Giudice, L.D., Wolf, K., Sassone-Corsi, P. & Mazza, A. Mol. gen. Genet. 172, 165-169 (1979). Hsu, W.H., Magee, P.T., Magee, B.B. & Reddy, C.A. E; Bacteriol. (Submitted, 1983). Beach, D. & Nurse, P. Nature 290, 140-142 (1981). Laskey, R.A. & Mills, D. FEBS Lett. 82, 314-316 (1977). CHAPTER I I I Construction of New Cloning Vectors for Gene Transfer in Saccharomyces cerevisiae Abstract We have constructed several new, relatively small, yeast-Escherichia coli shuttle vectors which carry the 2 u DNA replication origin, an autonomous replication sequence (E£§)r and a centromere gene (gggB), either singly or in combination, from Saccharogyces cerevisiae» Each of the vectors additionally carries a kanamycin determinant from the S. 99;; transposon Tn601 (Tn903) which codes for an aminoglycoside phospotransferase that inactivates the antibiotics G418 and kanamycin. All the newly constructed vectors contain a: unique restriction site for BamHI, transform G418-sensitive yeast strains to G418 resistance with a high frequency and replicate autonomously in the host yeast. The transformation efficiency was 103-104 transformants per ug DNA. It appears that the smaller the molecular size of the vector the greater is its mitotic stability in S. cerevisiae. The presence of a centromere element (9323) appears to further enhance the mitotic stability of the vector (pHR2). 146 147 INTRODUCTION The recent advances in recombinant DNA methodology have led to a rapid increase in investigations on yeast genetics and have made possible the detailed analysis and manipulation of a number of genes in Sgggggggmyggg cerevisiae at the molecular level (l,3,4,9,12,15). These cloning techniques have been used to isolate and amplify the desired DNA sequences for basic genetic studies (12,21,24) and for cloning industrially important genes (11). However, most of the yeast cloning systems are presently restricted to specific yeast genotypes (mutants) which have an appropriate genetic marker for yeast transformation (10,12). The lack of stable mutants of known genotype for many industrially important yeasts has limited the application of recombinant DNA methodology, in improving the yield and productivity of various fermenta- tion processes or in constructing new yeast strains capable of producing new products. Therefore, the development of cloning systems suitable for wild type yeast would be very useful. Jimenez and Davis (18) recently developed a new cloning system for S. cerevisiae in which a cloning vector carrying a kggr determinant from Escherichia 991; transposon Tn601 and a G418-sensitive recipient yeast strain were employed. The 1532‘ determinant codes for an aminoglycoside phosphotransferase that inactivates the aminoglycoside antibiotics G418 and kanamycin. Since 148 selection based on drug resistance would eliminate the requirement for the construction of a stable yeast mutant of a specific genotype to serve as the recipient, it appeared that this type of selection system may be valuable for the genetic manipulation of wild type strains of S. cerevisiae and perhaps other industrially important yeasts. Hollenberg (12) constructed a plasmid, pMp81, which carries Tn601 and has been used for the direct transformation of G418-sensitive S. cerevisiae to G418 resistance. However, the usefulness of this plasmid as a vector for cloning yeast genes has not been demonstrated. A plasmid pTY14 r which carries a kan determinant form the transposon Tn601 has recently been constructed by J. Marmur (unpublished r determinant to data). However, plasmids carrying the £22 date are relatively large in size ( 12 kb) and, therefore, would permit cloning of only a relatively small amount of foreign DNA. Additionally, these types of plasmids were reported to be mitotically unstable (12,18). In this paper we describe the construction of several new, relatively small and stable yeast cloning vectors for wild type S. cerevisiae which carry 532‘ from Tn601 and stabilizing elements (segments of DNA capable of conferring mitotic stability on the autonomously replicating vectors in yeast) 2 p DNA, an autonomous replication sequence (SSS) and a centromere gene (9333), either singly or in combina- tion. 149 MATERIALS AND METHODS Strains,4p1asmids and media. The yeast and bacterial strains and plasmids used in this study are listed in Table 1. The yeast medium (YPD) was described by Sherman et a1. (22). The LB medium used for growing S. 99;; was described by Maniatis et a1.(20). DNA preparation. Plasmid DNA was prepared from S. gel; as previously described (14). Total yeast DNA from both transformed and untransformed cells was isolated as described earlier (17). DNA fragments to be labeled by nick translation and recloned into other vectors were isolated from agarose gels by electroelution as described by Maniatis et a1.(20). Transformation of yeast and bacteria. Yeast strains and S. coli were transformed as previously described (22). Yeast transformants were selected on plates of YPD containing 350 ug of antibiotic G418 per ml. Southern transfer and hybridization to nitrocellulose paper. The procedures used for Southern hybridization were described previously (17). Determination of mitotic stabilities of the hybrid plasmids in yeast. Cells from a single transformant colony were grown in YPD medium for 20 generations at 30°C. Cell density was measured by the absorbance at 600 nm and the cultures were diluted in sterile distilled water to yield 100-500 colonies when suitable aliquots were spread on YPD agar plates. After overnight incubation at 30°C, 96 150 randomly picked colonies were tested for the presence of the transforming plasmid using the yeast colony hybridiza- tion technique (22). Yeast DNA immobilized on nitrocellulose paper was hybridized with 32P-labeled pBR322 DNA by the method described previously (17). 151 Table l. Microbial strains and plasmids used and their sources Host Plasmid References Strains Plasmids markers or source g. 99;; HBlOl pTY14-SSSS 533‘, 335‘, 933‘ Marmura g. 99;; JA221 pYE(ggSB)4l $332+ 5 g. 99;; 33101 YRp12 5£21+, g£§3+ 21,23 S. coli HBlOl pBR322 535‘, Sgpr 2 S. cerevisiae 23 YNN27 aMarmur, J., Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY. 152 RESULTS Construction of cloning vectors. Both plasmid pTYl4- SSQS and YRp12 (21,23) are S. cerevisiae-S. pgli shuttle vectors. pTY14-SSSS contains the S. _cp_l__i_ plasmid pBR325, the 533‘ determinant from transposon Tn601, and yeast 2 )1 DNA (Fig. 1). YRp12 carries functional 3533+, _t__r;p1+ and _a_r__s_l sequences from S. cerevisiae and the S. 55ng plasmid pBR322. To construct a new yeast-S. 53ng shuttle vector containing §£§1r 2 n DNA, the S. 99;; replication origin and a kanr determinant, plasmids pTYl4-SSSS and YRp12 were digested with restriction endonuclease PstI and then ligated with T4 DNA ligase (Fig. 1). The pool of hybrid DNA was then used to transform S. 53ng HBlOl and transformants resistant to tetracycline and kanamycin and sensitive to chloramphenicol and ampicillin were selected. One of the plasmids, pHR40 (Fig. 1), containing DNA fragments from both pTYl4-SSSS and YRp12 was isolated and its restriction enzyme map was determined. pHR40 (13.5 kb) was subsequently reduced in size by deleting a 4.1 kb EcoRI fragment which does not contain DNA sequences essential for the selection and replication of this plasmid either in yeast or in S. gel}. The resulting smaller plasmid, pHR7l (9L4ldn, carries all the useful determinants originally present in pHR40 (Fig. 1). It had been reported earlier that the addition of a centromere gene from S. cerevisiae effectively stabilizes the ars plasmid through both mitosis and meiosis in yeast 153 Fig. 1. Construction of the yeast cloning vectors pHR40 and pHR7l. Symbols:«-qu yeast 2 u plasmid DNA;——+—-, EcoRI;-Hh— , HindIII; —A- , BamHI; a , PstI; + , Sa1I; —E]— 1‘ pvuII. 154 155 (5,8,16L. Therefore, we constructed a new yeast cloning vector (pHR2; Fig. 2) which contains 2£§1I 9333 and the S. ggSS_replication.origin from plasmid pYE(gng)4l fin, and 533‘ and 2 p DNA from plasmid pTYl4-Sgp5. Plasmid pYE(ggp3)4l and pTYl4-SSSS were digested with EcoRI and then ligated with T4 DNA ligase. The hybrid DNA was used to transform S. 53ng HBlOl and transformants resistant to kanamycin and sensitive to tetracycline were selected. One of the several plasmids isolated from S. 99;; transformants was designated pHR2. It contained the desired sequences from pYE(gng)4l and pTYl4-SSSS. To confirm that pHR2 carries a 9333 element from pYE(gng)4l, both these plasmids were cleaved with EcoRI and PstI individually and the resulting fragments were separated by agarose gel electrophoresis. Two DNA sequences, a 7.1 kb EcoRI fragment (Fig. 3, lanes D and F) and a 5.0 kb PstI fragment (Fig. 3, lanes G and H), both of which contain the gng element were generated from the restriction enzyme diges- tion of either pYE(ggp3)4l or pHR2. These results clearly indicated the pHR2 carries 2323 element from pYE(ggp3)4l. Plasmid pHR31 (9.8 kb) was constructed by deleting a 4.1 kb EcoRI fragment of 2 11 DNA from pTYl4-Sgp5 (Fig. 2). The orientation of the EcoRI fragment carrying the tetracycline determinant was reversed in the plasmid pHR31, resulting in inactivation of the chloramphenicol determinant. All of the newly constructed plasmids, which contain one or more of the stabilizing elements 2 u DNA, 156 Fig. 2. Construction of the yeast cloning vectors pHR2 and pHR31. Symbols are the same as in Fig. l. 157 pTY14-k n5 (13.9Kb Fig. 3. 158 Restriction analysis of the pHR2 DNA containing cen3 gene of S. cerevisiae. pHR2 DNA digested with the indicated restriction enzymes was run on agarose gel parallel to similarly digested pYE(ggg3)4l and pTYl4-SSSS. Lanes A [pYE(ggg3)4l], lane B (pTYl4-SSSS), and lane C (pHR2), contained undigested DNAs; lane D [pYE(ggSB)41], lane E (pTYl4-SSSS), and lane F (pHR2), contained DNAs digested with EcoRI; lane G [pYE(ggg3)4l], and lane H (pHR2), contained DNAs digested with PstI; lane I (pHR2), contained DNA digested with BamHI. Lane J contained size markers of A DNA-HindIII fragments. 159 ABCDEFGHIJ 160 gggl and £323, transform G418-sensitive S. cerevisiae (strain YNN27) to G418-resistance with high efficiency (103-104 transformants per pg DNA). Mitotic stability of new cloning vectors in S, cerevisiae. The data on mitotic stability of the newly constructed plasmids and that for pTYl4-SSSS are presented in Table 2. The results suggest that mitotic stability is influenced by the size of the transforming plasmids. Plasmid pTY14-SSSS (13.9 kb) which carries a: 2 u stabilizing element is relatively unstable in that 34% of the host cells retain this plasmid after growth on a nonselective medium for 20 generations. Presence of both aggl and 2 u DNA in the same plasmid, as in pHR 40 (13.5 kb), resulted in no detectable increase in mitotic stability in comparison with that of pTYl4-Sggs. However, plasmids pHR31 and pHR7l, which were obtained, respectively, by the deletion of a 4.1 kb EcoRI fragment from pTY4l-SSQS and pHR40 (Fig. l and 2), displayed greater mitotic stability than the parent plasmids, with about 50% of the cells harboring the transforming plasmid after 20 generations (Table 2). Although plasmid pHR2 (11.5 kb), which carries a gggB gene besides aggl and 2 u DNA elements, is larger than pHR71 (9.4 kb) and pHR31 (9.8 kb), its mitotic stability is comparable to these two plasmids and considerably higher than of pHR40 and pTYl4-Sgg5. This result suggested that 2323 gene does confer a certain degree of mitotic stability on the plasmid carrying it. 161 Table 2. Mitotic stability of different cloning vectors in S. cerevisiae Mitotic Size Stabilizing stabilityb Vectors (kb) elements (%) pTY14-kan5 13.9 34 pHR31 9.8 48 pHR40 13.5 arsl, 31 pHR7l 9.4 arsl, 52 pHR2 11.5 cen3, arsl, 51 aMitotic stability was determined by the yeast colony hybridization technique (22) as described in Materials and Methods. b Stability is expressed as the precentage of cells containing the transforming plasmid after 20 generations in a non-selective medium. 162 Autonomous replication of the new cloning vectors ingyeast. Several lines of evidence indicate that all the new cloning vectors described here replicate autonomously without integrating into yeast genome. High frequency transforma- tion of S. cerevisiae YNN27 to G418-resistance with the cloning vectors indicates autonomous replication because integration of the plasmids into the yeast chromosome is known to result in a low frequency transformation (9). Furthermore, the pHR plasmids were easily recoverable from S. 9911 transformed with total DNA isolated from the yeast transformants. Yeast integration plasmids, such as YIp5 (23), are not expected to behave in this way (9). The restriction map of pHR plasmids recovered from S. 99;; revealed that these plasmids are structurally intact after yeast transformation. Southern blot hybridization was then employed to demonstrate the presence of autonomously replicating pHR plasmids in the G418-resistant yeast transformants. Total yeast DNA was prepared from the transformed and untransformed S. cerevisiae YNN27 and each sample was completely digested with BamHI. All the pHR plasmids have a single restriction site for BamHI. The digested DNA samples were fractionated on an agarose gel, blotted onto nitrocellulose paper and probed with 32P- labeled pBR322 (Fig. 4). DNA from untransformed YNN27 (control) did not show hybridization with the pBR322 DNA (laneln. However, DNA from the transformants contained bands homologous to pBR322 with electrophoretic mobility Fig. 4. 163 Hybridization of total yeast DNA to 32P- labeled pBR322 DNA. Total DNA preparation (10 ug each) from untransformed yeast (lane A) and yeast transformed with pHR40 (lane B and F), pHR7l (lane C and G), pHR31 (lane D and H), and pHR2 (lane E and I) were fractionated by electrophoresis on an agarose gel, blotted onto nitrocellulose and hybridized with 32P-1abeled pBR322 DNA under high stringency conditions. Lanes B, C, D and E represent uncut DNA and lanes F, G, H and I represent DNA completely digested with BamHI. 164 BCWD'EHFGH A t” 'r r~ . . Si 9 ! A. ‘, --.... -..A'L._'_._._L-.___. 165 corresponding to the supercoiled, open circular and multimer forms of the transforming plasmids (lane B, C, D and EL. Restriction of DNA from the transformed yeast with BamHI showed a single band of hybridization with pBR322 DNA indicating that the plasmids are not integrated into the S. cerevisiae chromosomal DNA (lane F, G, H and I). 166 DISCUSSION The mitotic stability of a composite plasmid under nonselective conditions is reported to be an indication of the copy number of that plasmid (1,12), becauSe composite plasmids maintained at a high copy number per cell are segregated out less frequently that those that have a low copy number. The copy number of a composite plasmid, carrying SSSl or 2 )1 DNA as the stabilizing elements, was shown to increase by decreasing the size of this plasmid (12,27). Thus, the higher stability of pHR31 and pHR7l, as compared to pTYl4-SSSS and pHR40, may probably be attributed to their relatively small size (Table 2) and consequently higher copy number. Plasmids which contain 2£§ and 9323 as stabilizing elements were previously shown to be stably maintained in host yeast for many generations even in the absence of selection pressure (5,8,16). pHR2 which was shown to contain stabilizing elements 2 u, §£§1 and 9393, as evidenced by restriction enzyme digestion and agarose gel electrophoresis (Fig. 3), was relatively stable in host yeast, with 51% of the cells carrying this plasmid after 20 generations in a nonselective medium (Table 2). This stability value is somewhat lower than that previously reported (5,8) for BEE-£22 plasmids (GO-100% of stability). The reason for the lower mitotic stability of pHR2 is not 167 clear, since it has been shown previously that 9323 plays a dominant role in determining the mitotic stability of a cloning vector containing either EEE‘ESB3 or 2 u-ggp3 (16). Earlier studies (1,25) showed that certain DNA sequences on a plasmid may be responsible for the lower mitotic stability of that plasmid (6&L, pBR325 sequence on the plasmid pMP78). Thus, the relatively lower mitotic stability of pHR2 may also be due to certain DNA sequences (such as the 532‘ determinant) on this plasmid. Additional features like strain specificity of the host yeast (5,12) and structures of the chimeric plasmid have been reported ho be of importance in determining mitotic stability (1,12). It would be of interest to delete certain nonessential DNA sequences from pHR2 and look for the stability of the plasmid obtained in several different strains of G418-sensitive S. cerevisiae. Drug resistance determinants such as Saar, are capable of functioning in a wide variety of bacterial (7) and yeast species (6,12,13,18). The SSS element of eukaryotes was also reported to be widely distributed and have a broad host range. For example, the SSSS from several phylogenetically distant eukaryotes were known to confer on a yeast integration plasmid (YIp5) the ability to replicate autonomously in S. cerevisiae (19,23,26). (A question of considerable interest is whether plasmids pHR7l and pHR2. which contain arsl and kanr would serve as broad host-range cloning vectors for a wide variety of G418-sensitive yeasts 168 other then S. cerevisiae. Studies are being undertaken to test this possibility. The new cloning vectors, pHR31, pHR71 and pHR2 are relatively small in size, contain a unique BamHI site, transform G418-sensitive yeast strains with a high efficiency, replicate autonomously in the host yeast without losing any of the useful functional determinants and are relatively stably maintained. These properties make the pHR plasmids useful vectors for cloning genes in industrially important G418-sensitive S. ggpgyigigg strains. 10. 11. 169 LITERATURE CITED Beggs, JxD. 1981. Multiple-copy yeast plasmid vectors. Alfred Benzon Symp. SS:383-395. Bolivar, F., R.L. Rodriguez, P.J. Greene, M.C. Betlach, H.L. Heynecker, H.W. Boyer, J.H. Crosa, and S. Falkow. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:75-93. Broach, J.R., and J.B. Hicks. 1980. Replication and recombination functions associated with the yeast plasmid 2 u circle. Cell Sl:501-508. Broach, J.R., J.N. Strathern, and J.B. Hicks. 1979. Transformation in yeast: development of a hybrid cloning vector and isolation of the CANl gene. Gene S:121-133. Clarke, L., and J. Carbon. 1980. Isolation of a yeast centromere and construction of a functional small circular chromosome. Nature 287:504-509. Cohen, J.D., T.R. Eccleshall, R.B. Needleman, H. Federoff, B.A. Buchferer, and J. Marmur. 1980. Functional expression in yeast of the Escherichia coli plasmid gene coding for chloramphenicol acetyl transferase. Proc. Natl. Acad. Sci. USA 11:1078-1082. Ehrlich, S.D., B. Niaudet, and B. Michel. 1982. Use of plasmids from Staphylococcus aureus for cloning of DNA in Bacillus subtilis. Curr. Top. Microbiol. Immunol. SS:l9-29. Fitzgerald-Hayes, M., L. Clarke, and J. Carbon. 1982. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 22:235—244. Hinnen, A., J.B. Hicks, and G.R. Fink. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. USA 1S:1929-1933. Hinnen, A., and B. Meyhack. 1982. 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Characterization of a yeast replication origin (EEEZ) and construc- tion of stable minichromosome containing cloned yeast centromere DNA (SSS3). Gene SS:157-166. Hsu, W.H., P.T. Magee, B.B. Magee, and C.A. Reddy. 1983. Construction of a new yeast cloning vector containing autonomous replication sequences from Candida utilis. J. Bacteriol. (Submitted). Jimenez, A., and J. Davis. 1980. Expression of a transposable antibiotic resistance element in Saccharomyces cerevisiae: a potential selection for eukaryotic cloning vectors. Nature SS1:869-87l. Kiss, G.B., A.A. Amin, and R.B. Pearlman. 1982. Regions of Tetrahymena rDNA allowing autonomous replication of plasmids in yeast. ICN-UCLA Symp. Mol. Cell. Biol. SS:607-614. Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Scherer, S., and R.W. Davis. 1979. Replacement of chromosome segments with altered DNA sequence constructed Sp vitro. Proc. Natl. 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Construction, replication and chromatin structure of TRPI Rl circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol. Cell. Biol. S:221-232. APPENDIX APPENDIX Alkaline Hydrogen Peroxide Pretreatment of Wheat Straw for Enhancing Cellulase Hydrolysis and Ethanol Production 172 173 Summary The effectiveness of the HZOZ—NaOH pretreatment (0.5% NaOH plus 0.5% H202) in enhancing the susceptibility of wheat straw to cellulase digestion was investigated. H202- NaOH pretreatment of wheat straw for 8 hr at 30°C was very effective in giving saccharification equivalent to 86% of the theoretical maximum. Glucose yields were considerably lower when wheat straw was pretreated with 0.5% NaOH alone. There was no appreciable increase in the efficiency of pretreatment when HZOZ—NaOH was supplemented with 0.44 mM FeSO4. When wheat straw pretreated with NaOH alone or with HZOZ-NaOH was saccharified with Sgigggggpmg Sggggi cellulase, and the resulting hybrolysates were fermented anaerobically with Saccharomyces cerevisiae, ethanol production was appreciably higher from the HZOZ—NaOH treated wheat straw compared to that treated with NaOH alone. This result in turn indicates that more cellulose is converted to glucose when wheat straw is treated with HZOZ-NaOH than when treated with NaOH alone» In conclu- sion, HZOZ-NaOH pretreatment of wheat straw appears to be relatively faster, more effective and allows enhanced saccharification and greater ethanol yields than those obtained with NaOH treatment alone. 174 INTRODUCTION Lignocellulosic biomass is the most abundant renewable resource in the biosphere and is composed of approximately 40-50% cellulose, 20-30% hemicellulose and 15-30% ligninl. An estimated 2-3 billion tons of lignocellulosic residues (wet weight) such as wheat straw are produced annually in the United Statesz. Bioconversion of lignocellulosic materials to sugars for the production of fuels and chemical feed stocks have been receiving increasing atten- tion in recent years3. However, lignin, which is recalcitrant to biological degradation, exists in close physical and chemical association with cellulose and limits the efficiency and extent of its utilization in various bioconversion processes4. Chemically, lignin-carbohydrate bonds form metabiolic blocks that greatly limit the action of microbial cellulases and physically lignin forms a barrier suppressing the penetration by cellulases. To some extent, the degree of crystallinity of the cellulose also limits the extent of its hydrolysis by cellulasess'e. Therefore, to increase the yield of sugars from the enzymatic hydrolysis of lignocellulosic materials these substrates must be pretreated to depolymerize or remove lignin and to reduce the crystallinity of cellulose so that more cellulase would be accessible for cellulase action. Physical, chemical and biological pretreatment processes, either singly or in combination have been used to increase the efficiency of utilization of 175 lignocellulosic materials. These methods included ball milling7'8, wet millingg'lo, steam-explosionllllz, irradiation13, alkali swellingl4'15, partial acid hydrolysis16'17, ozonationle, solvent extractionlg'20 and biological delignification21. Many of these processes are, however, relatively expensive, energy intensive or inefficient. Hydrogen peroxide in alkaline medium is widely used as a bleaching agent in the pulping industry22'23. In the absence of a stabilizing agent such as sodium silicate, NaOH-H202 is known to result in extensive delignification and depolymerization of cellulose23. Most recently, Forney et al24 reported that hydroxyl radical, derived from H202 in a Fenton-type reaction, plays an integral role in lignin degradation by Phanerochaete chrysosporium (a white-rot fungus). Koenig (1975) also reported that hydrogen peroxide plus FeSO4 alters the structure of lignin and also depolymerizes cellulose25. These results indicate that H202 pretreatment may be effective in reducing the lignin content and crystallinity of the cellulose in lignocellulosic biomass. However, there has been little published information to date on utilizing NaOH-HZOZ for pretreating lignocellulosic materials to enhance the enzymatic hydrolysis of these substrates for obtaining greater yield of sugars or ethanol. In this paper we investigated the effectiveness of Hzoz-NaOH pretreatment on the enzymatic hydrolysis of wheat 176 straw, one of the major lignocellulosic residues generated in the ELSNA. and worldwide26. The efficiency of this process is compared with that of NaOH pretreatment process which is one of the more efficient and widely used pretreatment procedure526'27. Our results showed that pretreatment with HZOZ-NaOH will yield more glucose inzi shorter time and under milder conditions, for ethanol production as compared to the pretreatment with NaOH alone. 177 MATERIALS AND METHODS Substrate Wheat straw which was chopped to 2.5-5.0 cm length and Wiley-milled to about 40 mesh, prior to pretreatment process, was used as the substrate. The average composi- tion of wheat straw was 10% moisture, 37% cellulose, 65% total carbohydrate and 20% lignin. Sigma Cell, a microcrystalline cellulose was purchased from Sigma Chemical Co. (St. Louis, Missouri) and was used as a sub- strate ix: the control experiments for evaluating the efficiency of different preatreatment processes. Pretreatment and Enzymatic Hydrolysis Cellulase was prepared by the method of Mandels and Sternberg utilizing Trichoderma _r__e_gS_e_i QM 941428. Wheat straw was pretreated with HZOZ-NaOH which contained 0 to 1% NaOH and 0 to 0.5% H202 by weight in different experiments as noted in the results. In some experiments HZOZ-NaOH was supplemented with 0.44 mM FeSO4. The cellulosic substrate was suspended in the HZOZ-NaOH solution “L5 9/45 ml) at 30°C for 24 hr, unless otherwise indicated. Citrate buffer (2.5 ml of a l M solution; pH 4.8),HO. + 05. + H20 (2) H202 + M->M+ + H0. + H0“ (3) H202 + w“ + 20H'—>M + 02.- + 21120 . (4) 02': + H0. +02 + HO" (5) The hydroxyl radicals and/or other oxygen radicals produced from H202 decomposition under alkaline conditions may cause oxidative degradation and depolymerization of 22,23 41 lignin and cellulose . Previous investigators have found that high alkalinity accelerates the decomposition of hydrogen peroxide to give oxygen23. The partial pressure of oxygen derived from the 195 decomposition of H202 also increased with increasing H202 concentration in the reaction mixture. Moreover, high alkalinity and H202 favor the formation of carbanions, the substrates for the oxygenation reactionzz. Accordingly, the extent of delignification appears to increase with increasing alkalinity and H202 concentration and subsequently increase the accessibility of cellulose in wheat straw to cellulase action. Pretreatment with HZOZ-NaOH and FeSO4 significantly increases the weight loss of wheat straw after cellulase digestion. However, this resulted in a decrease in sugar yield but no change in the extent of delignification, as compared with HZOZ-NaOH pretreatment without FeSO4. It has been demonstrated that the presence of FeSO4 increases the rate of decomposition of hydrogen peroxide, and the forma- tion of both hydroxyl radicals and superoxide ions (equation 3 and 4) in HZOZ-NaOH reaction37'38'42. The results reflected that the addition of the FeSO4 does accelerate the reaction as shown by the increase in loss of dry weight. An increase in temperature also results in an enhanced rate of decomposition of H202 to produce various oxygen radicals23'43. Increased dry weight loss obtained on HZOZ-NaOH pretreatment at elevated temperature (50°C) is consistent with this explanation. However, both in the presence of added FeSO4 and at elevated temperature, H202- NaOH pretreatment followed by cellulase digestion resulted in decreased sugar yield, which may be due to the oxidative 196 breakdown of the carbohydrates by oxygen radicals44. As glucose is the common substrate desired for various industrial processes such as the fermentative production of alcohol, any system which can result in a greater conversion of the cellulose in the substrate to glucose is more desirable. The results of this study (Table 2) show that pretreatment of wheat straw with HZOZ-NaOH results in more release of glucose from cellulose and consequently more ethanol production than that treated with NaOH alone. Recently Detroy et al. reported relatively low alcohol yields on fermentation wheat straw that was pretreated with NaOH and hydrolysed with cellulase15'45. These low yields are perhaps attributable to the generation of inhibitory substances during the pretreatment process or due to other reasons not clear at this time. In this study we obtained ethanol yield of about 0.3 g per g of glucose consumed (59% of the theoretical yield) which is comparable to that reported by other investigatorsls'45. 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