—I—I -_IU1-‘ I ICDNU1 309'! 6 b ‘1 H Li WIWW"?[NW/[WWWWWW LIBRARY Michigan State University This is to certify that the thesis entitled CLONING AND EXPRESSION OF THE RAS HOMOLOGUES 0F MUCOR RACEMOSUS presented by YIH—JIHN LEE has been accepted towards fulfillment of the requirements for _M...S_._degree in Food Science / r / Major professor f Date 7-28-88 0.7639 MS U i: an Affirmative Action/Equal Opportunity Institution MSU‘ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. CLONING AND EXPRESSION O? m m HOMOLOGUES 01' £19.93 W BY Yih-Jihn Lee A THESIS Summitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1988 ABSTRACT CLONING AND EXPRESSION OF THE RAE: HOMOLOGUES 01" MHQQB W BY Yih-Jihn Lee The rag proteins (GTP-binding regulatory proteins) are thought to play a role in signal transduction pathways in eukaryotic cells by regulating the production of "second messengers" via cyclic adenosine 3'5’monophosphate (cAMP) or phosphatidylinositol (PI) metabolism. PI and cAMP metabolism are correlated to the morphogenetic response in Hugo: rams, a dimorphic filamentous fungus. The focus of this research was to characterize the m; Lag genes to initiate our investigation of the molecular mechanisms which regulate morphogenesis. Three DNA restriction fragments were cloned from MI gene libraries by hybridization to Saggharomyces We M gene probes. Southern hybridization data and restriction endonuclease analysis suggest that these 3 clones represent 3 My rag genes (:4 , L18, and E10. Northern blot analysis of Myc_or RNA suggests that the three 113,192]: ms genes are expressed and that one of these genes (L18) is expressed in a morphology-specific manner. I would like to dedicate this work to my parents, Shing-Mole and Swae-Siang Chou Lee, my sisters, and brothers. ii ACKNOWLEDGES I would like to thank Dr. John E. Linz for all of advice, inspiration, and friendship he has given to me during my career in academics. He has help me to increase my knowledge of Microbial Genetics and their applications in Food Science through our discussions and has been a great life and source of stimulation in laboratory. I would also like to thank the members of my graduate committee: Dr. James J. Pestka, Dr. Maurice R. Bennink, and Dr. Everett S. Beneke for their timely advice and constructive criticisms of my work. Finally, I would like to thank my uncles, aunts, and cousins for their loves, support in my work. iii TABLE OF CONTENTS Page LIST OF FIGURES --------------------------------------- Vi INTRODUCTION Bag genes and gas related genes -------------------- 1 Properties of {as genes ---------------------------- 2 Hugo; racemosus: A model system for study of fungal food spoilage, and extracellular enzyme biosynthesis ----------------------------- 5 Correlates of morphogenesis ------------------------ 6 Research hypotheses -------------------------------- 7 Genetic analysis of Hugo; -------------------------- 9 Preliminary data ----------------------------------- 11 MATERIALS AND METHODS Organism, growth media, and culture conditions ----- 12 Spore production ----------------------------------- 12 Germling production -------------------------------- 13 Yeast cell production ------------------------------ 13 Yeast-to-hypha cell production --------------------- 14 Purification of RNA and DNA from M; :acemosus cells ------------------------------ 14 Bacterial strains and plasmids --------------------- 16 Construction of an n; :acemgsgs gene bank in bacteriophage lambda ------------------------- 17 Preparation of bacteriophage DNA ------------------- 17 Radiolabelling of DNA restriction fragments -------- 18 IRAQI. we, and ERAS DNA probes ------------------ 19 Selection and screening of recombinant clones ------ 19 Restriction endonuclease analysis and agarose gel electrophoresis --------------------- 21 Southern and Northern blot analyses of nucleic acids -------------------------------- 21 RESULTS Identification of {as-related sequences in M; racemosus genomic DNA --------------------- 24 Screening lambda/u; racemosgs DNA libraries for gas homologous sequences -------------------- 25 Grouping lambda phage clones by restriction patterns ---------------------------------------- 26 iv Table of contents (Cont.) Page Subcloning of n; raggmgsus [as genes (8C4, 8L18, SE10, and P810) into the EL 9911 plasmid vector, pUC9 ---------------------------- 2? Restriction endonuclease maps of 8C4, 8L18, and P810 sequences ------------------------------ 28 A comparison of the hybridization pattern of original phage clones (C4, L18, and E10) with recombinant plasmids (pHC4, p8L18, pPElO, and pSE10) ------------------------ 28 Determination the location of three gas homologues in the u; ragemosus genome ----------- 29 Northern analysis of u; :gggmgsus RNA with gene-specific probes ---------------------------- 30 DISCUSSION -------------------------------------------- 32 SUMMARY ----------------------------------------------- 38 APPENDIX ---------------------------------------------- 39 LIST OF REFERENCES ------------------------------------ 57 LIST OF FIGURES Figure Page 1 Southern blot analysis of L W genomic DNA with 32P-labelled YRAS DNA probes ----------------------------------- 39 2 Autoradiograms of recombinant DNA library screening by in situ plaque hybridization ------------------------------- 41 3 Southern blot analysis of purified recombinant phage DNA with a 32P- labelled m1 DNA probe --------------------- 43 4 Autoradiograms of colony screening by in situ colony hybridization using 32P-labelled ms; DNA probe ----------------- 45 5 Restriction endonuclease maps of L W ras homologues subcloned into pUC9 ----------------------------------- 47 6 Southern blot analysis of L W as genes in recombinant lambda clones using 32P-labelled DNA probes --------------- 49 7 Southern blot analysis of L W genomic DNA with 32P-labelled DNA probes from the as homologous regions of pHC4 , pBLIB, pSElO -------------------------------- 51 8 Cross hybridization of three L zagemgsus {as homologous genes by Southern blot analysis ------------------------------------ 53 9 Northern analysis of L :acemosus RNA with three L W gene-specific probes -------------------------------------- 55 vi INTRODUCTION 33; genes and m-rolated genes During the past 15 years, a series of oncogenes (cancer associated genes) has been identified through analyses of transforming genes in tumorigenic viruses and in tumor tissue. The transforming genes of Harvey and Kirsten strains of murine sarcoma viruses for example (Harvey, 1964; Kirsten and Mayer, 1967) have been named ES oncogenes. This acronym is derived from the words r_a,t sarcoma. Cellular versions of these transforming genes, called c-La_s_, have been identified through their nucleotide sequence homology to the retroviral oncogenes. At least three genes are found in the mammalian Lag oncogene family: Harvey gas (Ii-Lag) and Kirsten gas (K- gas) which were isolated from their respective murine sarcoma viruses (Ellis et al . , 1981) , and N-zas which was found as an activated oncogene in a human neuroblastoma (Taparowsky, et al . , 1983) . Genes exhibiting limited sequence homology to the La_s gene family are called pas-related genes that share 36% — 55% sequence homology. La_s homologues are at least 55% homologous to the as gene family. In view of the difficulty of assigning a cellular role for the as protein, p21, great excitement attended the discovery that genes with remarkable homology to human as are 1 2 widely distributed throughout a variety of different organisms. Bag homologues have been identified and characterized in simple eukaryotic organisms such as the yeast Saccharomyces ggrgyisiag (DeFeo-Jones et al., 1983; Power et al.,1984), ngsgpnilg (Neuman-Silberberg et al., 1984) and pigtyostelium (Raymond et al., 1984). However, only in yeast has the cellular function of a {as gene been determined (see below). Properties of :33 genes The most important goal in as research is to identify the biological function of as proteins and to determine the connection between mutations in ms and tumor development in tissue. The presence of ms genes in normal cells and in evolutionarily diverse organisms suggests that these genes have a fundamental role in cellular functions such as proliferation (Noda et al. , 1985; Bar-Sagi and Feramisco, 1985; Guerrero, 1986) and terminal differentiation. All forms of mammalian {as proteins, independent of their phylogenetic origin, effectively bind GTP and GDP (Shih et al . , 1980; Papageorge et al . , 1982) , have GTPase activity (McGrath et a1. , 1984; Gibbs, Sigal, and Scolnick, 1984), and are associated with the inner surface of the plasma membrane (Willingham et al. , 1980; Willumsen et al. , 1984) . In addition to these properties, the significant sequence homology of :35 and G proteins (Hurley et al. , 1984; Itoh et al. , 1986) has led to speculation that rag proteins, like G proteins, may 3 participate in the transduction of signals across the cytoplasmic membrane. Little else is known about the biochemical function of the mammalian ras proteins. The basic biological functions of {as proteins, although unknown, appear to be conserved in evolution. For example: 1) the human ERAS protein can complement yeast strains lacking {as genes: 2) the yeast RAS; gene product can, in a derivative form, transform mammalian cells: and 3) the GTPase activity of the yeast proteins is impaired by mutations analogous to those present in the oncogenic mammalian rag variants (DeFeo—Jones et al., 1985: Kataoka et al., 1985: Gibbs et al., 1985). other experimental data support the concept that rag genes of mammals play a dual role in proliferation (Mulcahy and Smith, 1985) and in certain differentiation processes (Noda et al., 1985: Bar- Sagi and Feramisco, 1985). One experimental approach to understanding rag protein function involves the study of rag-related genes in simple organisms such as Yeast (W W) . W, and Qigtyggtglium which are more easily manipulated experimentally than mammalian cells. By a combined genetic and molecular approach, efforts to identify the role of these gene products were initiated in yeast. The results of these studies revealed much about the two encoded yeast RAS proteins, 135g; and IBASZ proteins. The N-terminal 164 amino acids of each protein are 70% homologous to mammalian res proteins. XBAS proteins bind and hydrolyze GTP, and they are essential for 4 yeast proliferation and mating (DeFeo-Jones et al.,1983; Tamanoi et al.,1984: Telemeles et al., 1985: Kataobu et al., 1984: Tatchell et al., 1984). In yeast, functional rag genes are required for the maintenance of growth and cell viability (Kataobu et al., 1984: Tatchell et al., 1984). Taken together these observations predict that the biochemical function of gas in yeast may be similar to the function of [as in mammalian cells. These results also suggest that the yeast RAS proteins could be a good model for understanding the function of mammalian gas proteins. Recent experimental evidence demonstrates that one of the two gas homologous genes of S; cerevisiae (XBASZ) activates yeast adenylate cyclase in the presence of guanine nucleotides (Broek et al., 1985: Toda et al., 1985). In spite of the conserved structural and functional properties of gags; and mammalian {as genes, mammalian {as proteins do not appear to activate adenylate cyclase (Beckner et al., 1985: Birchmeier et al., 1985). Recent studies have suggested that yeast BA§1 protein and vertebrate rag proteins may play an indirect role in the regulation of phosphatidylinositol bisphosphate (PIP2) hydrolysis (Kaibuchi et al. , 1986: Fleischman et al. , 1986) . These observations have led to the proposal that each of the _r_a_s genes in a gene family play different cellular roles in spite of their sequence homology and genetic complementarity. Therefore, model systems other than yeast may be useful in understanding gas protein function and gas interactions with other proteins. £139.91 W: A model system for study of cellular morphogenesis, fungal food spoilage, and extracellular enzyme biosynthesis Mum is a filamentous fungus, belonging to the Class W. The genus Hum has been and continues to be explored by a great number of microbiologists and mycologists in both the basic and applied areas of biological research. Most of the Hugo: spp have properties similar to other W. There is one characteristic, however, which makes several mpg: spp quite distinct from other W and the subject of biochemical study. This is the property of dimorphism. Early in the nineteenth century, the morphological variability of Hugo; was first noted. Environmental conditions such as growth medium composition, Sparging gas, or temperature are factors which are most often controlled to study M299; morphogenesis. Dimorphic m spp are those which can grow in a variety of differentiated hyphal morphologies and also in the form of multipolar budding yeasts. Examples of dimorphic M_u_c_o_r_ spp include IL. means. :11 mm. 141 W. :41 Wis and L mums (some strains) . These organisms have the ability to undergo a morphogenetic change from hyphal to budding yeast-like growth in response to various environmental stimuli (Bartnicki-Garcia, 1963) . Members of the genus M929: are saprophytes and generally produce high levels of extracellular enzymes resulting in their 6 wide distribution as food spoilage agents. Foods associated with Hugo; spoilage include meat, poultry, bakery goods, dairy products and citrus fruits. finger spp are also used to produce ethanol or wines, and several fermented food products in the orient such as sufu and tempeh (Beuchat, 1978). The production of a variety of extracellular enzymes by Hugo; also results in industrial interest. Two species of M2991, ML pusillus and u; mighei are used to produce many useful extracellular enzymes such as lipases, amylases, cellulases, and acid proteases (microbial rennin) which are commonly used in the food industry (Van Heeswijck, 1984). Hugo; is usually not a pathogen, but some species are opportunistic pathogens which cause a rare disease , mucormycosis or zygomycosis, which ultimately attack the central nervous system and is often fatal. Correlates of morphogenesis 11299:: is an especially attractive model system for study of morphogenesis because of the ease of manipulations to alter cell morphology, either yeast or mycelial growth, as a function of thegrowth environment. For example, the organism can be induced to undergo a morphogenetic change from hyphae to budding yeast-like growth by shifting the growth environment from an aerobic to an anaerobic atmosphere. The reverse is also true (Cihlar, 1985) . This ability provides an advantage for the researcher to investigate the biochemical and molecular mechanisms that regulate morphogenesis. Morphogenesis in Hugo; 7 represents a useful model for study of cell differentiation in higher eukaryotes. Hugo; Iguxii and M299: zaggmgsgs are well suited for such studies. They have been more closely studied and perhaps better understood than the other species of Hugo; in terms of the biochemical and molecular events that accompany morphogenesis. Several physiological parameters have been found which are correlated to cellular morphogenesis in M; racemosus. For example, changes in intracellular cyclic AMP levels and specific rates of protein synthesis were correlated to yeast- to-hypha conversion in this fungus (Orlowski and Rose, 1981). The specific activity of ornithine decarboxylase (CDC), the first enzyme leading to polyamine biosynthesis in n; e sus, was also found to increase when the incubation atmosphere was shifted from C02 to air (Inderlied et al., 1980) resulting in a yeast-to-hypha transition. Finally a change in the rate of synthesis of lipids and a change in the turnover of phospholipids (including PIPZ, Ito et al. , 1982) accompany morphogenesis in this fungus. Based on these observations, it is possible that as gene products play an important role in cell morphogenesis in M; by interacting with cellular components (membranes) and physiological effectors as part of a signal transducing system. Research hypotheses The goal of this research was to test 2 hypotheses. These 8 are: 1) The n; :aggmgsns genome contains {as homologues. To determine the validity of this hypothesis the rag genes will be cloned in huge; ragemgsus. Initial work in this area will involve using known :35 genes such as ERAS (human cellular {as gene), XBASL and XBASZ (yeast RAS genes) to screen a Huge; genomic DNA library. The cloned Hugo: genes will be further studied by nucleotide sequence analysis. Once {gs-related genes are cloned in Muggr, then it is possible to continue the study on their functions by overexpressing normal {as genes in £999; or by expressing a gas gene with missense mutations and to observe the effects of these in vitro manipulations on cAMP, PI metabolism and morphogenesis in vivo. 2) There are different expression levels of the rag gene products in different cell morphologies in u; Iggemggus. This hypothesis will be tested by studying the level of ras gene transcripts which accumulate during normal growth and cellular morphogenesis in Hugo; by dot blot or Northern analysis (see Methods). These 2 testable hypotheses were based on the following data: 1. as proteins, in comparison with G proteins, are similar in nucleotide sequence and putative functional domains; 2. In yeast, data suggest that as may function in regulation of the production of second messengers cAMP and PIP; in signal transduction pathways: 3 . In §_,_ W, ras activity is correlated with cellular proliferation, differentiation and intracellular cAMP levels 3 4. In LJQQI, 9 morphogenesis, in response to an external signal (02) is correlated to cAMP and PIP; metabolism : 5. ms genes are ubiquitous in eukaryotes, implying a fundamental role for gas in growth and development. Genetic analysis 0151993: The development of recombinant DNA technology provides us with the tools for engineering of organisms used for production of biochemicals by direct manipulation of their genomes. However, before we can successfully undertake genetic programming of industrial microorganisms such as m, we must learn more about the basic genetics and biology of these organisms. A basic understanding of the biochemical and molecular events which regulate growth of Hugo; will help to increase production of industrial enzymes and control food spoilage by this genus of filamentous fungi. The methodologies of molecular genetics can help us to generate a greater understanding of the factors that regulate morphogenesis. For example the contribution of a particular gene product (such as rag) to the morphogenetic process in m: can be studied by altering the cloned gene by in vitro mutagenesis and reintroducing the gene back into cell . This protocol requires a cloned gene and a transformation vector. We have available DNA probes (Y_RA;§;, 1mg, M) which will be useful in isolation of gas genes from the Huger. In addition we are developing vectors will be designed and constructed to 10 contain selectable markers for use in transformation of H1 :gggmgsufi. Of course, an effective transformation protocol must also be developed in order to introduce recombinant DNA into the cell. Preliminary work in this area was done by Van Heeswijck in Denmark (Van Heeswijck, 1984, 1986: Van Heeswijck and Roncero, 1984) who demonstrated that protoplasts can be efficiently generated from u; Iaggmgsus by using chitosanase and chitinase. These protoplasts were transformed at a high frequency (600 leu+ transformants/ug DNA) using the plasmid vector pLeu4 (see Materials and Methods) . Recently, a leucine requiring strain, L W R78, was transformed with the aspartic protease gene (rennin) from L mighgi. The resulting heterologous protease was secreted in active form and had the same apparent molecular weight as the aspartic protease produced in L 111131391. However, the level of enzyme produced in L W was low (Dickinson et al . , 1987) . Possible reason for this low level of expression will be examined thoroughly in our laboratory. MI gene products from one species can be expressed and secreted in a different M9}: species. These data suggest that we can develop an expression/secretion vector of general use in different species of 119,991. One additional factor which makes L W potentially a very useful organism is its ability to undergo morphogenesis. We propose to combine experimental tools (i.e. transformation systems, and expression vectors) with an understanding of Hugo; growth and development to increase the 11 ability of M229: cells grow in high density continuous culture. We also hope to use knowledge gained in our studies to develop an efficient heterologous genes expression system in Mucor niehsi and Muse: musings- Preliminary data The following preliminary data were supplied by Dr. J. Linz as a background to this research. The primary evidence for the existence of ms genes in Muggr was provided by Southern blot analysis of restriction endonuclease digested L W genomicDNA using yeast RAS genes as probes to detect as related sequences (as described in detail in Materials and Methods) . These analyses revealed several DNA fragments with strong similarity to the heterologous m probes and encouraged us to do further isolation of rag related genes. The ERAS}, DNA probe was used by Dr. Linz to screen two lambda/Mg; W libraries (E and L) for ms- related genes. Several phage clones were isolated from the E and L libraries. Two of the stronger hybridizing clones (L18, E10) were subcloned and analyzed. The results are reported in this thesis. In addition, C bank was screened with the ms]. DNA probe to isolate ras homologues in L W with more similarity to the IRAS]. gene, which shares some sequence similarity with 1&5}. but differs significantly at 3’ end of the gene. This experiment resulted in the isolation of 4 lambda clones, C1-C4, one of which (C4) was also analyzed and reported here. MATERIALS AND METHODS Organism, growth media, and culture conditions m W ( Llygiflnigus) ATCC 12168 was the source of DNA and RNA used in cloning and analysis of the ras homologues in this research. Sporangiospore stock cultures were obtained from Paul Sypherd (University of California, Irvine) . Mum cultures were maintained on YPG agar [ 2% (wt. /vol. ) glucose, 1% (wt. /vol . ) Bacto-Peptone, 0. 3% (wt. /vol.) Bacto- Yeast Extract, and 3% (wt. /vol.) Bacto-Agar (Difco Laboratories, Detroit, Michigan) ] . The medium was adjusted to PH 4 . 5 with H2804 . Glucose was always autoclaved separately from the other components of the medium to prevent carmalization. A small amount of a stock spore culture (see below) was inoculated on the center of YPG agar plates (100 mm) which were incubated at room temperature (22°C) for 7 days to produce a confluent growth of mycelium. YPG liquid growth medium was the same as described above except that it did not contain agar. Spore production Sporangiospores for germination experiments were produced in 150 mm petri dishes containing 45ml of YPG agar. A small 12 13 - quantity of pure spore suspension (50 ul of a frozen stored spore culture) was inoculated in the center of YPG agar plates which were incubated at 28°C for a period of 7 to 10 days. At this time the agar surface was completely covered with aerial hyphae bearing the sporangia containing the grey- black sporangiospores. Spores were harvested with ice cold sterile water by scraping the mycel ium with a sterile glass rod. Spores were collected by centrifugation at 6, 000 xg for 10 min at 4°C and used in RNA preparations (see below) . Germl ing production Germl ings were prepared by germinating sporangiospores (2 x 105 spores/ml) in YPG liquid medium with shaking (180 RPM) at 28°C in a rotatory shaker water bath. The culture was sparged with 2 volumes of sterile air per volume of growth medium per minute. When germ tubes reached 10-12 spore diameters, the germl ings were cooled down in a salt ice bath and collected by filtration (Whatman no. 1 filter) . The cells were frozen immediately in liquid nitrogen and stored at - 70°C, or nucleic acids were extracted immediately, as described below. Yeast cell production Yeast cells of Hugo]: W were prepared by inoculating spore suspensions (2 x 105 spores/ml) into YPG broth and incubating with shaking (100 RPM) at 28°C while bubbling C02 gas through the culture at a flow rate of 0. 5 14 volume of gas per volume of culture fluid per min. These anaerobic conditions resulted in the germination of spores to yeast cells which continued growth by budding. After a 21 hr incubation period, the culture reached mid-log growth phase, (A600 = 0.8: yeast cells doubling time, approximately 4 hr). Yeast cells were harvested by filtration, frozen in liquid nitrogen and stored at -80°C until RNA extraction was carried out. Yeast-to-hypha cell production Yeast-to-hypha transition stage cells were obtained from growing yeast cells. Yeasts were grown in YPG with shaking under C02 until early log-phase (A500 = 0.22) . The culture was shifted to an atmosphere of air until 10% of the cells had germ tubes (approximately 3 hr. ) . The cells were used directly at this time for cellular RNA preparations. Purification of RNA and DNA from mm mm; cells Total RNA was extracted from L W cells by the procedure of Maramatsu (Maramatsu, 1973) as modified by Horst Domdey (personal communication) . The collected cells were broken with grinding for 5 to 10 min in a sterile mortar containing liquid nitrogen. The ground material was transferred to cold 30 ml corex tubes and suspended in 5 volumes of warm (65°C) SOS-RNA extraction buffer ( 50mM NaOAc, 1. 0 mM EDTA, 1% SDS: adjusted to PH 5. 0 with acetic acid) treated with 15 diethylpyrocarbonate (DEPC). The cell suspension was extracted 2 times with 5 volumes of warm RNA extraction buffer-saturated redistilled phenol (65°C) containing 0.1% (wt./vol.) 8- hydroxyquinoline. The aqueous phase was reextracted with 1 volume phenol (RNA extraction buffer saturated) : chloroform : isoamyl alcohol (25:24:1) and then with 1 volume diethyl ether (water-saturated) . Finally, the upper phase (ether) was removed and 1/ 6 volume of 3 M NaOAc (pH 5. 2) was added to the aqueous phase followed by 2 . 5 volumes ethanol to precipitate the RNA (- 20°C overnight) . High-molecular-weight L ragemosus genomic DNA was extracted from germl ings by the procedure of Cihlar and Sypherd (Cihlar and Sypherd, 1980) . L we germlings were grown, harvested as described earlier, and ground in a mortar under liquid nitrogen for 10 to 15 min to break the cells. The ground material was suspended in 8 volumes of TES buffer (100 mM PH 8. 3 Tris, 150 mM NaCl, 100 mM EDTA) . SDS (sodium dodecyl sulfate) was added to a final concentration of 0. 1% to solubilize the cell membranes. The broken cell suspension was extracted gently (to avoid shearing) with 8 volumes of phenol (TES buffer saturated) and reextracted with 8 volumes phenol (Tris saturated) / CHCl3 : isoamyl alcohol (24:1) . The DNA was precipitated by the addition of 2 volumes of absolute ethanol (-20°C overnight) . The pellet was dissolved in 1X TE (10 mM Tris-Cl , 1 mM EDTA PH 8 . 0) and treated with RNase (50 ug/ml) and proteinase K (100 ug/ml) for 2 hr at 37°C. The DNA was 16 extracted with an equal volume of phenol / CHC13 : isoamyl alcohol and ethanol precipitated (2 volumes) at -20°C overnight. RNA and DNA were quantitated by measuring the absorbance at wavelengths of 260 nm and 280 nm. The ratio between the readings at 260 nm and 280 nm (00260/00230) provides an estimate for the purity of the nucleic acid. Pure preparations of DNA and RNA have OD260/0D280 of 1. 8 and 2 . 0, respectively. Bacterial strains and plasmids L 9911 LE392 (F' hst514 supE44 supF58 lach galK2 galT22 met81 trpR55 lambda') (Murray et al. , 1977) , L 9211 CESZOO (Rec 8C") and L 9211 P2392 (P22 lysogen) were used to propagate the M3952: W gene library in bacteriophage lambda. L 5&1; JM83 [ara (proA,8-lac) rpsL th180 dlacz M15] (Messing, 1979) was the host strain for plasmids pUC8, pUC9 (Viera and Messing, 1982) , and p8R322 (Bolivar, 1977) , which were used in subcloning the ras genes. These plasmids were grown and amplified in LB medium (tryptone [Difco] , 10 g/liter: yeast extract [Difco] 5 g/liter: NaCl, 10 g/liter [PH 7.5]) and purified by equilibrium centrifugation in CsCl-ethidium bromide gradients (Birnboim and Doly, 1979: Maniatis et al. , 1982) . L £911 JM83 was made competent for transformation with recombinant plasmids by a CaClz procedure reported previously (Mandel and Higa, 1970) . The transformed cells were selected and maintained on LB medium supplemented with the chromogenic 17 substrate 5-bromo, 4-chrolo, 3- indolyl, 8-D-galactoside (X— gal, 20 ug/ml) and ampicillin (50 ug/ml, final concentration) for pUC recombinant plasmids. The alkaline lysis method was used in rapid, small-scale or large scale plasmid DNA preparations (Maniatis et al., 1982). Construction of an M; xgggmgggg gene bank in bacteriophage lambda M1192: W gene banks, were prepared by Dr. Linz . Three M1192]: W gene banks, C bank, L bank, and E bank, were propagated in L £911 CESZOO, L 95211 LE392 , and L Egg LE392 respectively. C bank and L bank were established from mI-digested m; W genomic DNA[ 10 to 15 Kilobases (kb) fragments] which were purified on sucrose gradients (Maniatis et al. , 1978) and ligated to the WI arms of the lambda vector EM8L4 . My DNA, cut with A1131 , was ligated to the S911 arms of lambda EM8L3A to construct the E bank. The procedure for library construction is described in more detail by Maniatis et al (1978) . Preparation of bacteriophage DNA Lambda DNA was purified from the phage clones (plagues) for further analyses by a small-scale, plate lysate method (Maniatis et al . , 1982) or large-scale CsCl density gradient method of Carlock (1986) . 18 Radiolabeling of DNA restriction fragments DNA probes for hybridization were purified by resolving DNA restriction fragments on agarose gels and electroeluting the DNA fragments from gel slices using an electroelution unit from International Biotechnologies Inc (181) . DNA fragments were then denatured and labeled as described below. We used [alpha-32p] dGTP in a random primer labeling procedure (Feinberg and Vogelstein, 1984) which allowed DNA probes to be labeled to high specific activity ( > 1 x 108 CPM/ug DNA) . The high specific activity could be achieved with quantities of DNA as low as 10 ng per reaction. A 25-ul mixture was prepared as follows: 10 ul oligo-labeling buffer (0L8 buffer, see below) , 2 ul of (1 mg/ml) bovine serum albumin, DNA 10-30 ng, 1 ul Of 1 deATP, 1 ul of 1 mM dC‘I‘P, 1 ul of 1 mM dTTP (each triphosphate previously disolved in TE with MgClz PH 7.0), 5 ul of [32P1dGTP (DUPONT/NEN, 3000 Ci/mmole, 10 uCi/ul) , 2 units of large fragment of EM; Eli DNA polymerase I (8M8 klenow fragment of DNA polymerase) . The complete reaction was incubated at room temperature for at least 2 hrs. The reaction was stopped by addition of 2 ul EDTA (0. 5 M) and the labeled DNA was purified by a gel filtration column (sephadex G-50-80, 5 ml packed volume) eluted with TE buffer (pH 8 . 0) . The labeled DNA was eluted from the column and collected in the first peak response detected with a Geiger counter. The specific activity of the labeled DNA was measured by liquid scintillation spectroscopy of a 5 ul sample spotted onto a 19 glass fiber filter. Ml: tease. and me D” P303333 Three different heterologous ras probes, including DNA fragments from two yeast ras genes (m1, M2) and a human cellular ras homologue (MS) , were used in this study. The 1.33.5.1 gene is carried on a 2 . 2 kb MRI/WI fragment cloned into p8R322 also cut with MRI/MRI (total 6. 6 kb) . The IRAS; DNA probe is a 1. 6 kb DNA fragment generated in a SindIII digest of the 6. 6 kb mgr-p8R322 DNA. THe XRAS; gene is carried on a 3 . 0 kb SggRI/SindIII fragment cloned into a pUC plasmid cut with MRI/HindIII (total 5.7 kb) . The LRASZ, probe is a 1. 2 kb fragment generated from a Spa} digest of the 5.7 kb YRAsz-pUC DNA. The HRAS gene is carried on a 6. 6 kb 8amHI fragment from the human genome cloned into WI site of pSVZNEO. The M probe is a 2 .9 kb S191 fragment which contains all of the exons of the HRAS gene. Selection and screening of recombinant plasmid clones The task of isolating a desired recombinant from a population of bacteria or phage was carried out by genetic selection and nucleic acid hybridization. 1.) Genetic selection: The pUC9 vector carries an ampicillin resistance gene and a multiple cloning site in a 8-galactosidase gene. These properties made it possible to screen transformed L 9211 cells 20 for recombinant plasmids by resistance to the antibiotic ampicillin and insertional inactivation of the 8- galactosidase gene. Transformed cells containing the recombinant plasmids grew on LB agar plates supplemented with ampicillin as white colonies (hydrolysis of the chromogenic substrate, X-Gal, generates a blue color). 2.) DNA hybridization methods: Two DNA hybridization methods were used to screen recombinant clones in this study. For in situ plaque hybridization (Benton and Davis, 1977), recombinant DNA from phage in plaques was transferred to a nitrocellulose (N.C.) filter. The N.C. filter was placed face-up (DNA side up) onto a sheet of filter paper saturated with Southern base (0.5M NaOH, 1.5M NaCl) for 5 minutes to disrupt the phage coat and denature the DNA, and then placed on a second filter saturated with neutralization solution (1M Tris-HCl pH 8.0, 1.5M NaCl). Nitrocellulose filters were baked for 2 hr at 80°C in a vacuum oven. The second method was in situ colony hybridization (Gruntan and Hogness, 1975) . Bacteria containing plasmids were spotted onto nitrocellulose filters and grown overnight at 37°C on L8 ampicillin medium. The N.C. filters were placed onto a sheet of 3 mm paper saturated with Southern base (5 minute) , and then placed on by a second filter saturated with neutralization solution. The filters were air dried and baked at 80°C for 2 hr under vacuum. The DNA on the filters was 21 hybridized to radioactive probes to detect recombinant clones (see below). Restriction endonuclease analysis and agarose gel electrophoresis The optimal reaction conditions for restriction endonuclease digests supplied by the manufacturer were followed. DNA restriction fragments were resolved by electrophoresis through 0. 8 to 2 . 0 % agarose gels (Maniatis et al. , 1982, P. 150) with a Tris acetate buffer (TAE) system. Fragments of DNA in the gel were visualized by staining with the fluorescent dye ethidium bromide (0. 5 ug/ml) . Stained gels were photographed by transillumination with UV light (260 nm) . RNA samples were resolved by electrophoresis through denaturing formaldehyde-agarose gels with a diethyl pyrocarbonate (DEPC) -treated autoclaved MOPS/EDTA buffer: 0. 2 M MOPS [3-(N-morpholino) propanesulfonic acid], 50 mM sodium acetate, 10 mM EDTA adjusted to PH 7 . 0 . Ethidium bromide was added to the sample prior to electrophoresis in order to visualize RNA in agarose gels by transillumination. This method for efficient RNA staining was described by Fourney et al (1988). Southern and Northern blot analyses of nucleic acids DNA fragments, separated by electrophoresis through agarose gels, were denatured, transferred and immobilized. 22 Nitrocellulose filters (Schleicher and Schuell) were used to immobilize DNA fragment sizes greater than 300 bp and nylon membranes (3&8 Nytran) were used to immobilize smaller DNA fragments (Southern, 1975). The DNA transfer buffer was 20X SSC (1X SSC is 0.15M NaCl plus 0.015M sodium citrate, PH 7.0). Transfer of DNA was allowed to proceed for 12-24 hr, after which the filter was air dried and baked for 2 hr at 80°C under vacuum prior to hybridization. Transfer of RNA from formaldehyde-agarose gels was carried out by the procedure of Maniatis (1982) . The typical hybridization conditions for nitrocellulose filters were described by Maniatis (1982) . Nitrocellulose filters were soaked for 2 hr in prehybridization solution (40% deionized formamide for low stringency or 50% formamide for high stringency, 5X Denhardt solution [1X Denhardt solution is 1% Ficoll {Pharmacia Fine Chemicals) , 1% bovine serum albumin, 1% polyvinylpyrrolidone] , 6X SSC, 0. 1% sodium dodecyl sulfate, denatured salmon testes DNA [100 ug/ml] , SmM EDTA) at room temperature for 2-4 hr. Then a radiolabelled DNA probe was added to the prehybridization solution (5 x 105 to 1 x 106 CPM per ml) . Hybridizations were allowed to proceed for 16-24 hr at 37°C for low stringency or 42°C for high stringency in a shaking water bath. Following hybridization, the nitrocellulose filters were washed twice for 15 min in 2X SSC 0. 1% sodium dodecyl sulfate at room temperature and then once for 60 min in 2X SSC at 37°C for a low stringency wash or 0. 1X SSC at 65°C 23 for a high stringency wash. Filters were dried in air and exposed to Kodak X-ray film at -70°C with an intensifier screen for 4 hr to several days. RESULTS Identification of Lag-related sequences in 11922! M genomic DNA In order to detect the presence of ms homologues in the Mm; W genome, Southern blot analysis of L W genomic DNA was conducted using gel purified, random primer- labelled mg, m (Figure 1) and HRAS DNA probes (data not shown) . These three DNA probes were previously described in the Materials and Methods section. L W genomic DNA was digested to completion with SingII, EggRI or SamHI to identify genomic DNA fragments which might contain the m-related sequence. Two strongly hybridizing restriction fragments of approximately 6. 5 and 3 . 5 kb and three lightly hybridizing restriction fragments of approximately 24 , 9. 6 and 7 . 0 kb were detected in the EQQRI- digested DNA using the Y_RAS2_ probe. SamHI-digested genomic DNA showed three strongly hybridizing restriction fragments of approximately 3 .9, 3 . 8 and 3 . 6 kb and seven weakly hybridizing restriction fragments. The IRAS]. probe hybridized strongly with a 3 . 8 kb restriction fragment and weakly with a 24 kb (lighter band) fragment in SamHI-digested genomic DNA, and hybridized also with 23 , 9. 6 and 7 . 0 kb 24 25 restriction fragments in EQQRI-digested DNA. Similarly, nindIII-digested genomic DNA had two restriction fragments (4.7, 3.5 kb) which hybridized with the XBASl DNA probe and three restriction fragments (4.7, 4.2, 3.5 kb) which hybridized with the XEASZ DNA probe (data not shown). The HRAS DNA probe also hybridized strongly with one restriction fragment (7.0 kb) and weakly with one restriction fragment (4.1 kb) in EQQRI- digested ML zgggmgggg genomic DNA.This probe also hybridized strongly with a 3.9 kb restriction fragment and weakly with a 14.5 kb restriction fragment in ngHI-digested genomic DNA (data not shown). The results in this experiment provided the initial evidence for the existence of {SS-related genes in 3399; and encouraged us to screen an M; :gggmgggs DNA library in order to clone the putative {SS homologues. Screening lambda/M; rgcemgsus DNA libraries for ggg homologous sequences The C bank of L ragemgsus DNA in lambda phage contained 1 x 106 plaque forming units per m1 (pfu/ml) . 1-5 x 103 pfu of the lambda library were plated on 3 separate agar plates and the resulting plaques transferred to nitrocellulose as described in Materials and Methods. The radiolabelled YRASl DNA probe was used to hybridize to the phage DNA which was fixed nitrocellulose circles (82 mm) . Four positive recombinant phage clones (plaques) C1-C4 (Figure 2A) were isolated and subjected to a second in situ hybridization screening. Only 2 26 recombinants, C3 and C4, showed strong hybridization with XRAS; (Figure 28). In similar fashion, Dr. J. Linz also screened the L and E libraries with the use YEASZ DNA probe and isolated nineteen phage clones - called L1-L9, and El-Elo. At this time, I selected two strongly hybridizing phage clones (L18 and E10) from Dr. Linz analysis along with C4 for further cloning and nucleotide sequence studies. These clones were selected because they strongly hybridized with XRAS DNA probes and showed different restriction patterns (see below) in restriction endonuclease mapping analysis. Grouping lambda phage clones by restriction patterns DNA purified from the phage clones C4 , L18, and 810 were digested to completion with MRI, SamHI, HindIII, or MRI/HindIII. Restriction fragments were resolved by 1% agarose gel electrophoresis and subjected to Southern blot analysis. The M1 probe hybridized with 10. 5 kb EQQRI, 9. 4 kb SamHI, and 4 .7 kb andIII restriction fragments in the C4 phage clone. In the L18 phage clone, 7.4 kb MRI, 3.8 kb WI, and 3.2 kb flindIII restriction fragments hybridized with MS; DNA probe. The E10 phage clone which was screened from the E bank by the ma DNA probe showed no detectable hybridization with MS]. DNA probe (Figure 3) . ESAI and Sug3A digests of the phage clones L18 and 810 were also analyzed by Southern hybridization with YRASZ DNA probe and showed 27 different hybridization patterns (data not shown). Based on these data, the phage clones which contained {SS homologues were grouped into several unique restriction patterns by Southern analysis. These results suggested that more than one m gene was present in the L W genome. Subcloning of L 1322828113. ras genes (HC4, 81.18, 8810, and P810) into the L 9311 plasmid vector, pUC9 The 4.7 kb morn (HC4) , 3.8 kb8_a_mHI (8L18) , 1.25 kb S111 (SE10) , and 1.65 kb MIT (P810) restriction fragments containing putative mg genes were cloned individually into pUC9 cut with SingII, S_a_mHI, S111, and m1, respectively. These recombinant plasmids were transformed into JM83 . The transformation efficiency of L 95211 JM83 by recombinant plasmids containing ras-related gene was low ( 400 transformants/ug DNA) while the transformation efficiency of control plasmid, pUC9 was 1 x 104 transformants/ug DNA. The reason for the low efficiency was not clear. We screened transformants by in situ colony hybridization with YRASl (for HC4) or XBASZ probes (for 8L18, SE10 and PE10) . Only one of two putative transformants included a 4 .7 kb C4 flindIII restriction fragment HC4 which was able to hybridize with YES; (Figure 4A, plate a) . Restriction mapping and Southern hybridization analysis confirmed that this recombinant plasmid contained the 4.7 kb MIII restriction fragment (HC4) (Figure 48) . The recombinant plasmids containing 8810 and P810 and 8L18 were identified in a similar manner through restriction analysis and 28 Southern blot analysis with the XEASZ DNA probe (data not shown). Dr. J. Linz subcloned the 8L18 restriction fragment ( 3.8 kb ngHI) into pUC9 ngHI site and isolated the recombinant plasmid in the same fashion Restriction endonuclease maps of HC4, 8818, and P810 sequences HC4 , 8L18 and P810 were digested with several restriction endonucleases singly and in combination to produce the restriction maps shown in Figure 5. These data show that the pattern of restriction enzyme recognition sites is unique to each of three clones. The S111 site of HC4 appears to be located at critical sequence for hybridization with the mg; DNA because the probe did not hybridize to any restriction fragments whenever S111 was used to cut HC4 . A comparison of the hybridization pattern of original phage clones (C4, 1.18 and 810) with recombinant plasmids (pHC4, p8L18, pPElO and p8810) Southern blot analysis of restriction endonuclease digested phage clones with BASIL, m and ES DNA probes (Fig. 6.) showed that each recombinant phage contained a restriction fragment also found in the recombinant plasmid (Figure 5) . The C4 phage clone and pHC4 contain a 0. 7 kb deII/KpnI segment which hybridizes to the w; DNA probe. The L18 phage clone and p8L18 contain a 2 . 0 kb SindIII/MII segment which hybridizes to m. The 810 phage clone and pSElO (or pP810) contain a 1.25 kb S111 (or 1.65 kb MII) 29 fragment which hybridizes to XBA_2, but did not hybridize detectably with YBASl. Therefore, each recombinant plasmid contained a subcloned :15 gene in the same form as originally isolated from the lambda/M199; xgggmgggg library. Determining the location of three :13 homologues in the Mucor M 931101“ In order to specifically measure the expression of individual as genes in M91, we sought to isolate gene- specific DNA probes. Small DNA restriction fragments from each subclone, which hybridize to heterologous 1:15 probes, were identified and gel purified (see Figure 6) . These restriction fragments were resolved on an agarose gel and analyzed by Southern hybridization to look for cross hybridization (Figure 8) . This experiment shows that the three DNA segments (0. 7 kb MdIII/KEQI digest pHC4 , 0. 69 kb EggRI/S111 digest M13Sau3A,and 1. 25 kb S111 digest pSElO) did not hybridize to each other. These three DNA restriction fragments were then used as probes in Southern blot analysis of L racemgsus genomic DNA digested to completion with flindIII, S1mHI, or MRI . The restriction fragments were resolved by agarose gel electrophoresis (10 ug of DNA per lane) and transferred to a nitrocellulose filter. Replica filters were then incubated individually with one of the DNA probes shown in Figure 5. Each of the specific probes hybridized to a unique L W genomic DNA restriction fragment (Figure 7) corresponding to one of the hybridizing restriction fragments which were 3O detected by Southern analysis of M1 r1ggmg§n§ using the heterologous yeast RAS probes (Figure 1). These data demonstrated that the three ras homologues reside at unique locations in the L W genome. Northern analysis of I; {12331131 RNA with gene-specific probes The three gene-specific probes (A, 8, and C from Figure 5) were labelled to high specific activity with [alpha-32P] dGTP and used to analyze the accumulated levels of ras- related mRNA from several cell morphologies of L W to determine whether there was a morphology-specific pattern of gene expression for M31952; ras genes. RNA was purified from sporangiospores, germlings, yeasts, and yeast cells which had been induced to undergo morphogenesis to hyphae. Total RNA (20 ug/lane) was resolved on formaldehyde-agarose gels and analyzed by Northern analysis. The pattern of transcript accumulation detected by each probe was quantitated visually. In this analysis only the 810 probe hybridized at detectable levels to an mRNA (Figure 9) . In the control experiment (data not shown) an identical nitrocellulose filter was probed with TEF-l, a gene encoding elongation factor 1 alpha, in L m. This gene is expressed at constant levels in each of morphologies tested (Linz and Sypherd, 1987) . In this experiment the TEF-l probe hybridized at significantly higher levels with germlings mRNA than with any of other three morphologies. These data suggest that the higher signal intensity observed in germling 31 mRNA with the 810 probe was an artifact in the procedure. This artifact may have arisen as a result of an measuring error The other two probes (C4 and L18) did not hybridize to mRNA which indicates that the genes were either not transcribed or transcribed at levels too low to be detected (data not shown). These data suggested that there was differential expression of the three genes encoding M199; ras proteins. In a recent experiment, the polyadenylated mRNA fraction from M; Igggmgggg cells was purified by oligo(dT) cellulose column chromatography. The mRNA was resolved on a formaldehyde- agarose gel, transferred to nitrocellulose and analyzed by Northern analysis with the gene-specific probes (data not shown). There were different gene transcript levels with each probe. For example, the levels of putative 810 mRNA was several fold higher than the level of putative C4 mRNA. The level of putative C4 and 810 mRNAs did not vary significantly when observing the different cell morphologies suggesting that they are expressed constitutively (i.e., they are always expressed). However, the putative L18 mRNA was detected only in sporangiospores. These preliminary data suggested strongly that there are three genes encoding 115 protein in M1 nggmgggg and that at least one gene, L18, showed a morphology-specific pattern of transcript accumulation. DISCUSSION During the past few years, the biological function of m proteins has been studied by comparing their amino acid sequence to other proteins whose function is known or by analysis of their biochemical activities in eukaryotic organisms. In the course of these studies it has become apparent that regardless of their phylogenetic origin, the 1:11 proteins have the ability to bind guanine nucleotides. They also have GTPase activity, and are bound to the cytoplasmic membrane. Their significant sequence homology with G proteins suggests that 111 proteins may participate in signal transduction pathways which allow cells to communicate with their surroundings. Mutations which alter the function of GTP exchange rate, GTP hydrolytic activity, or 1:11 transcription level were found to alter cell growth (proliferation) and development (cell differentiation). In L 9erevis111, the experimental data suggest that at least one functional m gene is required to maintain cell growth. Other experimental data indicate that the :11 gene products of S_,_ 9111111111 may be regulatory proteins that control cAMP and PI metabolism. In S_,_ 91mi1111 cells, the SEAS; gene is involved in regulation of the adenylate cyclase pathway. 32 33 However, the role of genes coding for :11 proteins in higher eukaryotic organisms is not clear. There are several things that we want to discover about :19 protein function using 1999; as a model system including the biological function of :11 and the identity of effector proteins, receptor proteins, and other components of a putative signal transduction pathway. These data will hopefully allow us to clarify the connection between expression of mutant :11 and tumor formation. M999; is a good model for the study of a possible correlation between signal transduction and cell differentiation because it can grow in 4 different morphologies with different growth rates in response to an external signal. These observations directly led to our hypothesis that one or more :11 proteins are involved in signal transduction pathways which allow M999; to respond to an external signal to change its morphology and growth rate. The identification and isolation of the :11 genes from M1 :191m9191 was accomplished by the use of three heterologous DNA probes, XBAS1, 131S;,and SEAS. The fiindIII, S1181, S111 and 21911 restriction fragments of phage clones C4, L18, and 810 which hybridized to these probes in Southern blot analysis, were subcloned into a pUC9 plasmid vector and used to generate restriction endonuclease maps (Figure 5). The restriction sites located in each of the three subclones did not overlap. Southern blot analysis of M1 :191m9191 genomic DNA with 32P-labelled gene-specific DNA probes from each of the 34 subcloned M999; :19 genes (Figure 7), provided additional evidence for the presence of three genes in the M1 119119191 genome. These gene-specific probes hybridized to unique restriction fragments in the genomic DNA but did not hybridize to each other suggesting that the M2921 :19 genes are located at unique locations in the genome. The three H3921 :11 gene-specific DNA probes were also used in standard Northern analysis of RNA purified for several cell morphologies of £1 919119991. These preliminary data suggest that all three genes are expressed in the cell morphologies studied. However, the accumulated level of transcript derived from each gene varied considerably, with 810 mRNA present in several fold greater levels than either the C4 transcript or L18 transcript. The level of putative 810 and C4 mRNAs varied little among the cell morphologies studied suggesting that they are expressed constitutively. The putative L18 mRNA was only present in detectable quantity in sporangiospores. These data suggest that there is differential expression of the genes encoding :11 proteins in 11 {19119191. At least one gene, L18, shows a morphology- specific pattern of transcript accumulation. These data are preliminary and must be confirmed by further experiments. Expression of each of the 1999; 111 genes may be regulated during cellular proliferation such that changes in the requirements for individual :11 proteins results in changes the level of each these genes' activity. This may provide a crude 35 mechanism to regulate the :11 protein level and activity in the cell. The :19 genes in M1 919119199 may represent a gene family in which the individual genes are developmentally regulated. Because we do not know the exact location for each of the :11 gene in the three gene-specific probes, the data must be regarded as preliminary. We do know that each of these £999; probes hybridizes with more than one heterologous :11 gene. However, only the L18 gene containing the 690 bp (base pair) gene-specific probe has been subjected to nucleotide sequence analysis. The L18 gene fragment has been completely sequenced in our laboratory. These data showed that the 690 bp L18 probe contains an open reading frame of 390 bp which shares 70-80% homology at the amino acid level with the 5' end of the yeast :19 genes. We speculate that the remaining 300 bp in the L18 gene fragment are flanking sequences, which represent the LES promoter sequence and an intergenic sequence which is rich in adenine and thymine residues. We currently are unable to determine if the :11 sequences of the L18 probe hybridize with mRNA or if it is the flanking sequence. To clarify the data from Northern analysis, we must identify internal restriction fragments from C4, L18, and 810 to use as probes. After more nucleotide sequence data from the three 1999; :11 genes is available, we will repeat these experiments with new DNA probes. In future work, we plan to analyze the regulation of transcription of the :11 genes using molecular genetic 36 technology. We also will conduct Sl nuclease mapping to locate the :11 promoter and terminator sequences and confirm the presence of intervening sequences which have been tentatively located in the nucleotide sequence of L18. These experiments are designed to carefully explore if this gene family is being regulated differentially during cellular morphogenesis. Now that we have cloned several :19 genes in M9221. we can use these genes as tools to initiate studies which are designed to reveal the biological function of :11 genes in u99_;. We will change the activity of these genes through in vitro mutagenesis or to introduce multiple copies of :11 gene into M999; cells to overexpress :11 gene and observe any differences in the phenotype of the cells and any effects of altered :11 expression/activity on cAMP or PIP; metabolism. Finally we plan to generate a series of monoclonal antibodies (MAb) which will bind specifically to each of the Lzm 1:11 proteins. These MAb can be used to 1) confirm changes in the quantity of :11 proteins: 2) determine the location of :11 proteins in 11999: cells: and 3) inhibit or alter 111 protein activity by binding to the protein in cell extracts of membrane preparations. We can then measure any effect of MAb binding on cAMP or PIP; metabolism. The dimorphic fungus 11999: 1191199199 is a useful model for studying the possible relationship between 111 genes expression and cell differentiation. A basic understanding of growth and development in M199; 1191199191 should help us to more 37 effectively use the industrial M922: spp to improve yield of food products such as organic acids, fermented food, enzymes, or to prevent food spoilage. SUMMARY Two hypotheses were established at the beginning of the research: 1) M1 919119199 contains :19 homologues. 2) There are different expression levels of the :11 gene products. The experimental data suggest that these two hypotheses are correct. Southern hybridization data and restriction endonuclease analysis suggest that the 3 clones represent 3 M999; 911 genes. These genes are C4, L18, and 810. Northern blot analysis of £999; RNA suggests that the three 1999; 111 genes are expressed at different levels and that one of these genes (L18) is expressed in a morphology- specific manner. 38 APPENDIX 39 Figure 1. Southern blot analysis of M1 Lacemosus genomic DNA with 32P-labelled XBAS DNA probes. 20 ug of M1 za9emosus genomic DNA were fragmented with the indicated restriction endonucleases and resolved by agarose gel electrophoresis. Lanes: 33111, B: we used mSl as a probe to detect putative La_s homologues in a S111HI digest of L gacemosus genomic DNA: $11: $11 probe, SQRI digest: ESLB: 3&1; probe, MHI digest: ESLE: gm; probe, _L:9RI digest: kb: molecular size markers (H_indIII digest of lambda DNA) . (Autoradiogram provided by Dr. J. Linz) . Figure 1. YRAIl ' E 40 "“32 ‘probe B E ......_.. Rb ,, , <14 - 46.6 44.4 C 2.3 41 Figure 2: Autoradiograms of recombinant DNA library screening by in situ plaque hybridization. A radiolabelled XBAS1 DNA probe was used to hybridize duplicate nitrocellulose filters using the low stringency conditions which are outlined in Materials and Methods. A: 1. Control, S1 9911 only. 2. First round screening of approximately 3,000 recombinant lambda plaques (C bank). 8: Duplicate filters of second round screening of one positive recombinant phage (C3) isolated from first round screening. C: Duplicate filters of second round screening of a second positive recombinant phage (C4) isolated from first round screening. 42 43 Figure 3: Southern blot analysis of purified recombinant phage DNA with a 32P-labelled XBAS1 DNA probe. DNA purified from the phage clones C4 , L18, and 810 was digested with the indicated restriction endonucleases and analyzed by Southern hybridization under low stringency conditions (see Materials and Methods) . Hybridization was performed at 37° for 12-18 hours. Lanes: 1-3: E99RI digest of C4 , 810, L18 phage clones DNA. 4-6: Sa_mHI digest. 7-9: madIII digest. 10-12: S91RI/H_i_n_d111 digest. kb: molecular size markers (LingII digest of lambda DNA) . Approximate DNA fragment sizes are indicated in kbp. (E = 119121, 8 = mm", H = morn, E/H = EcoRI/HindIII) 44 Figure 3 . E B H El", 45 Figure 4: A: Autoradiograms of colony screening by in situ colony hybridization using 32P labelled XBASl DNA probe. Two colonies from a primary in situ colony hybridization thought to contain a 4 .7 kb _H_i_n_dIII fragment from C4 recombinant phage were streaked onto agar plates containing LB medium, X-Gal, and Ampicillin. 12 single colonies from each plate were then patched onto nitrocellulose filters on new agar plates. In situ hybridization was carried out on these filters with a radiolabelled 13111 DNA probe under low stringency conditions (see Materials and Methods) . In plat a, the colonies strongly hybridized with the YRASl probe: In plate b, the colonies did not hybridize, including the control colony which represents a pUC9 plasmid with no insert. 8: Autoradiogram of Southern blot analyses of the subcloned C4 gene. Plasmid DNA was prepared from five colonies from plate a using the alkaline lysis minipreparation procedure (see Materials and Methods) and cut with H_i_r_1dIII to generate a 4 .7 kb 3318111 insert fragment from the C4 lambda clone and a 2 . 7 kb pUC9 vector fragment. The DNA was resolved on a 1% agarose gel transferred to nitrocellulose filter. Southern blot analysis was conducted with the 332.181 DNA probe under low stringency conditions. 46 Figure 4. 47 Figure 5: Restriction endonuclease maps of M1 :acemosus 911 homologues subcloned into pUC9. the 911 containing recombinant plasmids pHC4, p8L18, pSElO, and pPElO were cut with several restriction endonucleases. The DNA restriction fragments were resolved by electrophoresis through a 1% agarose gel and their sizes were analyzed to generate restriction maps. Only the M1 racemosug DNA inserts are shown in the maps. The :11 homologous region in each clone as determined by Southern blot analysis of this gel with XRAS1 and XBAS; probes (data not shown) is indi- cated by the highlighted line segment. 48 Figure 5. lkb =35: .. _o_uz .. 2.08 r 23 .. .3x .. >¢oou i as. :6 .A :35: M k 7. 4 1L "F4 ( _ 15am J :_vch.. 52.4.... lkb =am C zam M m k E s 5 Mn =a>m am ,2 Sum find .5. :1 :6 7mm :2,“— PEIO (1.65kb) 49 Figure 6: Southern blot analysis of L W £618. genes in recombinant lambda clones using 32P-labelled DNA probes. Three different 32P-labelled probes were used to hybridize to replicate sets of DNA restriction fragments to generate the autoradiograms shown in panel a, b and c. These restriction fragments were resolved by agarose gel electrophoresie and transferred to nitrocellulose filters. Low stringency hybridization conditions were used. a: A 32P-labelled probe prepared from M1 DNA was used in hybridizations against: 1ane2 , C4 digested with 11i_ndIII/1$p_nI: lane3 , L18 digested with mun/mu: lane4, ElO digested with 1111: lane5, 310 digested with MII . b: A m 32P-labelled probe was used in hybridizations against C4 lambda, L18 lambda, 810 lambda restriction enzyme digested segment. c: A SfiS 32P-labelled probe was used. The DNA in lanes 2 through 5 in b and c are replica sets of the DNA in panel a. The hybridizations were performed under low stringency conditions. 50 51 Figure 7: Southern blot analysis of M1 919119191 genomic DNA with 32P-labelled DNA probes from the :11 homologous regions of pHC4 , p8L18, pSElO. In panels a, b and c, three different 32P- labelled probes were used to probe 3 replica sets of L racemosug genomic DNA digested with restriction endonucleases. High stringency hybridization conditions (42°C, 50% formamide, 5X SSC) and high stringency wash conditions (65°C, 0. 1X SSC for 1 hr)were used. Panel a, A 32P-labelled probe was prepared from a gel purified deII/KQQI restriction fragment from pHC4 (0.7 kb) : panel b, Gel purified SL13A restriction fragment from p8L18 (0.69 kb) : Panel c, Gel purified S111 restriction fragment from pSElO (1.25 kb) (See Figure 5. the regions of highlighted line segment) . Lanes: S, molecular size standard (deII digest of lambda DNA) . lanel: H_i_r1dIII digest of L racemosus DNA. lane2: MRI digest of L racemosus DNA. lane3: 899RI digest of L 9ac1mosus DNA. (H = 111ng11, 8 = _8_a_mHI, E = EcoRI) 52 53 Figure 8: Cross hybridization of three M999; r1cemosu1 :11 homologous genes by Southern blot analysis. DNA restriction fragments were purified by agarose gel electrophoresis and used as DNA probes. Lanes: kb, molecular size makers. In panel a, b, and c: three different DNA probes indicated by DNA fragment A, 8, and C see in Figure 5 were used to probe nitrocellulose filters. lanel: S111 digest pSElO. lane2: S11I/S99R1 digest M13S193A. lane3: HingII/KQQI digest pHC4. High stringency hybridization and wash conditions were used (65°C, 0.1 x ssc for 8 hr). 54 Figure 8. probe A 3. 2_ 1. 3. 2_ 1_ 0.6- 55 Figure 9: Northern analysis of H222: :ac1m0191 RNA with three M1 racemosgs gene-specific probes A, 8, C (shown in Figure 5). Total RNA was purified from sporangiospores (S), yeast cells (Y), germilings (G), and yeast cells which were induced to undergo morphogenesis (YH) as described in Materials and Methods. The purified total RNA (20 ug/lane) was resolved on formaldehyde- agarose gels and analyzed by Northern hybridization analysis with :11 gene specific probes A, B, C under high stringency conditions. Only the 810 probe hybridized detectablly with an mRNA. 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