M“ \“g‘ \N‘ggw “33!; EH3 llllluomau 1}" n u “h 3 LIBRARY l Michigan State University This is to certify that the thesis entitled CONSTRUCTION OF A GENE EXPRESSION MODEL IN MUCOR (TRANSFORMATION OF N. racemosus WITH A TEFl/hGH FUSION PLASMIDl presented by Yi-Yu Yen has been accepted towards fulfillment of the requirements for “-3- degree in Food Science QM” X ’ t¢;/7 Dr. John E. Linz Major professor Date II-8'83 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution vav PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. { DATE DUE DATE DUE DATE DUE l MSU Is An Affirmative Action/Equal Opportunity Institution cmWMS-Df CONSTRUCTION OF A GENE EXPRESSION MODEL IN MUCOR (TRANSFORMATION OF M. racemosus WITH A TEFl/hGH FUSION PLASMID) BY Yi-Yu Yen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Food Science and Human Nutrition 1989 ABSTRACT CONSTRUCTION OF A GENE EXPRESSION MODEL IN MUCOR (TRANSFORM M. racemosus WITH A TEFl/hGH FUSION PLASMID) BY Yi-Yu Yen The promoter is one very important element of a high level expression vector. To identify a suitable promoter for high level expression of heterologous genes, the function of the Mucor racemosus TEFl promoter was investigated in this model fungal protein expression system. A model plasmid was constructed by fusing the human growth hormone (hGH) gene with the TEFl promoter. The hGH gene was inserted into pMR1-3 at a position adjacent to the TEFl promoter. The correct structure of plasmid pMYYl was confirmed and used to transform 5. racemosus R7B. Southern analysis showed that pMYYl was present in the cells with no DNA rearrangements. However, Northern analysis did not detect an hGH gene fusion transcript in Mucor cells. These results suggested that the TEFl promoter was not capable of driving the high level expression of a heterologous gene. Future research will investigate additional factors required for the function of TEFl promoter. Dedicated to my dear parents, R.C. Yen and F.H. Yen. ii ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation and deepest gratitude to her major adviser, Dr. John E. Linz for his continuous encouragement, assistance and understanding in the course of her graduate study. The author also wishes to extend her thanks to the members of her Master's program committee, Dr. James J. Pestka, Dr. William G. Helferich and Dr. C.N. Shih for their helpful suggestion and involvement in her research. Sincere thanks and appreciation are given to Dr. William L. Casale and Jason Horng for their assistance in research and valuable technical advice. Special thanks are extended to Roscoe Warner, Cheng-shaun Chen and Lin-Bin Su for their help, particularly in the figure, photograph and slide production. Many thanks go to her laboratory coworkers: Sung-Yuan Wang, Fumin Chiu, Juili L. Lin and Yih- Jihn Lee for their caring and friendship. Last but not least, the author wishes to express her deepest appreciation and love to her parents, Mr. R.C. Yen and Mrs. F. H. Yen, her sisters, and brother, for their love, support and encouragement. iii LIST 0! LIST OF I. II. III. TABLE OF CONTENTS Tums 0.0000000000000000...IOOOOOOOOOOOOOOOOO. rIGma COO...OOOOCCCOOOOOOOOOOOOO000......I... INTRODUflION 00......OOOOOOOOOOIOOOOOOOOOOOOO HISTORICAL m1" 0..OOOOOOOOOOOOOOOOOOOOOOOO Genetic Modification of Enzymes Used in the road Industry OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Enzyme Uses in Food Areas ................ Conventional sources of Enzymes .......... Enzymes production via Recombinant DNA T.Chn°1°gy 0.00.0.00...OOOOOOOOOOOOOOOOOO Heterologous Gene Expression Systems ........ E. coli as a host ........................ fiacillus subtilis as a host .............. Yeast asahOSt OOOOOOOOOOOOOOOOOOOOOOOOOO Filamentous fungi as a host .............. Truafomation cyst“ OOOOOOOOOOOOOOOOOOOOOOO Transformation of Bacterial Cells ........ Transformation of g. ggrgyigiag .......... Transformation of Filamentous Fungi ...... 3992; Eeterologous Protein Expression/secretion ayat“ 0.0.00.00...OOOOOOOOOOOOOOOOOCOOOOOOO MATERIALS AND METHODS ....................... Microbial strains ........................... Plasmid, OOOOOOOOO...OOOOOOOOOOOOOOOOOOOOOOOO Chuic‘la OOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOO Growth Medium and Growth Conditions for 3. Transformation of E. coli .................... Colony Hybridization for the Identification of Recombinant Plasmids in E. 9211 .......... Isolation of Plasmid DNA from 3. 9911 ........ Growth Conditions for Mggg; .................. Tru.t°n.t1°n 01m eeeeeeeeeeeeeeeeeeeeee single Colony Isolation of finger Transformants. Isolation of Genomic and Plasmid DNA from Restriction Endonuclease Analysis and Gel Electrophoresis of DNA ...................... Preparation of RNA from Mace; ................ Gel Electrophoresis of RNA ................... southern and Northern Analyses of Nucleic Acid. Nuclease-81 Mapping of RNA ................... fi-Galactcsidase Assay ........................ iv PAGE vi vii 1 29 29 29 30 37 37 39 40 41 42 44 45 47 47 49 49 51 52 IVO “BULTB OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 5‘ (1) TEPl/lacz Fusion Plasmid ................. 54 Construction of Gene Pusion Plasmids ..... 54 Southern Analysis of DNA Isolated from 8222: Transformants ..................... 57 Northern Analysis of RNA Extracted from Mucor Transformants- pnrzs and pMP29 .... 67 Sl-NUclease Mapping of RNA Extracted from Transformants ........................... 7o p-Galactosidase Assay .................... 73 (2) TEEl/hGE Pusion Plasmid .................. 74 Construction of Gene Fusion Plasmids ..... 74 Isolation and Analysis of Constructed Plasmids OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 77 Transformation of 3392; racemosus R73 with pXYYI and pal-3 OOOOOOOOOOOOOOOOOOO 87 Southern Analysis of DNA Extracted from Mucor Transformants ..................... 88 Northern Analysis of RNA Extracted from Muco; Transformants ..................... 89 v O DISCUSSIO“ 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 O O O O O O O O O O 97 VI O 80W! 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 O O O O O O O O O O O O O 10‘ VII O “rauncn 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 O O O O O O O O O O O 105 LIST OF TABLES Table page 1. Major enzymes used by the food industry . .......... 5 2. Food processing enzymes and food additives used in the U.S. food industry that benefit from genetic engineering teChnOIOgy OOOOOOOOOOOOOOOOOOOOO OOOOOO 11 vi LIST OF FIGURES figure page 1. Restriction map of plasmid pLeu4 .... ........ ..... 31 2. Restriction map of plasmid pMRl-B .... ............ 33 3. Restriction map of plasmid pOGH ... ............... 35 4. Construction of plasmid pMFl4 .................... 55 5. Construction of plasmid pMFZS and pMF29 .......... 58 6. Restriction map of plasmid pMFZS ... ..... . ....... . 60 7. Restriction map of plasmid pMF29 ........... ...... 62 8. Southern analysis of genomic DNA isolated from N- 10. 11. 12. 13. 14. 15. 16. racemosus R78, pLeu4 tgansformants, pMF25 and pMF29 transformants using a 3P-labeled 6.2 Kb Sal; fragment (lacZ) and pUC8 fragment as probes ..... 65 Northern analysis of total RNA purified from u. racemosus R78, a pLeu4 transformant, a pMFZS gransformant and a pMF29 transformant using a P-labeled 6.2 Kb Sal; fragment containing the lacz gene as the probe ........... ..... ..... ..... 68 Sl-nuclease analysis of total RNA purified from M- racemosus R78, a pLeu4 transformant, a pMFZS transformant, and a pMF29 transformant .......... 71 Construction of plasmid pMYYl .................... 75 Construction of plasmid pMYYl (nucleotide sequence). 78 Colony hybridization with an hGH gene fragment as prObe OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 8]. Restriction analysis of plasmid pMYYl ............ 83 Restriction map of plasmid pMYYl ................. 85 Southern analysis of genomic DNA isolated from four pMYYl transformantfz and pMR1-3 transformant (control) using a P labeled 1.8 Kb Aatll fragment and 1.9 Kb HIDQIII fragment as probes ........... 9O vii 17. Northern analysis of total RNA isolated from four. pMYYl transformants and a pMR1-3 transformant u51ng a1”? labeled 1.8 Kb Aatll fragment containing the the hGH gene and a 1.9 Kb HindIII fragment containing the Leul gene as probes .... ....... ‘... viii INTRODUCTION Proteins play a very important role in industry. They have gained considerable attention in many industrial areas because enzyme-catalyzed reactions are clearly superior to purely chemical reactions in most cases. They are faster, more specific and safer. It was estimated that worldwide enzyme sales will reach $ 1.5 billion by 1990 (Knorr and Sinskey, 1985). Approximately 48% of all sales correspond to protease. Although the need for enzymes is continuously increasing, the quantity of enzymes produced by industrial microorganisms is too low to satisfy this demand. In order to increase the yields of enzymes and decrease the expense of producing industrial enzymes, areas of intensive research are microbial strain improvement, enzyme/cell immobilization and enzyme stability enhancement. Recombinant DNA technology is a significant new approach to strain improvement because it provides the tools to bring together genes from different organisms into one organism for increasing product yields or producing entirely new substances. Significant progress has been achieved using recombinant DNA technology for the production of nonmicrobial products in microbes. For example, insulin, somatostatin, human growth hormone, virus vaccines, and interferon have all been produced using recombinant DNA 2 technology (Bollon, 1983). There are several different microbial systems for heterologous protein expression under study such as §.coli , gagillus subtilis and Saccharomyces cerevisiae (Lin, 1986). Recently, considerable research effort has focused on the molecular genetics of filamentous fungi. The filamentous fungi are extremely attractive as potential hosts for expression of cloned genes for use in industry, since they naturally secrete vast quantities of protein and have the ability to successfully express a variety of heterologous genes without the need for extensive DNA manipulation (Dickinson, et a1., 1987 and Van Brunt, 1986). Muggr, a member of the filamentous fungi, is an excellent target for genetic engineering because it produces a number of extracellular enzymes such as rennin, lipase, amylase and cellulase which are very important for industry (Arima, 1986, Montenecourt,l985 and Somkuti, 1968,1974). One species of nuggr, Mugg; osus, has a haploid vegetative phase and has a small genome (107 bp) making it particularly convenient for genetic study. Mucor racemosus has been widely studied because it can be induced to undergo a morphogenetic change from hyphae to budding yeast-like growth in response to various environmental stimuli (Larsen, et a1., 1974). A wealth of biochemical data has been generated in studies of morphogenesis, contributing to the 3 usefulness of Mugg; racemosus as a successful model system for the study of cell growth and development. In our laboratory, we plan to use Nugg; racemosus as a model system to study the efficiency of expression and secretion of heterologous proteins from a fungus. The main goal of my research was to investigate the function of the TEFl promoter isolated from M. racemosus. The promoter plays a very important role in gene expression because the initiation of transcription is a critical point for controlling gene expression. Finding an efficient promoter is one important step for developing an efficient heterologous protein expression and secretion system. We plan to apply the technology developed in N. racemosus to more important industrial fungi such as the closely related Zygomycetes M. miegei and u. pusillus. HISTORICAL REVIEW ed a o o e sed i the cod Indust es ’ o : Enzymes, a major group of proteins, have catalytic activities and cause a multitude of essential chemical reactions to occur at reasonable rates and at standard temperature, pressure, and reactant concentrations (Haas, 1984). Thousands of enzymes have been identified, and several dozen are important in food processing (Table 1) (Wasserman, 1988). Food processors are the largest users of industrial enzymes, especially in starch processing and cheese making (Newell, 1986).A Enzymes have a long history of use in food processing, dating back to early use of calf stomachs for their rennin content to coagulate milk in cheese production. Nowadays, common uses of enzymes in the food industry include ingredient production and texture modification. Enzymes break down starch, tenderize meat, clarify wines, coagulate milk protein, and produce many other desirable changes (Potter, 1974). Some major food applications of enzymes include the production of high- fructose corn syrups, beverage clarification, brewing, baking, production of low-lactose milk, and meat tenderization (Wasserman, 1988). Table 1. Major Enzymes Used by the Food Industry Enzyme Application Proteases Papain, bromelain, ficin Rennin (chymosin) Glycosidases Amylases (a-, 8-, gluco-, and debranching) Cellulases/xylanases Glucanases Glucose isomerase Glucose oxidase/catalase Glycolytic enzymes Invertase Lactase (fi-galactosidase) Pectinases Lipases Acyl glyceride hydrolases and phospholipases Meat tenderization, haze removal, and chill proofing Cheesemaking Baking, brewing, sweetener production Biomass conversion, juice clarification Brewing High-fructose corn syrup (HFCS) production Desugaring of egg whites, oxygen removal Fermentation-carbon dioxide and ethanol production Candymaking Low-lactose dairy products, whey disposal Beverage clarification, texture modification Texture modification, flavor generation (Wasserman, 1988) 6 From an economic standpoint, approximately $ 160 million worth of enzymes are consumed annually by the food and detergent industries (OTA, 1981). However, only relatively few enzymes such as amylase, glucose oxidase, lipase, cellulase, protease, glucose isomerase, and invertase (Wasserman, 1984) are used in large quantities in food processing. The limited use of enzymes is related to low enzyme production and high enzyme purification cost. In order to overcome this limitation, it is required to develop economical new enzymes. Genetic engineering provides powerful tools to reach this goal. Conventional Sgnzggs 9f Enzynes: Enzymes are derived from animal, plant and microbial sources. Currently, the majority of the food processing enzymes and additives are produced by biological process (Lin, 1986). Enzymes obtained from animals are relatively expensive, and their availability usually depends on the market for those animals. For example, rennin, which is extracted from the stomach of calves, depends on the demand for lamb or beef for its availability (Loffler, 1986). Most of the commercial plant enzymes such as papain, bromelain, and ficin are easy to extract, but their supply is also governed by food demands for papaya, pineapple, and figs (Loffler, 1986). Supplies of enzymes produced by animals or 7 plants may fluctuate because of environmental, political, or economic factors (Haas,1984). For technical and economic reasons, microorganisms may be the best sources for enzymes. The main reason for the attractiveness of microorganisms as potential sources of enzymes is the ease with which enzyme yields may be increased by environmental and genetic manipulations. Microbial enzymes are produced by methods that are cheap and relatively easy to scale up. Microorganisms have a rapid doubling time, which allows their production to be adjusted more easily to market demands. Environmental conditions can also be monitored closely to provide a high level of consistency in enzyme purity, stability, and activity (Loffler, 1986). Microorganisms - bacteria, yeasts, and fungi - are all used extensively in various aspects of food processing. For instance, a number of filamentous fungi (9-9-.Eh15222§ chinensis and £222: nigngi) secrete aspartyl proteases (rennin) which are structurally related to each other and to the gastric aspartyl protease from calf stomach (Gray et al., 1986). In spite of the many advantages of using microbial enzymes, animal and plant enzymes are still preferred for many food processes. For example, papain, ficin, and bromelain are preferred for the chill-proofing of beer and 8 tenderization of meat; rennet and pepsin are the proteases of choice for the cheese industry, and plastein reactions have almost exclusively been carried out with papain, trypsin, chymotrypsin, and pepsin (Loffler, 1986). Ideally, we would like to combine the production characteristics of microbial cells (high yields, reproducibility, controllable conditions, etc.) with the use of well-known, generally recognized as safe (GRAS), plant and animal enzymes. This is now possible via recombinant DNA technology. Enzymes nngguction via Regombinnnt DNA Technology: The single greatest obstacle limiting new enzyme development is high enzyme cost. To reduce the expense of producing and using enzymes, three broad research areas have been established: microbial strain improvement, enzyme and cell immobilization, and enzyme stability enhancement (Wasserman, 1984). Historically, microbial strain improvement has been obtained in three ways: 1) the detection, within a population, of those individuals which have undergone desirable changes: this is an inefficient, slow process; 2) the enhancement of the nucleotide sequence of DNA and the screening of the treated organisms for variants displaying the desired trait; and 3) the discovery of new organisms which naturally possess the desired quality '(Haas, 1984). Significant contributions have been achieved 9 with these methods. However, they are time-consuming and inefficient because the mutation event is generally random across the entire genome. Since it is extremely difficult to design a random mutagenesis which generates a high proportion of the desired type of mutant, the examination of thousands of organisms is often required. This observation was the stimulus for the development of genetic engineering and related technologies. The elucidation of the structure of DNA and the understanding of DNA replication and protein synthesis, together with the discovery of plasmids and restriction enzymes, have now made it possible to genetically engineer microorganisms (and higher organisms) to produce biochemicals which are foreign to the microorganisms. A major technique used in genetic engineering is recombinant DNA, which is the joining of pieces of DNA from different organisms to produce a hybrid molecule. This technique could theoretically allow microorganisms to express high levels of any human, animal, or plant proteins by the transfer of the genes into microorganisms. Recombinant DNA technology has essentially widened the range of enzymes that can be considered for commercialization to include even those from unusual organisms, plants, and animals (Pitcher, 1986). In some cases, yields can be increased by inserting multiple copies of the desired gene into the host organism. 10 Secretion systems are also being developed to allow high production levels of extracellular enzymes, a necessity in many cases to achieve favorable economics by reducing purification costs. Production of unwanted proteins or other materials can be eliminated either by specific deletion or by use of a host organism without the disadvantages of the original organism. Recombinant DNA technology has been applied to bacteria and fungiand has contributed to the development of strong fermentation technologies in the pharmaceutical, food, and brewing industries (Haas, 1984). In 1977 it was shown that a gene from a higher eukaryote could be expressed in a microorganism, E. 991i, to produce a biologically active protein, somatostatin (Itekura et al., 1977). Numerous mammalian gene products, including antiviral, antitumor, and antidiabetic agents and growth-promoting factors, have been expressed in microbial cells (Lin, 1986). For enzymes in the food industries, the application of recombinant DNA technology is beginning to play a significant part in the corporate commercial product development process. As shown in Table 2, almost all the food processing enzymes and additives have been targeted by recombinant DNA techniques (Lin, 1986). Genes encoding calf rennin (Nishimori et al., 1981), thaumatin (Edens et al., 11 Table 2. Food Processing Enzymes and Food Additives Used in the U.S. Food Industry that Benefit from Genetic Engineering Technology CQLGQOEY Food Processing Enzymes Starch processing Dairy products Brewing Wine/fruit/vegetable processing Fuel alcohol Food Additives Low-calorie products Flavor enhancers Human and animal diet supplements Stabilizing agents Preservatives (Lin, 1986) njflflflflfl£L_ c-Amylase fi-Amylase Glucoamylase Glucose isomerase Pullulanase Rennin Lipase Lactase Amylases Proteases Pectinases Amylases Glucoamylase Aspartame Thaumatin Glutamic acid 5'-Ribonucleotides Amino acids Vitamins Xanthan gum Cecropin 12 1982: 1984), and cecropin(Hofsten et al.,1985) have been cloned and produced in suitable hosts, and the commercial production processes are currently under aggressive development (Lin, 1986). For example, rennin has received the most attention as a candidate for the commercialization of recombinant DNA technology in the food processing industry (Pitcher, 1986). The gene for calf rennin has been isolated and cloned into yeast (Mellor et al., 1983; Moir et al., 1985) and fungi (Cullen et al., 1987). At Genencor, much effort has been focused on the filamentous fungi for producing commercial rennin. Properly folded, active prochymosin has been secreted from Aspergillus nidulans, A§pezgilln§ aw o ', and Irighgdernn reesei (Pitcher, 1986). In the last few years, at least 19 cellulase or amylase gene have been molecularly cloned (Pitcher, 1986). Many of these have been expressed in heterologous microorganisms, such as Kluyyezomycgs Inngilig and §nccnanomyces cerevisiae (Pitcher, 1986). Another example is the lactose utilization genes which have been cloned into Sacchanomycgs ggrgyiging (Sreekrishna and Dickson, 1985; Farahnak et al., 1986), znngngnnnng camnenstris (Walsh et al., 1984), and other microorganisms so that the lactose in whey permeate can be converted into ethanol, single-cell protein, or xanthan gum. Recently Bacillus subtilis (GRAS) was utilized to produce a 13 thermostable a-amylase by expression of the gene from Bacillus Whine (Wasserman, 1984)- This enzyme has been accepted for use in the food industry opening the way for production of other food grade enzymes via recombinant DNA technology. The benefits of achieving commercially useful processing enzymes through genetic engineering are expected to include: 1) cost savings in enzyme production; 2) production of enzymes in GRAS organisms suitable for food processing: and 3) specific genetic modifications at the DNA level to improve enzyme properties such as thermal stability, ability to operate over a wide pH range and other performance characteristics (White, et al., 1984). e e 0 on cue ress on S stems Heterologous gene expression begins with the cloning of the gene(s) of interest and transfer of this genetic material to a suitable host organism. This procedure allows the investigation of the genetic activity of the DNA fragment in a characterized genetic background, the production of sufficient quantities of DNA for nucleotide Sequence analysis, and the isolation of significant amount of the proteins encoded by the DNA. 14 The suitability of selected organisms for expression of DNA from other sources has been enhanced by the isolation of variants with advantageous mutations. For example, one strain of E. 9911 has been isolated which lacks a normal complement of DNA-degrading enzymes. These bacteria accept foreign DNA more efficiently than their wild-type progenitors and are routinely used as DNA recipients (Haas, 1984). The choice of the proper host organism may greatly increase the production levels of the desired protein and, perhaps even more important, high secretion levels. For instance, bacterial secretion of calf rennin has been difficult to achieve, but scientists have succeeded in secreting properly processed prochymosin, a precursor of calf rennin, from filamentous fungi (Pitcher, 1986). The bacterium E. ggli is the most thoroughly characterized microorganism and has been the most frequently used in recombinant DNA studies. The advantages of using g. sgli are the rapid growth rate of cells and the high synthesis rate of the heterologous protein. Moreover, in g. 9911, several strong promoters, such as lac, lambda PL, lambda Par lambda OR/PL, ara 8, tac (combination of trp and lac), or combinations of trp, lambda.F§, and lac, have been Commonly used for overproduction of enzymes and food 15 additive products (Lin, 1986). Until now, the most widely used host for heterologous genes has been E. coli. In the past few years, achieving a high-level expression of foreign proteins in E. 9911 using various expression vectors has become a common practice. For example, rennin was produced in E. 9911 via recombinant DNA technology. A number of mammalian secretory proteins have been correctly processed and secreted by E. 9911 cells using either a natural mammalian or an E. 9911 signal sequence (Chang, 1987). These include rat proinsulin, human proinsulin, human immunoglobulin light chain, hGH and human epidermal growth factor (Chang, 1987). Although a high level expression of foreign proteins in E. 9911 has been achieved, eukaryotic proteins expressed in E. 9911 are generally insoluble products. The reason is that g. 9911 does not carry out post-translational modifications which are necessary for activity for many enzymes, therefore the proteins produced from eukaryotic genes in E. 9911 may differ from the normal gene product and lack the required biological action or be insoluble until appropriately modified chemically (e.g. calf prochymosin: interferon-gamma ) (Kingsman et al., 1985). For example, the rennin produced in E. 9911 is not secreted and forms refractile bodies of improperly folded protein. These 16 bodies must be solubilized and refolded to allow formation of the prOper disulfide bonds. This process is expensive and, under the most practical conditions, yields are relatively poor (Pitcher, 1986). Human growth hormone (Goeddel etal., 1979) and human leucocyte a-interferon (De Maeyer et al., 1982) are produced as soluble proteins to a certain extent, but there is no indication that these products are entirely soluble. The use of prokaryotic or eukaryotic signal sequences has increased the secretion level of heterologous protein, but the level of secreted protein is generally low (Chang, 1987). g. 9911 also produces endotoxins and is therefore not always the preferred organism for recombinant DNA experiments with genes of interest to the food industry (Haas, 1984). Efforts are underway to apply recombinant DNA methods to organisms accepted as safe by this industry. Bac111us subtilis as a nost: §a9111us §ubt1115 has the potential for becoming an extremely useful system for the cloning and expression of heterologous genes. The practical advantages of this organism include its ability to secrete proteins into the medium, its non-pathogenic nature to humans, its ability to Grow at relatively high temperatures, its relatively heat- stable proteins, its ability to grow on either simple or 1? complex growth media, and its historical use in the production of many industrial and food products (Doi, 1984). Transformation has been employed to introduce foreign genes into 9. 99991119 and to alter the rate of production of heterologous enzymes. Palva (1982) cloned the 9. nny191199999919n9 a-amylase into 9. subti119 by a shotgun method employing the plasmid pU8110. The amylase activity released into the extracellular fluid represented a 2,500- fold increase over the activity secreted by the untransformed host (Wasserman, 1984). Through a combination of mutation and transformation, Hitotsyanagi et al. (1979), developed a strain of 9. 99991119 which secreted 1,500-2,000 times greater a-amylase activity than the parent. The major problems with the 9. 99991119 system are the inability to carry out posttranslational modification that eukaryotic proteins seem to require to be fully functional (Van Brunt, 1986) and the low quantities of the protein product. So other host systems are being pursued. W: The Yeast. SW cererieiae. is one of the most useful eukaryotic organisms for the study of the regulation 0f gene expression. Because of its small genome (only four 18 times the size of E. 9911) and short generation time (a few hours), yeast can be experimentally manipulated as easily as most prokaryotes (Watson, 1983). Yeast are often considered superior to E. 9911 as expression hosts because they can glycosylate, acetylate, fold and perform many of the other post-translational modifications that eukaryotic proteins seem to require to be fully functional (Van Brunt, 1986). Many of the eukaryotic proteins which are formed by E. 9911 in an inactive state are produced as soluble, biologically active proteins in 9. 9999919199 (e.g. calf prochymosin, Mellor et al., 1983: IFN- gamma, Derynck, Singh and Goeddel, 1983). 9. 991ev1siae has an additional advantage over 9. 9911 in that it has a secretion system which is similar to that found in higher eukaryotic organisms (Schekman and Novick, 1982) and which can be manipulated to allow the secretion of heterologous proteins. Unlike 9. 9911, yeast produces no endotoxins, therefore enzymes made in yeast may be more acceptable as human or animal pharmaceutical and food products (Mellor et al., 1983). Recent studies have shown that heterologous eukaryotic genes can be expressed in the yeast 9. 9999919199 when fused to specific yeast expression signals. For example, rennin (Mellor et al., 1983), glucoamylase (Innis et al., 1985), 19 several c-interferon genes (Hitzeman et al., 1981: Tuite et al., 1982), gamma-interferon (Derynck et al., 1983), the hepatitis 8 surface antigen (Valenzuela et al., 1982; Miyanohara et al., 1983), superoxide dismutase (SOD), and epidermal growth factor (EGF) (Van Brunt, 1986) have been expressed in yeast. The study of heterologous expression in 9. 9999919199, detected several problems. Although several 9. 9999919199 genes have introns (e.g. actin, ribosomal proteins) heterologous introns are not reliably processed (Langford et al., 1983). Yeast cells appear to be very stringent about the sequence specificity for recognition of splice junctions (Dobson et al., 1982). In order to express any heterologous coding sequences it is therefore important to use a cDNA. The ability of 9. 9999v151ae to recognize transcriptional signals in heterologous DNA is variable. In many cases, as with rabbit fi-globin, there is some transcriptional activity but the mRNA initiates at the wrong site in 9. 9erevisiae (Kingsman et al., 1985). Experimental transfer of the rat growth hormone gene into yeast (Hitzeman et al., 1981 and Ammerer et al., 1981) showed that heterologous genes are not always accurately transcribed or properly translated. Although the secretion system of yeast cells is very similar to that in higher eukaryotic cells, the secretion level is not high even with the new "super secretor" mutants (Van 20 Brunt, 1986). These inherent problems of 9. cerevisiae led researchers to study the filamentous fungi. Eilamenteu§_fungi_a§_a_hest= The filamentous fungi have several characteristics which make them candidates for useful expression systems. Many filamentous fungi secrete large quantities of proteins into the culture medium. For example, certain 59999911199 9199: strains secrete over five grams of glucoamylase per liter of growth medium when grown under appropriate conditions (Cullen et al., 1987). Moreover, filamentous fungi have the ability to carry out the correct posttranslational modification on many eukaryotic proteins, including the proper glycosylation pattern. Many fungal genes and genes of higher eukaryotes can also be transcribed and their transcripts processed correctly without adding specific transcription regulatory signals (Van Brunt, 1986). The technology for gene expression in filamentous fungi is developing rapidly, building on the frame-work already established for yeast. A few examples of heterologous gene expression in filamentous fungi have been described - including the expression and secretion of M. mienei protease in A. n19er, Negregeera erases. ustilage margis. Ehreemrges blakesleeanus 21 and 999911999199 99999999999999 (Dickinson et al., 1987). Research also has been done in filamentous fungi such as production of human interferon in 959e99111us, yeast invertase in 219919, calf rennin in 99999, and cellulase in T91999999n9 (Enari and Paavola, 1987). The secretion of chymosin by 9. 91991999 represents a dramatic improvement over other microbial system such as yeast and 9. 9911 that have been used for this purpose. In 9. n1dulans cells transformed with the chymosin genes more than 90% of the chymosin synthesized was secreted into the culture medium and the majority of this material was enzymatically active (Cullen et al.,1987). The filamentous fungus 99999 is an excellent target for genetic engineering since it produces many extracellular enzymes, and it provides a useful system for the study of the biochemical basis of morphogenesis. In our laboratory, we use 9. 9a9emosus as a model system to study heterologous proteins expression and secretion in a filamentous fungus. Transformation System The optimal use of recombinant DNA technology in the genetic study of any organism requires the availability of an efficient genetic transformation system. The technique 22 of DNA transformation - more particularly, plasmid-mediated transformation - presents an opportunity to transfer selected DNA molecules (genes) of interest into the host cells. In transformation, the vector acts as a carrier to carry the genes of interest into a suitable host. Vectors are designed with particular marker genes which allow the foreign genes to be detected in the host, and ensure replication of the foreign DNA. The ability to detect the successful uptake of exogenous DNA depends on the presence of the genetic marker. One common procedure for fungal transformation is to use an auxotrophic host strain and incorporate the corresponding wild-type gene on the plasmid vector which can therefore be selected against the background of non-transformed auxotrophs (Kingsman et al., 1985). Any wild-type gene encoding an essential biosynthetic enzyme is useful as a selectable marker in cells bearing a mutation in this gene. Another useful procedure is to use dominant selectable markers whose presence can be selected for in wild-type cells (Rine and Carlson, 1986). This method is widely used in transforming bacteria with antibiotic resistance genes, especially strains which lack suitable auxotrophic mutations. 23 s o t'o f cte ' C l 5: Procedures for transformation and expression in some simple bacteria such as g. 99;; or Bacillus species and unicellular fungi such as yeast are now well established (Haas et al., 1984, Davis et al., 1980). Yeast cells are not naturally competent for DNA uptake because the cell wall is an effective barrier. This permeability barrier must be modified for DNA uptake to occur, usually by enzymatic treatment and the addition of calcium or lithium salts (Kingsman et al., 1985). These transformation systems involve enzymatic removal of the cell wall to produce sphaeroplasts which can take up DNA on treatment with polyethylene glycol and calcium ions (Kingsman et al., 1985). Under appropriate conditions the cell walls can regenerate to permit propagation and selection of transformants. Another popular method of transformation relies upon the observation that intact yeast cells treated with Idf ions will take up exogenous DNA (Rine and Carlson, 1985). Once the DNA enters the yeast cell, it may become integrated into one of the chromosomes or it may remain in an autonomous state, depending on the nature of the DNA molecules used in transformation (Boguslawski, 24 1985). The LEUZ and TRPl genes which encode 3-isopropylmalate dehydrogenase and N (5'- phosphoribosyl) anthranilate isomerase respectively, are commonly used as selectable markers in 9. 9999319199 (Kingsman, 1985). In addition, various dominant selectable markers which confer resistance to drugs are available, e.g. thymidine kinase, chloramphenicol acetyltransferase, dihydrofolate reductase, etc (Kingsman, 1985). I E !' E E°J m ! E .: Filamentous fungi in general have relatively low transformation efficiencies when compared with E. 99;; or yeast. This is possibly due in part to an increased number of chromosomes and quantity of DNA, and to more highly regulated mechanisms of transcription and translation (Wasserman, 1988). Protoplasts formation and regeneration of mycelium is a critical protocol for transformation of filamentous fungi. The commercially available enzyme Novozyme 234 is most convenient for this purpose by removing the cell wall. Among the filamentous fungi, the first to be transformed were a few ascomycetous species using recombinant plasmid 25 DNA. The transformation of 9999999999 99ass9 was accomplished by Case et al. (1979), while the transformation of Bedeénere angering and Aensrsillu§ nigglgns were first described by Stahl et a1. (1982), and Ballance et a1. (1983). The genetic analysis of 99999 species is hampered by the difficulties in utilizing the sexual cycle of these organisms. Mating of (+) and (-) strains does not always result in the formation of zygospores. In some species the number of spores produced is low while in other species zygospore development remains incomplete (Schipper, 1978). Moreover, the successful germination of zygospores under laboratory conditions is rare and is preceded by a considerable period of dormancy (Van Heeswijck, 1984). These characteristics of 99999 make a transformation system desirable. The transformation system in 99999 reported by Van Heeswijck (1984) is currently one of the most successful systems which have been developed in fungi. A high frequency transformation system was developed in this organism using protoplasts of a leucine auxotrophic mutant, M- 999999999 R78, and a shuttle vector, pLeu4, which is able to propagate in bacterial cells and in 5- 999999999 (Van Heeswijck, 1984). In our laboratory, we used derivatives of 26 pLeu4 to transform plasmid DNA into 9. race9osus R7B cells. n e o o o 0 es on See etion 8 stem: 99999 is a filamentous fungus of the class Zygomycetes which is of biological and industrial interest because of the production of a variety of extracellular enzymes and the ability of some species to undergo a morphogenetic change from hyphae to budding yeast-like growth in response to various environmental stimuli. One species, 99999 999999999, has been widely studied because of its ability to undergo cellular morphogenesis. These studies resulted in the collection of a large quantity of biochemical research data which contributed to our choice of 9. racemosus as a good model heterologous protein expression system. In our laboratory, we used 9. r9ce9os9s R7B (leucine auxotroph) as a host to receive heterologous genes. The transformation vector, pLeu4, is a shuttle vector that contains a putative M- I99999999 ARS (autonomous replication sequence), an 9. 9911 replication origin, and selectable markers for 9. 99;; (ampicillin resistance gene) and 99999 (Leul, leucine biosynthetic gene). In the study of gene regulation, it frequently is useful to join the promoter and controlling elements of one 27 gene to the structural part of another well-characterized reporter gene whose product is easy to assay. The promoter selected for use in our system is the 99999 TEFl promoter. TEFl is a constitutive, highly expressed gene encoding elongation factor In, one of the most abundant proteins present in 9.999999999 (approximately 1-5 % of total protein). Because of the high level of transcription of this gene, we hypothesized that the promoter of TEFl would be a very strong promoter (Linz et al., 1986) and useful to express heterologous genes in 99999. To test this hypothesis, plasmids were constructed by fusing a 1.9 Kb EcoRI/Hpal DNA fragment containing the TEFl promoter region with the lacz gene of E. 9911. These plasmids were named pMFZS and pMF29 and were transformed into 99999 cells. The level of fi-galactosidase activity from the lacz gene would indirectly measure the level of transcription driven by the TEFl promoter. Southern analysis, Northern analysis, S-l nuclease mapping and fi-galactosidase assays showed that 9. I32999999 was successfully transformed with the fusion plasmids but the level of the fusion transcript detected in Northern analysis was extremely low possibly due to mRNA instability. One possible explanation was that no transcription termination signal was included in these constructs. 28 Since the lacz gene proved unsatisfactory as a reporter gene to measure TEFl promoter activity, we decided to generate a TEFl/hGH (human growth hormone) gene fusion (pMYYl) to detect the function of the TEFl promoter. The advantages of using the hGH gene in 99999 (Chang, et al., 1987 and Selden, et al., 1986) instead of the lacz gene are: 1) It produces stable mRNA in many eukaryotic cell types: 2) It has its own polyadenlylation site (transcription termination signal); 3) Its splice junctions are similar to those in 99999; 4) It contains a signal peptide potentially allowing secretion from the fungus; 5) Southern and Northern analyses conducted previously showed there is no similar gene or transcript in 99999 (Wang, S.Y. 1988. unpublished); 6) Polyclonal antibody is available commercially for use in Western analysis. Expression of the hGH gene provides an excellent model system for the study of the evolution of related genes and host specific gene expression. The goal for this research was to construct a plasmid containing the TEFl/hGH gene fusion and to transform the resulting recombinant DNA into 9. 9a99m9sus R7B cells. The level of transcription of this gene fusion would be measured to detect the function of TEFl promoter. MATERIALS AND METHODS Microbial Strains: Es999ri9hia 99;; strain DHSa (BRL), genotype: Fkflu. 80dlacZAMlSA(lacZYA-argF)U169 recAl endAl hst17(r{, mK‘)supE44lambda'thi-1 gyrA relAl, was modified from the parental strain DHS (Hanahan, 1983). The strain was used for amplification and isolation of plasmid DNA (pMRl-3, pOGH and pMYY-l) and also as the recipient strain in bacterial transformation experiments. This bacterial strain is sensitive to ampicillin, so plasmid encoded ampicillin resistance can be used to screen transformants. 99cor racemosus ATCC 1216b R78, a leucine auxotroph, was derived from Mucor 9i99199il91999 f. lusitanicus CBS 277.49 (Syn. Mu909 9acemos99 ATCC 1216b) (VanHeeswijck, 1984). It was used as the recipient strain in the Mucor transformation experiment. Plasmids: The plasmid pLeu4 (which was kindly donated by Carlsberg laboratory, Denmark) carries the B-lactamase gene (MFR) in pUC13 for selection in the ampicillin sensitive strain, 9. 991; DHSa, plus a 4.4 Kb PstI restriction 29 3O fragment of 99999 999999999 genomic DNA which contains a leucine biosynthetic gene as a selectable marker in Mucor 999999999 ATCC 1216b R78, (Van Heeswijck and Roncero, 1984). This restriction fragment also carries a putative Mucor autonomous replication sequence (ARS) (Roncero et al., 1988) (Fig. 1, pp.31, 32). The plasmid pMR1-3 was derived from pLeu4 by inserting a 1.9 Kb EcoRI-HpaI fragment from TEF-l containing a putative strong promoter (Fig. 2, pp.33, 34). The plasmid pOGH, donated by R.Selden (Harvard Medical School), is a pUC19 based vector which contains a promoterless gene encoding human growth hormone (hGH) (Fig. 3! pp-BS’ 36) 0 Chemicals: Most chemicals used in the experiments were purchased from Sigma chemical company with the exception of those described specifically in the text. Restriction endonucleases, T4 DNA ligase, DNA polymeraseI (Klenow fragment), 2'-deoxynucleotides, and the buffers utilized in DNA manipulations were obtained from Boehringer Mannheim Biochemicals. (aJRP)dGTP (>6000 Ci/mmole) was purchased from New England Nuclear (USA). Polyethylene glycol 4000 31 Figure 1. Restriction map of plasmid pLeu4. 32 amwm :1: m msww. E. z smmm HS? .mmmv :I .83. u—u—n ....‘10 % 3&1 8mm: . 1E 29 _ _ 3:: n3 8:. VDMJQ Bama._m§8 mNH«._w&K 3m. 3% 9mm. .. :1: mm H :mu @fidmg :9”. .39 ism. mm? 55.9 moms 39.. .89.. _\mw mmmw Figure 2. 33 Restriction map of plasmid pMRl-3. 34 msvv._m_ncwr mmmv _wym Pfixzm . a _ “newt .wmfim . 7&9an -mmN.H:ew~ p/ . firm... 2 ... . mwww . “2?,“ H 3m... .m 1551/ an swam m-Hmzl . a 2.15 fifi mmmmn SEN _ :33 \ :1 romhzfi .wm. . Hump .ag. _ Sam H many... mad : r mmmf mama H .. 25?. mam. . 25¢ N/m!!! MB\ 3cm. ..wmmm 9mm . HI: rum . ... . . n. HIQm. .wuumm H C rnx H ficamrxu pummx . .wmwmm" H 1:5“. Emumm mfipux. . .0“me : mum. wUmm 35 Figure 3. Restriction map of plasmid pOGH. 36 83 s: mm“ : man. Gem. ““5 tom Q 8mm 392. .889 2:8.i amt“. :mmi Nomv :r... .89. 7.9.9... Emv 35% Saw 39.x. .mmmm 37 (PEG 4000) was obtained from Fluka AG (Switzerland). Novozyme 234 (prepared from 29199999999 narzianum: mainly containing a-1,3-glucanase activity) was from Novo Industries. Crude chitosanase (isolated from Myxobacter AL- 1) was prepared in our laboratory. Growth Medium and Growth Conditions for Escher9chia coli: 9. 9919i, cells were grown at 37°C in Luria-Bertani (LB) medium (10 g 8acto-tryptone, 5 g 8acto-yeast extract, and 10 9 NaCl per liter, pH 7.5) with constant shaking or on solid medium containing 1.5% (wt./vol.) Bacto-agar. 9. 99;; strains containing a plasmid which carries the ampicillin resistance gene were grown on LB medium supplemented with ampicillin (50 ug/ml). YT medium (8 g 8acto-tryptone, 5 g 8acto-yeast extract and 5 g NaCl per liter, pH 7.2-7.4; 1.5% wt./vol. 8acto-agar for solid medium) was used in the bacterial transformation experiments. Transformation of Bgche9ic919 9911: E. 99;; strain DHSa was used as a host strain in bacterial transformation. Competent cells for plasmid transformation were prepared by the Calcium Chloride procedure (Maniatis et al., 1982). Calcium chloride (CaClz) and magnesium chloride (MgClz) were used to introduce cracks 38 in the bacterial cell walls. A single colony of 9. 99;; strain DHSa was inoculated into 3 m1 YT broth at 37°C with shaking (ca. 225 rpm), overnight. An appropriate amount of the overnight culture was diluted 1 to 200 with YT broth (100 ml is sufficient for 20 transformation reactions) and incubated with shaking at 37°C until mid log phase (A590=0.4-0.6; ca. 2-3 hours). The cells were chilled on ice, pelleted by centrifugation (7000X g, 5 minutes at 4%”, gently resuspended in ice-cold 0.1 M MgC12 (1/5 of the original volume) and kept on ice for 15 minutes. The cells were pelleted again by centrifugation (7000X g, 5 minutes at 43C), gently resuspend in ice-cold 0.1 M CaCl2 (1/50 of the original volume) and kept on ice for 60 minutes to generate competent cells. ~ An aliquot of ligation mixture (5 ul ~ 10 ng)was added to 100 ul competent cells at.4PC. The tube was shaken gently and then kept on ice for 30 minutes. The cell mixture was heated to 42°C for 2 minutes without shaking (heat shock) and then placed on ice for 2 minutes. Nine volumes (900 ul) of SOC (2% wt./vol. 8acto-tryptone, 0.5% wt./vol. 8acto-yeast extract, 10 mM NaCl 2.5 mM KCl, 10 mM MgC12, 10 mM 9950, and 20 mM glucose) held at room temperature were added to the cell mixture which was then ' l incubated by shaking at 37°C for 60 minutes. One hundred u aliquots of transformed cells were spread onto YT medium 39 containing ampicillin (50 ug/ml), and incubated at.37°C for 24 hours. Colony Hybridization for the Identification of Recombinant Plasmids in E. coli: Transformants containing the desired recombinant plasmid were identified by the colony hybridization procedure (Maniatis T., et a1, 1982, Grunstein and Hogness, 1975) using 3ZP-labeled gene-specific DNA probes. This method was used to screen small numbers (100-200) of colonies isolated from several agar plates. Transformant colonies, which grew on selective plates, were consolidated onto a master agar plate and onto a nitrocellulose filter laid on the surface of a second agar plate. After a period of growth (ca.24 hr), the colonies on the nitrocellulose filter were lysed and the DNA denatured with Southern base (1.5 M NaCl and 0.5 M NaOH) and then neutralized with Southern neutralizer (l M Tris-C1 and 1.5 M NaCl, pH 8.0). The DNA liberated from these colonies was fixed to the filter by baking for 2 hours at 80°C under vacuum. The master plate was stored at 4°C until the results of the screening procedure were available. The baked filters were floated on the surface of 6x SSC solution until throughly wetted from beneath, then submerged 40 into the solution for 5 min. The wet filters were put into 100-300 ml prewashing solution (50 mM Tris.Cl [pH 8.0], 1 M NaCl, 1 mM EDTA and 0.1% SDS) and incubated for 2 hours at 42°C. The filters were incubated in prehybridization solution (6x SSC, 5X Denhardt's solution [Maniatis et al., 1982], 50% formamide, 0.1% SDS, 100 ug/ml denatured salmon sperm DNA , 5 mM EDTA) for 2 hours at 42°C. An appropriate amount of denaturated probe was added to the prehybridization solution, and the hybridization was performed at 42°C for 12 to 36 hours. After two non- specific washes (1x SSC, 0.1% SDS, 15 min at room temperature) and one high stringency final wash (0.1x SSC, 0.1% SDS, 1 hr at 65°C) , the filters were analyzed by autoradiography. Colonies which gave positive autoradiography results were recovered from the master plates. Isolation of Plasmid DNA from 9.999;: Plasmid DNA was obtained from small cultures of 9. C011 by the alkaline lysis "miniprep" method (Maniatis et al., 1982). Plasmid DNA purified in this way was treated with DNase-free RNase (20 ug/ml) for one hour at.37°C during restriction enzyme digestion. 41 For large-scale preparation of plasmid DNA (Maniatis et al., 1982), a small volume of overnight culture (0.1 ml) was added to 25 ml of LB medium containing ampicillin (50 ug/ml) in a 100 ml flask and incubated at 37°C with vigorous shaking until the culture reached late log phase (oqwo== 0.6). Twenty-five m1 of the late log phase culture was inoculated into 500 ml of LB medium with ampicillin (50 ug/ml) in a 2-liter flask, incubated exactly 2.5 hr at 37%: with vigorous shaking (00600 ~ 0.4) followed by addition of 2.5 m1 chloramphenicol (34 mg/ml in ethanol) to amplify the plasmids. Alkaline lysis was performed on the cells (Maniatis et al., 1982). The plasmid DNA was purified by equilibrium centrifugation of the lysate in cesium chloride - ethidium bromide gradients. Ethidium bromide was removed from the purified plasmid DNA by isoamyl alcohol extraction followed by dialysis to remove cesium chloride. The DNA was stored in TE buffer (10 mM Tris.Cl [pH 8.0] and 1mM EDTA )at 4°C. Growth Conditions for Muco; : Sporangiospores of Mucor 9acemosus ATCC 1216b and the leucine auxotroph strain R78 were produced on YPG agar plates (0.3% yeast extract, 1% peptone and 2% glucose: the pH of medium was adjusted to 4.5 with cone. sulfuric acid) which were inoculated with a small amount of stock 42 sporangiospores in the center and incubated for 5-7 days at 28°C. The mature spores were harvested with ice-cold distilled water by scraping the surface of the medium with a sterile glass rod. The harvested spores were inoculated into fresh medium immediately or stored as a spore stock at -20°C in sterile distilled water containing 20% (vol/vol) glycerol. 99999 9acemosus ATCC 1216b was also grown in YNB minimal medium (0.5% Difco yeast nitrogen base [without amino acids and ammonium sulfate], 1.5% ammonium sulfate, 1.5% glutamic acid: pH was adjusted to 4.5 with conc. sulfuric acid). After steam sterilization, the medium was supplemented with 1% (w/v) sterile glucose, 1 ug/ml thiamine and 1 ug/ml niacin. 99999 999999999 ATCC 1216b R78 (leucine auxotroph) was also grown in YN8 minimal medium supplemented with 1 mM leucine. 99999 9ac9moS99 R78, transformed with plasmids pMF25, pMF29, pMR1-3 or pMYY-l, was grown on YN8 minimal medium without leucine to provide constant selective pressure for the presence of the plasmids. Transformation of 9uco9: Fresh sporangiospores of 99999 999999999 1216b R78 were suspended in 50 ml YPG medium (at a concentration of 2x105 spores/ml) and germinated about 5.5 hours at 28°C with 43 vigorous shaking. The germlings, whose germ tubes were 3 to 5 spore diameters in length, were harvested by filtration through nylon cloth (mesh size 30 um) and washed twice with 0.5 M sorbitol. The germlings were incubated 1 hour at 28%: with mild shaking in 1 ml 0.01 M sodium phosphate buffer (pH 6.5) containing 0.5 M sorbitol, Chitosanase (200 units/ml) and Novozyme (2 mg/ml) to produce protoplasts. The protoplasts and undigested cells were harvested by centrifugation (400x g, 8 minutes at room temperature), washed twice with 0.5 M sorbitol, washed once with 0.5 M sorbitol in Mops(3-N-Morpholine propane sulphonic acid) buffer (10 m9 Mops pH 6.3, 50 m9 CaClz) , and then centrifuged (400x 9, 8 minutes at room temperature) to harvest the protoplasts. The cell pellet was suspended in 2 ml of 0.5 M sorbitol in Mops buffer. The number of protoplasts was counted with a hemacytometer under a light microscope. The protoplasts were observed to burst after adding distilled water. The plasmid mixture (containing 0.1-50 ug plasmid DNA) was pretreated with 1 mg heparin in 20 ul of 0.5 M sorbitol in Mops buffer for 20 minutes on ice and added to 0.2 ml of the protoplast suspension. A 20 ul aliquot of 40% PEG 4000 in Mops buffer was added to this reaction mixture which was incubated on ice for 30 minutes. After incubation, 2.5 ml of 44 40% PEG 4000 in Mops buffer was added and the incubation continued at room temperature for 25 minutes to complete the transformation. The cell suspensions were diluted with 20 ml of 0.5 M sorbitol in Mops buffer and centrifuged (400x g, 5 minutes at room temperature). The cell pellet was resuspended in 5 ml of YPG broth (pH 4.5) with 0.5 M sorbitol and incubated at room temperature for 30 minutes to allow the cells to recover. The pellet was harvested by centrifugation (400x g, 5 minutes at room temperature) and resuspended in 5 m1 of YNB (pH 4.5) broth with 0.5 M sorbitol. Aliquots of these cell suspensions (0.1 ml and 1 ml) were added to a 9 ml YNB agar overlay (containing 0.5 M sorbitol, pH 3.0: 1% w/v agar) and inoculated onto YN8 (containing 0.5 M sorbitol, pH 3.0) agar plates. The lower pH of the medium limited the size of transformant colonies. The plates were incubated at room temperature for 2-3 days before colonies were counted. Single Colony Isolation of 9ucor Transformants: Sporangiospores of the putative Mucor transformants were harvested with 200ul ice-cold distilled water by scraping the surface of the mycelia of each single isolated colony with a sterile loop. 45 The harvested spore aliquots were diluted with YNB broth (1 to 10°) and inoculated onto YNB agar plates which were incubated in an anaerobic jar (88L GasPak-anaerobic system) at room temperature for about 5 days to generate isolated yeast colonies. Four colonies of each transformant were picked and reinoculated onto YNB medium to produce sporangiospore stocks. Isolation of Genomic and Plasmid DNA from 9990r: DNA was isolated from 99999 strains as described by Van Heeswijck (1984) with the following modification. Fresh sporangiospores (107 spores/ml) were inoculated into 300 ml of YNB (pH 4.5) minimal broth at 28°C with constant shaking. The germinated mycelia, 12 to 15 spore diameters in length, were harvested by filtration through filter paper (Whatman 1 mm), frozen in liquid nitrogen and ground to a powder with a mortar and pestle. The ground cells were suspended in 8 m1 TES buffer ( 100 mM Tris.Cl [pH 8.0] , 150 mM NaCl, 100 mM EDTA and 0.1% SDS) and mixed for one minute at room temperature. An equal volume of phenol equilibrated with TES buffer was added and mixed gently for 1 hour on a rocker platform. The aqueous phase was separated from the organic phase by centrifugation (3000X g, 10 min at.4°C) and the aqueous phase was recovered. This process was repeated. The aqueous phase was extracted with an equal volume of 46 phenol (equilibrated with TE buffer)/chloroform:isoamyl alcohol (ZS/24:1) by mixing gently for 30 min on a rocker platform. After the aqueous phase was recovered, the DNA was precipitated with two volumes of ethanol for 30 minutes at ~20%:. The precipitated DNA was collected by centrifugation (12000x g, 20 min at 4°C) , dried under vacuum, and dissolved in 1 ml of TE buffer. RNase (50 ug/ml) was added to the solution which was incubated for 2 hours at 37°C followed by addition of proteinase K (100 ug/ml). The incubation was continued for 2 hours at 37%:. The DNA mixture was extracted with an equal volumes of phenol (saturated with TE) and chloroform:isoamyl alcohol (ZS/24:1) by mixing for 5 min on a rocker platform. The aqueous phase was recovered by centrifugation (3000X g, 10 min at.4PC), and the DNA was precipitated by addition of NaOAC (final concentration is 0.25 M) and two volumes of ethanol and stored overnight at -20°C. The DNA was collected by centrifugation and vacuum dried. This DNA pellet was dissolved in TE buffer and quantitated by measuring the absorbance of the solution at 260 nm with a Spectrophotometer. An 00260 of 1 corresponds to approximately 50 ug/ml for double-stranded DNA. A pure preparation of DNA has ODzw/ODZ.80 of 1.8. 47 Restriction Bndonuclease Analysis and Gel Electrophoresis of DNA: DNA was digested with restriction enzymes according to the conditions recommended by the manufacturer. The DNA fragments were separated according to size by electrophoresis through agarose gels (0.8 to 1.5 %) together with lambda-DNA/HindIII size marker. Gels were stained with ethidium bromide (0.5 ug/ml) and photographed by transillumination with UV light. DNA restriction fragments for use in cloning or for preparing DNA probes were purified by agarose gel electrophoresis followed by electroelution using an apparatus from International Biotechnologies, Inc. (IBI). Preparation of RNA from 99999: RNA was extracted from 99999 cells by the hot phenol method described by Maramatsu (1973). Fresh sporangiospores (107 spores/ml) were inoculated into 300 m1 of YNB (pH 4.5) minimal broth at 28°C with constant shaking. The mycelia, which were 12 to 15 spore diameters in length, were harvested by filtration through filter paper (Whatman 1mm), frozen in liquid nitrogen and ground to a powder with a mortar and pestle. The ground cells were transferred to a 48 cold 30 m1 corex tube and suspended in 1 ml of cold SDS-RNA- x buffer (50 mM NaOAC, 1 mM EDTA, 1% SDS, pH 5.0) treated with diethyl pyrocarbonate (DEPC). Four ml of hot SDS-RNA-X buffer (65°C) was added to the solution followed immediately by 5 ml of hot phenol saturated with RNA-X buffer (65%». The mixture was vortexed for 30 sec. The extraction mixture was incubated for 15 min at 65°C . The aqueous phase was recovered by centrifugation (4300x g, 5 min at room temperature) and reextracted with an equal volume of hot phenol saturated with RNA-X buffer. The aqueous phase was recovered and extracted with one volume of phenol and chloroform:isoamyl alcohol (ZS/24:1). The aqueous phase was recovered, extracted with an equal volume of chloroform : isoamyl alcohol (24:1) and then extracted two times with one volume of diethyl ether (water saturated). The upper phase (ether phase) was removed. The RNA was precipitated from the aqueous phase by addition of l/6 volume of 3 M NaOAC and 2.5 volumes of ethanol and stored overnight at -20°C. The RNA was recovered by centrifugation (12000x g, 10 min at 43C) and the pellet was washed with 70% ethanol. The RNA pellet was dried under vacuum and dissolved in H20 treated with DEPC. The concentration and purity of RNA was measured with a spectrophotometer. An ODZ,so of 1 corresponds to approximately 40 ug/ml RNA. A pure preparation of RNA has ODzw/OD2130 of 2 . The RNA samples were stored at -70°C in a freezer. 49 Gel Electrophoresis of RNA: RNA was denatured by treating the sample with formamide/ formaldehyde and size fractionated by electrophoresis through 1% to 1.5% (w/v) formaldehyde- agarose gels (Maniatis et al., 1982, Fourney et al., 1988) with a RNase-free Mops/EDTA buffer system (10x stock solution: 0.2 M Mops [3-(N-morpholinolino)-propanesulfonic acid], 50 mM sodium acetate, 10 mM EDTA, pH 7.0: treated with diethyl pyrocarbonate (DEPC) at least 12 hr and then autoclaved). After electrophoresis, the lane containing the RNA size standards was separated from the rest of the gel and stained in a solution containing 10 mM MgSO, and 0.5 ug/ml ethidium bromide for 45 min. The excess ethidium bromide and formaldehyde were removed by soaking the gel in 1 mM MgSO, solution for 1 hour. The image of RNA ladder was photographed by transillumination with UV light. Southern and Northern Analyses of Nucleic Acid: Identification of similarities between sequences of DNA was accomplished by the hybridization technique described by Southern (1975). DNA fragments were separated according to size by electrophoresis through an agarose gel, denatured with Southern base, and neutralized with Southern neutralizer. The denatured DNA fragments were transferred 50 and immobilized to a nitrocellulose filter (BA 85, from Schleicher and Schuell FRG) using the protocol of Southern (1975) described in Maniatis (1982). A 10X SSC buffer system was used to transfer DNA by capillary action. The filter with transferred DNA was air dried and baked for 2 hours at 80°C under vacuum. The quantities of specific RNA transcripts were analyzed by Northern analysis (Maniatis, 1982). RNA was denatured with formamide/formaldehyde and size fractionated by formaldehyde-agarose gel electrophoresis. The gel was prepared for transfer by soaking it for two 20 min periods in 10X SSC solution at room temperature with gentle shaking. The RNA was transferred to a nitrocellulose filter using the same protocol as for Southern blotting. DNA fragments which were used as probes were labeled with a4RP-dGTP by the random primer method (Feinberg, and Vogelstein, 1983). Nitrocellulose filters carrying target nucleic acid were hybridized to the denatured gene—specific lap-labeled DNA probes. Filters were prehybridized for 2 hours in prehybridization solution (6X SSC, 5X Denhardt's solution, 50% formamide, 100 ug/ml of denatured salmon sperm DNA, 0.1% SDS and 5 mM EDTA). An appropriate amount of probe (1-5 x 10° cpm/ml) was added and hybridizations were carried out at 429C for the required hybridization period 51 (usually 16-24 hr). After hybridization, the nitrocellulose filter was washed twice in 1X SSC/0.1% SDS for 15 min and once in 0.1x SSC/0.1% SDS for 1 hr at 42°C for low stringency or 65°C for high stringency. The washed filter was air dried, sealed with Saran Wrap, and autoradiographed with Kodak x-ray film with or without an intensifying screen for the required exposure time. The position of target bands complementary to the radioactive probe was observed by soaking the film in Kodak developer for 5 min and rapid fixer for 5 min. Nuclease-81 Mapping of RNA: Nuclease-81 mapping was used to map the ends of RNA molecules and the location of any splice points in relation to specific restriction sites within the template DNA. Total RNA (100 ug) was mixed with 0.1 ug template DNA, and the nucleic acids were coprecipitated with ethanol at -70%: for 15 min. The nucleic acid pellet was resuspended in 0.03 ml of hybridization buffer (40 mM PIPES [piperazine-N, N'- 5 bis {2-ethanesulfonic acid}] [pH 6.4], 1 mM EDTA [pH 8.0], 0.4 M NaCl and 80% formamide) and heated to 80°C for 10 min to denature the DNA. The tubes were transferred rapidly to a 49°C water bath and incubated for 3 hours. After incubation, 0.3 ml of ice-cold 81 nuclease buffer (0.28 M NaCl, 0.05 M sodium acetate [pH 4.6] and 4.5 mM ZnSQd 52 containing 400 units of S1 nuclease was added. The tubes were mixed well and incubated at 20°C for 30 min followed by 30 min at.4PC. The DNA/RNA hybrids resistant to $1 digestion were precipitated with 2.5 volumes of ethanol after addition of 10 ug of yeast tRNA. The precipitate was dissolved in TE buffer and resolved on a neutral agarose gel. Gels were further analyzed by Southern analysis as described above. fi-Galactosidase Assay: Fresh sporangiospores (107 spores/ml) were inoculated into 300 ml of YNB (pH 4.5) broth at 28°C with constant shaking. The mycelia, 8 to 10 spore diameters in length, were harvested by filtration through a filter paper (Whatman 1 mm), frozen in liquid nitrogen and ground to a powder with a mortar and pestle. The ground cells were resuspended in 1 ml of Z buffer (per liter, 16.1 g of NazHPO‘JHZO, 5.5 g of NaH2P04.H20, 0.75 g of KCl, 0.246 g of MgSO4.7H20, pH adjusted to 7.0, autoclaved, cooled, then 2.7 ml of Z-mercaptoethanol was added ). Three drops (pasteur pipette) of CHCl3 and 2 drops of 0.1% SDS were added. The cell suspension was vortexed at high speed for 10 sec. A 0.2 ml aliquot of ONPG (o-Nitrophenyl-fi-D-ga1actoside, 4 mg/ml solution [dissolved iJ)I§O]) was added to samples which were incubated at 28%: for up to 2hr. The reaction was stopped by adding 0.5 m1 of 53 1 M Na2C03, and the cell debris was removed by centrifugation (7000x g, 5 min). The enzyme activity of the reaction mixture was determined by spectrophotometry (ODQD). Because of the low level of fi-galactosidase activity observed in extracts, the protein concentration of the extract was not determined (J.H. Miller, ed., 1972). RESULTS (1) TEFl/lacz Fusion Plasmid: Gene fusions were initially constructed between the Hugo; TEFl promoter and the lacz gene of £.§gli to measure the activity of this Huge; promoter. TEFl encodes elongation factor la and was predicted to generate large quantities of the fusion transcript. The construction of the original fusion plasmid pMF14 is outlined in Figure 4 (pp.55. 56). Since pMFl4 did not contain a selectable marker for transformation of nuggr, the 8.1 kb Sal; restriction fragment containing the TEFl/lacz gene fusion was gel purified from a restriction digest of plasmid pMF14 and ligated into the S311 site of pLeu4. Plasmids with two orientations of the TEFl/lacz fusion in pLeu4 were obtained after ligation and called pMF25 and pMF29. Hugo; racemosus ATCC 1216b R78 was transformed with pMF25 and pMF29 in order to demonstrate the function of the TEFl promoter and the expression of lacz gene in Mugg; cells. The transformants grew when transferred to fresh minimal media (YNB). The above work was conducted by Ms. Fumin Chiu. 54 55 Figure 1. Construction of plasmid pI‘lF14. The regions representing particular DNA fragments are labeled: . TERI. promoter region; lllllllllllllll. ampicillin resistance gene; . lac operon. 56 H H H mHHHmHfi DROP-log Gnome a ”$f?$?? T lac SmaI HpaI EcoRI EcoRI H H J m H H H“ I 042(9—10 0 O) O 6 0 Hm4mm Bani-II '1“ 1 means 10.0Kb EcoRI N; I” 11% Ligase H H mHHHm cacao £53235 TEF1 BamHI AmpR . r___J HpaI/SmaI Sell 57 Sou e a o ted from ucor Tr sformants Genomic DNA was extracted from Mucor racemosus ATCC 1216b R7B transformed with plasmids pMF25 and pMF29. The DNA was digested to completion with the restriction endonucleases gal; and 39931. The E9931 digest was performed to distinguish between the two possible orientations of TEFl/lacz gene insert (see restriction maps, Figure 5, 6, 7, pp.58, 59, 60, 61, 62, 63). The DNA restriction fragments were separated by electrophoresis through a 0.8% agarose gel and transferred to a nitrocellulose filter. A 6.2 Kb gal; fragment containing the lacz gene was gel purified from a §§ll restriction digest of plasmid pMC1843, radioactively labeled with 3’ZP, and used to hybridize to the nitrocellulose filter for 16 hours. The filter was washed under high stringency conditions and exposed to a X-ray film. The lacz gene probe hybridized to a 8.1 Kb gall restriction fragment and to a 11.2 Kb £993; restriction fragment in genomic DNA extracted from the putative pMF25 transformant. The lacz gene probe hybridized to a 8.1 Kb gall fragment and to a 8.1 Kb £993; fragment in DNA extracted from the pMF29 transformant. The lacz gene probe did not hybridize to DNA isolated from the nontransformed host strain or pLeu4 transformants. This data confirmed the entry of plasmids pMF25 and pMF29 into the host strain during transformation. 58 Figure 5. Construction of plasmids pI‘II'ZS and 1:11:29. The regions representing particular DNA fragments are labeled: , TEN. promoter region; lllllllllllllllll . ampicillin resistance gene (from pIIC1843); . lac operon.“ . ampicillin resistance gene (from pLeu4); If '''''''''''' J 1&9; ms; — . £111 0 Leul. SalI EcoRI S9 ;\\\\\\\\\\\‘ TEFl SalI 60 Figure 6. Restriction map of plasmid pMFZS. 61 NE. Baum mmmw. “mm E a: ems... 33m Hmsmnmfimm Efim N2. . seems... Nam cream swam \ a masc Hum» asvv.i_euce< mmmv 3mm . :mm memos nvnm.___u::{ an gamma mwaza ow_ 82m 38m 30.. meow. Es. mmmm A a? H .....e mac omme is...“ name scents: : 3.3 may. .. mN: Ema . madman .Hwhwwm 2 mm gamma H Hymn. 62 Figure 7. Restriction map of plasmid pMF29. 3 6 wmafizgm mm? an... amen. warm Asa. 83. H _ :EE wmmv 3mm nqnmgzfii an comm :mm .mva . mmuza 93m 33m 33 88.5% mmmm .. flaw Em: o ....d , mmq \\ . 33 8 E Emmm mmmmfi Em: HZENI .. E x285...“ wsmme Ema “3.x. .. Esme Emma mN: new? 1, H65". wag; mam ESQ $20 mama 8mm 2.35%. Hmuum.®mama H .wal 21:... vmamfi 2m... gamma 64 Plasmid pUC8 was also used as a probe in Southern analysis because it is nearly identical to the pUC13 backbone of the plasmid pLeu4 (containing the Leul gene), and has no homologous sequence in the host strain, M399; raggmggus ATCC 1216b R7B. Therefore, only DNA extracted from the transformants containing pLeu4, pMF25 or pMF29 was expected to hybridize with this probe. In DNA extracted from pMF25 transformants, the pUC8 DNA probe hybridized to a 7.1 Kb Sal; restriction fragment and to a 3.9 Kb £993; restriction fragments. In DNA extracted from the pMF29 transformant, the pUC8 probe hybridized to a 7.1 Kb gal; restriction fragment and a 3.9 Kb £993; restriction fragments. No hybridization was detected in DNA extracted from the nontransformed host strain DNA or the pLeu4 transformant DNA (Figure 8, pp.65, 66). The explanation for this unexpected lack of hybridization to DNA extracted from pLeu4 transformants was because the pLeu4 transformant was grown under conditions without selective pressure which resulted in lower levels of plasmid DNA compared with DNA from pMF25 and pMF29 transformants which were grown under selective pressure. Since approximately equal amounts of DNA were loaded in all lanes, the plasmid pLeu4 is underrepresented and did not show up upon a short exposure of the autoradiography. In a previous experiment a 7.1 Kb gall band was present after longer exposure of the Southern blot (Figure 8, pp.65, 66). Because these putative pMF25 Figure 8. Panel A Panel B 65 Southern analysis of genomic DNA isolated from H- ragemosus R7B, pLeu4 transfo ants, pMF25 and pMF29 transformants using a 2P-labeled 6.2 Kb gall fragment (lacZ) and pUC8 fragment as probes. Lane 1: Genomic DNA (20 ug) of pLeu4 transformant was digested with Sall. The restriction fragments were separated by electrophoresis on a 0.8% agarose gel, transferred to a nitrocellulose filter and hybridized with aznP labeled pUC8 probe. The filter was washed at high stringency and exposed to X-ray film for 3 days at -70°C. Genomic DNA (20 ug) was digested with gall and Ecagl endonucleases. The restriction fragments were separated by electrophoresis on a 0.8% agarose gel, transferred to a nitrocellulose filter and hybridized with a 32P-labeled 6.2 Kb gall fragment containing the lacZ gene (from plasmid pMC1843) and pUC8 probe. The filter was washed at high stringency and exposed to X-ray film for 22 hours at room temperature. Lane 1 and 6: genomic DNA of u. raaamaaaa R7B digested with gall and £9931 respectively. Lane 2, 3, 7 and 8: genomic DNA of two different pLeu4 transformants digested with gall and EcoRl respectively. Lane 4 and 9: genomic DNA of pMF25 transformant digested with gall and gcaRI respectively. Lane 5 and 10: genomic DNA of pMF29 transformant digested with SalI and EgoRI respectively. 66 «7.1 as 4.36— I 2.32» 2.03— 67 and pMF29 transformants could grow on YNB minimal medium Combined with the result of Southern analysis suggested that the plasmids pMF25 and pMF29 were stable inside the host strain under selective growth conditions. Since the probes hybridized to the predicted restriction fragments, there were no obvious gene rearrangements after the plasmids were transformed and propagated in the Hagar host strain. Northern Aaalysis of RNA gatraagea from Nuco; Transformants- 25 2 Northern analysis was carried out to detect the level of'transcription from the putative TEFl promoter. RNA was extracted from the transformants. RNA samples (20 ug) were separated by electrophoresis on a 1% agarose-formaldehyde denaturing gel, transferred to a nitrocellulose filter, and hybridized to a 6.2 Kb lacZ gene probe (Figure 9, pp.68, 69). The probe hybridized to a heterogenous pepulation of transcripts in RNA extracted from pMFZS transformant (lane 3) after 17 days of exposure but not to the other RNA samples. This hybridization data suggested that transcription of the lacZ gene occurred inside the M222; host transformed with pMF25 but not pMF29. But, the level of transcript accumulation was so low that it was detectable only after a long exposure time (17 days). One possible explanation for this data is that there was no eukaryotic Figure 9. 68 Northern analysis of total RNA purified from u.racem0§as R7B, a pLeu4 transformant, a pMF25 transformant and a pMF29 transformant using aznP labeled 6.2 Kb gall fragment containing the lacZ gene as the probe (from pMC1843). Total RNA (20 ug) was denatured with formamide/ formaldehyde, separated by electrophoresis on a 1% agarose formaldehyde gel, transferred to a nitrocellulose filter, and hybridized within? labeled 6.2 Kb gall fragment (lacZ). The filter was washed at high stringency and exposed to X- ray film for 17 days. Lane 1: total RNA of M. racemosas R7B. Lane 2: total RNA of pLeu4 transformant. Lane 3: total RNA of pMF25 transformant. Lane 4: total RNA of pMF29 transformant. 69 2.80 1.80 70 transcription termination signal included in plasmids pMF25 and pMF29 so transcription resulted in many sizes of mRNA. The lack of a processed 3' end of the transcript may also have contributed to the low level of transcript possibly because of mRNA instability. Because the data from Northern analysis were unclear, Sl Nuclease analysis and B- galactosidase assays were performed to detect transcription from the TEFl promoter, and functional expression of a lacZ protein (8 galactosidase). 81-Nuclease Mapping of 33A Bgtraatad from Transformants Nuclease-$1 mapping was used to localize the start site of transcription of the TEFl/lacz gene fusion. Total RNA was isolated from the Hagar transformants. The 3.2 Kb gaal fragment containing the TEFl promoter region was gel purified from a restriction digest of plasmid pMF25 and used as the target DNA. Total RNA (100 ug) of each transformant (n.raaaaaaaa transformed with pLeu4, pMF25, pMF29) and the nontransformed host strain (3.;agamgaaa ATCC 1216b R7B) was mixed with 0.1 ug of the 3.2 Kb template DNA. The nucleic acids were coprecipitated, heated to denature the DNA and allowed to hybridize and incubated with $1 nuclease. The DNA/RNA hybrids resistant to SI digestion were resolved on a 1% agarose gel, transferred to a nitrocellulose filter and hybridized to the 6.2 Kb lacz gene probe (Figure 10, pp.71). Figure 10. 71 Sl-nuclease analysis of total RNA purified from M- racemosus R7B, a pLeu4 transformant, a pMF25 transformant, and a pMF29 transformant. Total RNA (100 ug) was incubated with the 3.2 Kb gaal fragment containing the TEFl promoter region (from pMF25) under conditions favoring RNA/DNA hybridization. The nucleic acids were coprecipitated, heated to denature the DNA, allowed to hybridize, and incubated with 81 nuclease. The DNA/RNA hybrids resistant to $1 digestion were resolved on a 1% agarose gel, transferred to a nitrocellulose filter and hybridized to a‘nP-labeled 6.2 Kb lacz gene probe. Lane 1: total RNA from n. racemgsas R7B plus the 3.2 Kb Sacl DNA fragment. Lane 2: total RNA from pLeu4 transformant plus the 3.2 Kb gagl DNA fragment. Lane 3: total RNA from pMF25 transformant plus the 3.2 Kb gaal DNA fragment. Lane 4: total RNA from pMF29 transformant plus the 3.2 Kb SagI DNA fragment. Lane 5: tRNA plus the 3.2 Kb Sacl DNA fragment. Lane 6: total RNA from pMF25 transformant. Lane 7: 3.2 Kb Sacl DNA fragment. 72 73 A similar band pattern was present in RNA from pMF25, pMF29 transformants (lanes 3, 4) and from a pLeu4 transformant (lane 2) which did not contain the TEFl promoter region or lacz gene. In addition, the size of this band (z 3.2 Kb) was similar to the size of the DNA template but not to the size of expected fragment (1.9 Kb) if the TEFl promoter was functioning. These data suggested that the lacz gene probe hybridized to target DNA reannealed to itself and not with the target DNA protected by the fusion transcript. These data also suggested that either the fusion transcript detected in pMF 25 transformants was subject to degradation in the Hagar cells or was produced in insufficient levels to detect by S-l analysis. -Galacto a a Cell extracts of the pMF25, pMF29, and pLeu4 (control) transformants were analyzed to detect expression of 8- galactosidase activity by using a B-galactosidase enzyme assay. The level of B-galactosidase activity detected in transformants was not consistently higher than control cells in which there appears to be a low level of endogenous B- galactosidase activity. Since we were unable to conclusively demonstrate function of the TEFl promoter with the lacz fusion, further experiments were conducted to investigate the function of the TEFl promoter with a 74 different reporter gene, the human growth hormone gene (hGH). (2) TBFl/han Fusion Plasmids: Q2natrnsti2a.21_Qene_znaien_zlaamida A gene fusion was constructed between the Hagar promoter of TEFl, and the hGH (human growth hormone) gene in order to detect the level of transcription from the TEFl promoter. The construction of this model expression vector is outlined in Figure 11 (pp.75, 76). The backbone for this construct was pMR1-3, a plasmid consisting of a 1.9 Kb EgaBl-Haal restriction fragment containing the putative TEFl promoter sequence plus the first 8 amino acids from the EF- la protein, a pUC13 vector, and a Hagar leucine biosynthetic gene with a putative Hagar ARS (autonomous replication sequence). The plasmid pMRl-B was cut with gall endonuclease and the restriction sites were filled in with Klenow fragment of DNA polymeraseI to produce blunt ends. A 1.8 Kb Aagll fragment containing the hGH gene was gel purified from a restriction digest of plasmid pOGH and the restriction sites of this fragment were treated with T4 DNA polymerase to generate blunt ends. This blunt-ended Aagll fragment was ligated into the blunt ended gall site of pMRl- ' figure 11. '75 Construction of plasmid pIIYY1. The actual manipulations required for this construction are described in detail in . Results. The plasmids pun-3 and p063 are described in.neterials and Methods. The regions representing particular DNA fragments are labeled: , ‘I‘EFi promoter region; , ampicillin resistance gene: L; ------------ fl , IIuaor ARS; _ , hucor Leul; m , hGH (human growth hormone) gene. 76 H % HpaI/SmaI ;: BamHI H \ XbaI ‘3‘ ‘3'; SalI c>n hhn‘n TEFi PstI BamHI A/atII ~ gxnucor,uks ‘ If an1——3 nucor Leul SalI + AatII + , Klenow fragment T4 DNA.polymerase of DNA polymerase SalI XbaI $811 BQEHI l‘u o SmaI IHpaI 21.9.: ARS SmaI\ 395:; “El." SdCI\ WK SmaI/EcoRI MtII— 1’ 8Kb AatII SstI EcoRI HindIII PstI 1% DNA Ligase SalI/Antlfi 12671 AatII/SalI TEF1nucdrARS SmaI/EcoRI SstI 7 ROM ,- Hagar Leui AmpR \— HindIII PstI 77 3 adjacent to the TEFl promoter. This fusion generated a continuous open reading frame between the ATG initiation codon plus the first 8 amino acids of TEFl gene and the hGH gene beginning with amino acid number seven of the signal peptide of the pre-hGH protein. The transcription termination signal was therefore provided by the hGH gene while the transcription initiation signal was provided by the TEFl gene (Fig. 12, pp.78, 79, 80). an a s o s t d as 'ds Newly constructed plasmids were transformed into E. gali DH 5a to propagate the plasmid molecules. Transformants, thought to contain the desired recombinant plasmid, grew on LB plates supplemented with ampicillin (50 ug/ml) and were further identified by the colony hybridization procedure with a 32P-labeled 1.8 Kb gagil hGH gene probe (Fig. 13, pp.81). Fifteen of 330 colonies were found to contain hGH gene inserts (z 5% ligation efficiency). After single colony isolation, plasmid DNA containing the hGH gene was obtained from g. gall clones using the "miniprep" method. Samples of the resulting plasmid DNAs were digested with ggag; and gag; restriction endonucleases and analyzed by electrophoresis through a 1% agarose gel. gag; endonuclease was expected to distinguish between the two orientations of the insert by the generation 78 Figure 12. Construction of plasmid pMYYl. (nucleotide sequence) 79 pIIRi-3 -GGT AAA GAG AAG ACT CAC MT 666 GM cc'r CTA GAG 'rce Acc TAC CCA mm are we TGA G'I'G CAA ccc c'rA GGA cm are AGC TGG 3' S' l SalI 5' TEFI ' l 3' GGT AAA GAG AAG Ac'r CAc GTT 666 GM cc'r CTA GAG—1 'rce Acc TAc CCA TM are TTC TGA are CAA ccc CTA 665:6!“ c'rc Ag TLQG 3. I 5' Klenow fragment of DNA polymerase 5' TEFI 3' -eeIr AAAGAGAAsAc'r CAc GTTGGGGAT cg c'rAGAe-rcea 'rAc CCA 'r'r'r owe me rGA are CAA ccc on sea GAT at ABC 1' 3' 5- pOGH 5' 3. TCC CGG ACG TCC CTG C'l'C CTG G’I‘C GCT .......... TTTTGTCTGA AGG GCC TGC AGG GAC GAG GAC CGA CGA .......... AMACAGACT 3' 5' 11mm 5' 1 3' TCC CGG ACG C CTG CTC CTG arc GC'I' .......... TTTTGTCTGA AGG GCC TGC AGG GAC GAG GAC CGA CGA .......... AAAACAGACT 3' I 5' 1T4 DNA polymerase 5' 3' CC CTG CTC CTG Gd'C GCT ......... TTTTGTCTGA 66 GAC GAG GAC CGA CGA ......... AAAACAGACT 3' 5' 80 3 O 5. pIIYYi 5' prim-3 P°GH -. . . .cc'r CTA GAG mm A] [cc CTG c'rc CTG GCT. . . .TTTTG’I‘CTGA TAC....GGA an we we 'r “ GGGAC GAG GAC CGA....W flet Pro Leu Glu Ser Leu Leu Leu Ala 30 T4 Ligase 5| -. . . .cc'r CTA GAG TCG Acc CTG c'rc CTG GCT. . . .TTTTG’I‘CTGA 'rAc....GGA GA'r c'rc AGc TGG GAG GAG GAC CGA....AAAACAGAc'r Met Pro Leu Glu Ser Thr Leu Leu Leu Ala 30 Milli 81 Figure 13. Colony hybridization with an hGH gene fragment as probe. E. coli cells were grown on a nitrocellulose filter, lysed, denatured, neutralized and hybridized with the hGH gene probe to identify the cells which contained hGH gene inserts. Filters were washed under high stringency conditions and exposed to X-ray film for 2.5 hours at room temperature. "+" represents the colony containing the hGH gene insert as a positive control. "-" represents the colony containing plasmid pLeu4 as a negative control. 82 of three DNA fragments (8.7 Kb, 1.5 Kb, 0.6 Kb) in one orientation (in frame fusion) and three unique DNA fragments ( 7.3 Kb, 2.9 Kb, 0.6 Kb) in the other orientation. Restriction endonuclease analysis showed that two out of ten clones contained the correct orientation of the insert, 0 out of ten clones contained the insert in the opposite orientation and eight out of ten clones contained multiple inserts in several combinations. The two clones thought to contain the correct fusion construct were selected to perform detailed restriction analysis with different endonucleases including ggaBI, gagl, gaal, gaal & gagi, gaaH; and Hiaalgl (Fig. 14, pp.83, 84) to confirm the proper plasmid construction. The predicted restriction map (Fig. 15, pp.85, 86) of this constructed plasmid (named pMYYl) was confirmed according to the data of the restriction analysis. Plasmid pMYYl was amplified using the large-scale protocol and isolated by cesium chloride density gradient centrifugation. The purified plasmid DNA was used to transform protoplasts of Hacor ragemosus R7B. Figure 14. 83 Restriction analysis of plasmid pMYYl Plasmid DNA of pHYYl was digested with different restriction endonucleases and the restriction fragments were separated by electrophoresis on a 1% agarose gels. Lane 1: undigested plasmid pMYYl. Lane 2, 3, 4, 5, 6, 7: plasmid DNA of pMYYl digested with £9281. 539.11. $199.11. $221 and gaal, gaaHI and Hiaglll endonucleases respectively. 84 234567 1 85 Figure 15. Restriction map of plasmid pMYYl. 86 Ban? .. H H wail .wmwmfiH mel seem. a H :35 smfimgmuum ammw. $5.. 33 ssmmLcaa 9mm... E .< . @3115 Ema Zest: 39%.? mwss _Gsx 88.1.5.1 5mm Efim H Emu .88 mass. :0... mm? F E £23....de am...» .mmme an mamas size 1 Gem .mmmm Hume . é Efim mmmm H oEm\ “MRI . wwmm Io; Esme. smmm less mmmm . _ 5812...... mmmm l 74%”... «vmdm .. .33...» vwmm Salem vmmm Es. was? mmq .amm 23$: mm. amass H. ragaaaaag ATCC 1216b R7B was transformed with the model expression plasmid pMYYl to demonstrate the function of the TEFl promoter by measuring the expression of the hGH gene in Hagar cells. Approximately 50% of the sporangiospores of H. ragamagaa R7B inoculated in YPG broth germinated after 5.5 hours of incubation (3-5 spore diameters). Protoplasts were generated (about 40% of germlings) after 1 hour of cell wall digestion with Chitosanase and Novozyme 234. The protoplasts were transformed with the plasmid pMYYl. Eight putative transformants were obtained after 2 - 3 days growth on YNB (pH 3.0) minimal medium (without leucine). The protoplasts of H. raggaosus R7B were also transformed with pMR1-3 containing no hGH gene as the control. Two putative transformants were obtained after 2-3 days growth on YNB (pH 3.0) minimal plates. The frequency of protoplast regeneration of H. ragaaagag ATCC 1216b R7B was 40%. The frequency of transformation of pHYYl was 10 colonies/ug DNA/106 protoplasts, and the frequency of transformation of pMR1-3 was 2.5 colonies/ug DNA/10‘5 protoplasts. After single colony isolation, spore stocks were produced from four of the putative pMYYl transformants and two of the putative pMR1-3 transformants. 88 8o t n Anal 5 s of traoted r uc Transformants Genomic DNA of four putative pMYYl transformants and one pMR1-3 transformant (control) was extracted and digested completely with different restriction endonucleases (10 ug, per lane). DNA restriction fragments were separated by electrophoresis through a 0.8% agarose gel and transferred to a nitrocellulose filter. The 1.8 Kb Aa§;; fragment containing the hen gene was radioactively labeled with 32P and used to hybridize to the nitrocellulose filter for 23 hours. The filter was washed under high stringency conditions and exposed to X-ray film (Fig. 16, pp.90, 91). The 1.8 Kb Aag11_hGH gene probe hybridized to a 2.4 Kb gaaH; restriction fragment and to an 8.2 Kb Hiaggil fragment in DNA from all 4 putative pMYYl transformants. In the control transformant (pMRl-B), one very high molecular weight band was present on the X-ray film after a 23 hr exposure. One possible explanation for this unpredicted band is the non- specific binding of the probe to catenated pMR1-3 plasmids. Although the result of the control transformant was not as we expected, the data from Southern analysis confirmed the entry of pMYYl into the Hagar host strain during transformation. The same nitrocellulose filter was reprobed with a 1.9 Kb HinngI fragment from the pMR1-3 vector containing the 89 Leul gene (without removing the original hGH probe). The Leul gene probe hybridized to two gang; fragments (2.1 Kb, 6.0Kb), to one Hiaalll fragment (1.9 Kb) in DNA isolated from pMYYl transformants and to one xaal fragment (7.7 Kb) in DNA isolated from the pMRl-3 transformant (Fig. 16, pp.90, 91). The ability of these putative transformants to grow on YNB minimal medium combined with the Southern analysis data demonstrated that the plasmids pMYYl and pMRl- 3 were present and stable inside the host strain when grown under selective pressure. Because the hybridization patterns observed in Southern analysis were identical to the restriction pattern of pure plasmid DNA, no obvious gene rearrangements occurred during transformation or propagation in the Hagar host strain. Nort e a s s t ed on ucor ransformants Northern analysis was performed to detect the accumulated hGH transcript level to demonstrate the function of the putative TEFl promoter. Total RNA was prepared from cells transformed with pMYYl and pMR1-3. RNA samples (20 ug) were separated by electrophoresis on a 1% agarose- formaldehyde gel, and transferred to a nitrocellulose filter. The 1.8 Kb Aagll hGH gene fragment was radioactively labeled within? and used to hybridize to this Northern blot. After two non-specific washes and one high Figure 16. 90 Southern analysis of genomic DNA isolated from four pMYYl transformants and p 1-3 transformant (control) using a P labeled 1.8 Kb Aagll fragment (from pOGH) and 1.9 Kb Hiaalll fragment (from pMR1-3) as probes. Genomic DNA (10 ug) was digested with different restriction endonucleases. The restriction fragments were resolved by electrophoresis on a 0.8% agarose gel, transferred to a nitrocellulose filter and hybridized with a 32F labeled 1. 8 Kb Aarll fragment containing hGH gene (for Panel A), and a 1.9 Kb Hiaglll fragment containing the Leul gene (without removing original hGH probe: Panel B). The filter was washed at high stringency and exposed to X-ray film for 23 hours (Panel A) and 48 hours (Panel B) at room temperature. Lane 1, 2 of Panel A and Panel B: genomic DNA of pMRl-B transformant digested with Haa; endonuclease. Lane 3, 4, 5, 6 of Panel A and Panel B: genomic DNA of four pMYYl transformants digested with gaaHI endonuclease. Lane 7, 8, 9, 10 of Panel A and Panel B: genomic DNA of four pMYYl transformants digested with Hia_111 endonuclease. 91 A Xbal BamHl Hindlll 1 4 5 6‘ J 8 910 2.32-3 "’ "' " " B Xbal BamHl_I ,Hindlll , r12"3456'78910' 92 stringency final wash, the filter was analyzed by autoradiography (Fig. 17, pp.94, 95). After 9 days of exposure, several high molecular weight hybridization bands (30 - 100 Kb) were present on the lanes loaded with RNA extracted from the pMYYl transformants but not on the lane loaded with RNA from the pMR1-3 transformant. These large hybridization bands did not correspond to the predicted size (z 1.0-1.2 Kb) for the mRNA derived from the hGH gene. One possible explanation for these hybridization data was the lack of function of the hGH gene transcription termination signal inside Hagar cells which resulted in production of high molecular weight, heterogeneous transcripts. Another possible explanation was that the RNA samples extracted from pMYYl transformants were contaminated with plasmid DNA (pMYYl) and resulted in hybridization between concatamers of the plasmid DNA and the hGH gene probe. In order to distinguish between these possibilities and to show that the RNA samples were not degraded, a Leul gene probe was used to probe the same Northern blot. A previous study showed that the size for the mature mRNA derived from the Leul gene was about 2.5 Kb (Wang, S.Y., unpublished) which agrees closely with the size determined by Roncero et al., (unpublished). If only one hybridization band (z 2.5 Kb) appeared in the lane with pMR1-3 transformant RNA in Northern analysis (using Leul gene as the probe) it would mean that the high molecular weight bands observed previously were the result 93 of hybridization of transcripts of the hGH gene to the hGH gene probe. If hybridization to high molecular weight nucleic acid was also present in the lane with the RNA sample prepared from pMRl-B transformant, it would suggest that the previous data was due to plasmid DNA contamination of the RNA sample. The data from this experiment are seen in Figure 17 (pp .94, 95). Several high molecular weight bands were observed in the lane containing the RNA sample from the pMR1-3 transformant. In addition, the probe hybridized to a single transcript of approximately 2,600-nucleotides in lane 1 (containing the RNA sample from the pMR1-3 transformant) and lanes 3,4, 5 (RNA sample extracted from the pMYYl transformants). The size of this transcript corresponded to the expected size for the mature mRNA derived from the Leul gene. One possible reason for the absence of the 2.6 Kb transcript in lane 2 which contained RNA from one pMYYl transformant was because of low quantity of total RNA loaded in lane 2 (10 ug; compared with 20 ug in all other lanes). These data indicate that the large molecular weight hybridization signals resulted from contamination of the RNA samples with plasmid DNA and that the RNA samples were not degraded during the purification procedure. The data presented in Southern and Northern analyses suggested that the TEFl promoter sequence subcloned from the TEFl gene was Figure 17. 94 Northern analysis of total RNA isolated from four pMYYl transformaflts and a pMRl-B transformant using a P labeled 1.8 Kb Aarll fragment containing the hGH gene (from pOGH) and a 1.9 Kb HiaQIII fragment containing the Leul gene (from pMR1-3) as probes. Total RNA (10-20 ug) was denatured with formamide/formaldehyde, separated by electrophoresis on a 1% agarose-formaldehyde gel, transferred to a nitrocellulose filter, and hybridized with a 32p labeled 1.3 Kb M fragment (for Panel A), and 1.9 Kb Hiaglll fragment (without removing original hGH probe: Panel B). The filter was washed at high stringency and exposed to x-ray film for 9 days (Panel A) and 16 days (Panel B). Lane 1 of Panel A and Panel B: total RNA of pMRl-B transformant. Lane 2, 3, 4, 5 of Panel A and Panel B: total RNA of four pMYYl transformants. 95 A12345 B12345 4.40— 4-40 4-2.6 2.37 2.37— 1.35 1 .35— .24 96 not efficient to drive the high level expression of a heterologous gene. DISCUSSION Genetic transformation of Hagar ragaaagag was achieved by incubating protoplasts generated from a leucine requiring strain with recombinant plasmid DNA (pMYYl, pMR1-3, pMF25 or pMF29) in the presence of polyethylene glycol and CaClz. The transformation frequency of H.raggaagag with pMYY-l was 10 transformants / ug DNA / 10° protoplasts which was a much lower frequency than that obtained for pMCL1302 by Van Heeswijck (1984; 600 transformants / ug DNA / 3.2 x 106 protoplasts). This result might due to the fact that the mixtures containing protoplasts and plasmid pMYYl were plated at too high of a cell density or because glucose was inadvertently left out of the growth medium resulting in a decreased cell viability. Because the spontaneous reversion frequency of R7B to Leu+ is< 1.7x10'8 (Van Heeswijck, 1984) and the number of protoplasts used in transformation was so low (< 2 x 10°), the colonies harvested from the YNB medium after transformation were thought to be transformants. This hypothesis was confirmed by Southern analysis which showed that the plasmids pMYYl, pMRl-3, pMF25 or pMF29 were present in the fungal cells after transformation. The data also suggested that there was no obvious genetic rearrangement of the molecules after the plasmids were transferred into host 97 98 cells. There are three possible fates of a plasmid which enters a host cell. A plasmid may replicate autonomously. It may integrate into chromosomal DNA randomly by nonhomologous recombination or it may integrate into chromosomal DNA at specified sites via homologous recombination. The results of our Southern analysis suggested that the plasmids pHYYl, pMR1-3, pMF25 and pMF29 did not integrate into genome of the host cells, but existed as free plasmid molecules. These data agreed with similar results observed by Van Heeswijck (1986); i.e. transformed DNA plasmids are present as discrete extrachromosomal DNA in Hagar ragaaaaag. One experiment which will be useful to confirm our conclusion will be to isolate genomic DNA of Hagar transformants (pMF25, pMR1-3, pMF29 or pMYY-l) and utilize this directly to transform a. gall DH 5a. If the plasmid can be rescued from a. gall cell in its original form, it would support the notion that plasmid pMF25, pMF29, pMR1-3, or pMYYl exist as self-replicating molecules inside the Hugor cells. Other experimental data have demonstrated that pLeu4 transformants tend to lose their plasmids at a fast rate when they are grown without selective pressure (YPG rich medium containing leucine) (Juili Lin, M.S. Thesis). This data also agrees with results reported by Van Heeswijck (1986). Only 5% of viable sporangiospores isolated from 99 pMCL1302 transformants were Leu+ after the first transfer to complete medium, compared with 45% viability when growth of spores was continued under selective conditions (YNB medium). One possible reason for this observation could be that the plasmids do not integrate into genome of the host cell, so they would not be segregated equally to daughter cells during mitosis. Because gene stability is essential to an effective heterologous protein expression system, more data are required to determine the fate of transforming plasmid DNA inside H.ragamaga§ host cells. It would be advantageous from a stability standpoint if a certain percentage of vector molecules do integrate into the chromosome. The following experiment is currently being conducted to test for this possibility. Transformant cells were grown without selective pressure for several generations followed by replica plating of colonies onto selective minimal medium to generate isolated colonies. This procedure was repeated several times. If any plasmid DNA integrated into the genome of the host, it should be maintained stably and detected after inoculation of spores back onto YNB minimal medium. If the plasmids did not integrate into the genome of host, the high level of segregational instability in Hagar (Van Heeswijck, 1986) for cells grown in the absence of selective pressure would cause the cells to rapidly lose their plasmids resulting in the 100 inability to recover cells with the Leu+ phenotype upon plating spores onto YNB. The results of these experiments showed that one clone derived from the pHF25 transformant was stable after several transfers to YNB medium. One possible explanation for these data is that plasmid molecules integrated into the chromosomal DNA. Another explanation is that a wild type H. ragaaagag 1216b contaminant was isolated which resulted in extremely stable Leu+ phenotype. On the other hand, the clone derived from the pMF29 transformant was unable to grow on the YNB minimal plate even before passage on YPG medium (Linz, J., unpublished). The loss of the plasmid pMF29 during manipulations in generating a spore stock may explain the data of Northern analysis in which transcription of the TEFl/lacz gene occurred inside the Hagar host transformed with pMF25 but not in the pMF29 transformant. We must now perform Southern analysis on these clones to determine the location of plasmid DNA. The Hagar TEFl promoter was chosen in our model system because this gene is a constitutive, highly expressed gene encoding elongation factor 1a which is one of the most abundant proteins present in H. ragaaaaaa. Because of the high level transcription of this gene, the TEFl promoter was suspected to be a very strong promoter. In order to 101 determine the function of this promoter, lacz and hGH reporter genes were fused to TEFl promoter. The constructed plasmids were pMF25, pMF29 and pMYYl. The results of Southern analysis confirmed the presence of plasmids pMF25, pMF29 or pMYYl in transformants. The data of Northern analysis suggested that a low level of fusion transcript was produced inside the pMF25 transformant but not in pMF29 or pMYYl transformants. These data lead us to conclude that the TEFl promoter sequence subcloned from the TEFl gene was not efficient to drive the high level expression of the heterologous genes tested in this study. Some promoters seem to be recognized by RNA polymerase in a relatively general mechanism while other promoters are not adequate to support transcription by themselves. Specific ancillary factors such as enhancers are needed for initiation to occur efficiently (Lewin, 1983). We propose here that the TEFl promoter sequence utilized in this study lacks an important recognition site required for specific transcription factors required for initiation of transcription. A previous study (Linz, et al., 1986) determined that EF-la in Hagar raggaagag is encoded by three genes (TEFl, TEF2, TEF3) located at unique positions in the genome. Nucleotide sequence analysis of the genes indicated that these three genes share a high degree of similarity not only in the coding portion of the gene, but also in the 3' 102 intron. All three genes have a conserved intron near the 3' end of the coding region. In addition, TEF2 and TEFB, but not TEFl, each have an intron located near the 5' end of the coding region. Conservation of the nucleotide sequence within the 3' intron suggests that it might play an important role for facilitating transcription from the TEFl promoter. To investigate this hypothesis, the following experiments will be conducted. A portion of the hGH gene open reading frame (a 400-500 bp) will be ligated in frame into the intact TEF1 gene in the coding region downstream from the 3' intron. This construct will be ligated into the pLeu4 plasmid and used to transform H.ragaaagag R7B. Southern and Northern analyses will be carried out on nucleic acid purified from the transformants. If stable transcripts of this gene fusion are detected in Northern analysis using an hGH gene probe, it would suggest that specific nucleotides sequences between the ATG initiation codon and transcription termination signal are needed for the initiation of transcription from the TEFl promoter. Deletion of nucleotides sequences from the TEFl gene could be accomplished using several convenient restriction sites from upstream and downstream of the hGH gene insert to determine the position of important ancillary factors. If the intron is important in facilitating the initiation of 103 transcription and its position is not strictly limited, the nucleotide sequence which contains the promoter region (275 bp nucleotides sequence upstream from ATG initiation codon) and the intron will be used as a promoter for the further experiments. Studies such as heterologous protein expression/secretion in Hagar will be performed. If the presence of exonI or exonII of the TEFl gene is required for the function of TEFl promoter or if the distance between the promoter and the intron is fixed, the TEFl promoter will not be convenient to be a promoter in our model system. Another promoter (for example, Leul promoter) will be considered to perform further experiments. If no transcripts of this gene fusion are detected in Northern analysis using the hGH gene, it would suggest that transcription of the TEFl gene involves sequences at a distance from the gene itself. For example, nucleotide sequences a large distance upstream from the ATG initiation codon may be required in this construct. Experiments such as inclusion of additional nucleotides from the upstream region of the TEF1 promoter sequence could be conducted. Another possible reason that the TEFl promoter may not function is that the autonomously replicating vector lacks genomic context (ie chromatin, nucleosomes structure, etc.) required for TEFl gene expression. SUIHLR! Since the experimental results from the original TEFl/lacz fusion transformants did not conclusively demonstrate function of the Hagar TEFl promoter, this research project was designed to investigate the function of the TEFl promoter with the human growth hormone gene (hGH) as a reporter gene. The hGH gene was inserted into the vector pMR1-3 at a position adjacent to the TEFl promoter. The correct construction of the resulting plasmid pMYYl was confirmed by restriction endonuclease analysis. The plasmid was then used to transform H. ragaaagaa R7B. Plasmid pMYYl was observed to be present in transformants with no obvious DNA rearrangements. However, there was no detectable transcript of the hGH gene produced in Hagar cells. 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