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 r_—+_ [:]__ T—TL—J—T mm MSU In An Affirmative Action/Equal Opportunity Institution cWMS-o' CHARACTERIZATION OF SPORULATION-SPECIFIC GENES OF SACCHAROMYCES CEREVISIAE By Sheau-Huey Suhng A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1990 JOOX .4) 05L /, ABSTRACT CHARACTERIZATION OF SPORULATION-SPECIFIC GENES OF SACCHAROMYCES CEREVISIAE By Sheau-Huey Suhng In response to nutrient deprivation, a/a diploid strains of yeast Saccharomyces cerevisiae undergo meiosis and spore formation. During sporulation, several sets of sporulation-specific genes (SPR genes) are specifically transcribed, but their roles in sporulation are not clear. In this study, one sporulation-specific gene isolated previously by differential hybridization, SPRQ, was characterized molecularly and physiologically. SPRQ was transcribed 4 hours after transfer to sporulation medium and the transcript lasted at least 20 hours in the sporulation medium. The absence of SPRQ gene products did not block ascus formation; instead the mutant spores ger- minated less efficiently than the wild-type spores at high temperature. The protein predicted from the nucleotide sequence of SPRQ was 16.8 kD, and no homologous sequences have been found in a computer search. Two other abundantly transcribed SPR genes, SPRI and SPRQ, were studied to identify their phenotypes. Both mutant strains produced viable spores, but sprI mutant spores were less heat-resistant than the wild-type spores. There was no difference detected between spr? mutant spores and wild—type spores under the conditions used in this study. The double mutants, sprI/Q and sprQ/Q, were constructed to test for interaction among these three genes. The results indicate that the sprI/Q double mutant spores were very delicate and were very sensitive to heat, while the spr2/9 mutant spores were less sensitive to heat than spr1/9 spores but more sensitive to heat than the spr9 mutant spores. These observations suggest that the SPRI and SPR9 gene products are involved in thermotolerance and germination of spores. To my parents ii ACKNOWLEDGEMENTS I am grateful to my advisor Dr. P. T. Magee, for his guidance, patience, financial support and many other arrangements, and my committee members, Dr. B. Sears, Dr. L. Snyder, Dr. M. Pluck and Dr. R. Brubaker, who have given me many valuable suggestions throughout my research. I also would like to thank all the people in Dr. Magee’s and Dr. Jackson’s labs for their help and friendship. Among them, I am especially indebted to Dr. D. A. Primerano for his encouragement and many useful discussions, and Ms. Edith Schlemmer who prepared media and cleaned up the mess I made. Finally, my special thanks go to Dr. E. Wanner, for her kind and careful help in reading the manuscript, my husband, Jih-Shyr Yih, for his love and his expert assistance in preparing the manuscript for typesetting, and all my friends who have made my stay at East Lansing happier and easier. iii TABLE OF CONTENTS LIST OF TABLES ................................ vi LIST OF FIGURES ............................... vii CHAPTER I. Literature Survey ........................... 1 1.1 A short history of the yeast Saccharomyces cerevisiae . . . . . 1 1.1.1 Yeast as a model system for studying developmental processes of eukaryotic cells ............. 1 1.1.2 The life cycle of yeast ................. 2 1.2 Events in sporulation ...................... 4 1.2.1 Three developmental stages in sporulating cells . . . 4 1.2.2 Morphological development during sporulation . . . 5 1.2.3 Macromolecular synthesis and breakdown during sporu- lation ......................... 6 1.3 The regulation of sporulation .................. 10 1.3.1 Control of sporulation ................. 10 1.3.2 Genetic control of meiosis and spore formation . . . 11 1.4 Sporulation-specific genes and their mRNA and proteins . . . 13 1.4.1 Sporulation-regulated genes .............. 13 1.4.2 Identification of sporulation-specific proteins . . . . 15 1.5 Phenotypes of sporulation-specific genes ............ 19 1.6 Focus of this study ........................ 23 II. Materials and Methods ........................ 24 2.1 Strains and media ........................ 24 2.2 Culture conditions ........................ 24 2.3 Testing of free spores for thermoresistance and germination . 26 2.4 Construction of plasmids .................... 29 2.5 Yeast transformation ....................... 30 2.6 Tetrad analysis and micromanipulation ............ 32 2.7 Nucleic acid preparation ..................... 34 2.7.1 Preparation of plasmid DNA ............. 34 2.7.2 Preparation of yeast genomic DNA ......... 35 2.7.3 Preparation of RNA .................. 36 2.8 Contour-clamped homogeneous electric field gel electrophoresis 38 2.9 RN ase protection ......................... 39 2.10 DNA sequencing ......................... 40 III. Analysis of the Sporulation-Specific Gene, SPRQ ........ 43 3.1 Cloning and mapping of SPR9 ................. 43 3.2 Developmental regulation of SPRQ ............... 43 3.3 Kinetics of the transcription of SPR9 .............. 47 3.4 Chromosomal location of SPRQ ................. 48 3.5 Determination of the sense strand of SPRQ .......... 48 3.6 DNA sequencing of SPRQ locus ................. 51 IV. Phenotypic Analysis of Three SPR Genes, SPRI, SPRQ, and SPRQ ................................... 57 4.1 Disruptions of SPRI, 2, and .9 ................. 57 4.2 Detection of SPR messages in single mutants ......... 61 4.3 Phenotype detection of SPRQ .................. 61 4.4 Detection of the SPRI phenotype ................ 71 4.5 Phenotype detection of SPRQ .................. 73 V. Construction and Analysis of Double Mutants ......... 80 5.1 Construction of double mutants defective at two SPR loci . . 80 5.2 Phenotypes of the double mutants ............... 82 5.3 Multiple heat effect on germination ............... 88 5.4 Linkage between SPRQ and LY52 ................ 88 5.5 Linkages among SPRI, SPRQ, and ADE? ........... 90 VI. Discussion ................................ 97 BIBLIOGRAPHY ...... - .......................... 105 2.1 2.2 4.1 4.2 4.3 5.1 5.2 5.3 5.4 5.5 LIST OF TABLES List of strains ............................... List of media. .............................. Germination of wild-type and mutant spr9 spores ........... Germination of wild-type and sprl mutant spores ........... Germination of wild—type and spr? mutant spores ........... Thermotolerance of double mutants ................... Thermotolerance of double mutants by micromanipulation followed by incubation at 42°C .......................... Multiple temperature effect on germination. ............. Linkage analysis of three SPR genes. ................. The three types of asci generated from ADEQ/adc? ADEX/adar strain. KJ CD *1 m 96 Figure 1.1 2.1 2.2 2.3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 4.1 4.3 4.4 4.5 4.6 LIST OF FIGURES The life cycle of S. cerevisiae. ..................... 3 Free spore preparation for thermotolerance and germination. . . . . 28 Restriction maps of four plasmids .................... 31 Micromanipulation of tetrads and spore-spore matings. ....... 33 The restriction map of the SPRQ locus. ................ 44 Slot blot analysis of the SPRQ transcripts. .............. 46 The kinetics of the appearance of SPRQ. ............... 49 The chromosomal location of the SPRQ locus. ............ 50 Determination of the sense strand of SPRQ ............... 52 The sequencing strategies for the SPRQ locus and flanking regions. . 54 The nucleotide and deduced amino acid sequence of the SPRQ locus. 55 The homologous sequences in the upstream regions of the SPR genes. 56 One step gene disruption ......................... 58 Disruption of the three SPR genes. .................. 60 Southern blot analysis of the three SPR loci .............. 62 The growth curve of the wild-type and spr9 mutant strains. . . . . . 64 The sporulation of the wild-type and spr9 mutant strains at 30°C. . 65 The formation of small colonies during germination at temperature 2 37°C. ................................. 67 vii 4.7 4.8 4.9 4.10 4.11 4.12 5.1 5.2 5.3 5.4 Germination efficiency of wild-type and sprQ mutant spores. Germination efficiency of wild-type and sprl mutant spores. Thermotolerance of the wild-type and spr] mutant spores in SC MS-7 derivatives. ............................... Thermotolerance of the wild-type and sprl mutant spores in W 66—8A derivatives. ............................... Thermotolerance of the wild-type and sprI mutant spores of SPBI. Germination efficiency of wild-type and sprfi? mutant spores. Construction of mutants defective at two SPR loci. ......... Southern blot analysis of the heterozygous strains ........... Southern blot analysis of the spr double mutants. .......... Sporulation efficiency of double mutants. ............... viii CHAPTER I Literature Survey 1.1 A short history of the yeast Saccharomyces cerem’sz’ae 1.1.1 Yeast as a model system for studying developmental processes of eukaryotic cells In eukaryotes, the mechanisms involved in the sequential activation of genes dur- ing differentiation and development are not well understood. Among the eukaryotic systems currently in use, S. cerevisiae is one of the most amenable systems for this kind of study. The chromosome behavior of S. ccrevisiae is typical of higher eukary- otic cells, while it possesses all of the technical advantages of microbial systems. The landmark events of the sporulation process of S. cerevisiae have been well defined genetically, morphologically, and biochemically (Esposito and Klapholz, 1981). The recovery of all four meiotic products and the presence of well-characterized mitotic recombination events provide useful probes to analyze the interactions among gene functions involved in meiotic recombination and the mitotic process. Yeast is also easy to manipulate experimentally. Site-specific mutations can be easily created by gene disruption and replacement techniques. Large numbers of single cells can be stimulated to undergo meiosis in response to a simple change in medium. Meiosis can be interrupted and viable cells recovered at various stages of development. Another 2 important feature of using yeast as a model system is that there are many mutants of meiosis, cell cycle division, and metabolism available to be used in different studies. 1.1.2 The life cycle of yeast S. cerevisiae can grow vegetatively by budding as a haploid cell or as a diploid cell. There are three distinct cell types of S. cerevisiae: a haploid, a haploid, and a/a diploid (Figure 1.1). The type of any cell is determined by alleles, a or a, at the mating type locus, MAT (Herskowitz and Oshima, 1981). This MAT locus controls a variety of cellular processes including agglutination, phermone production, mating, and sporulation. The a haploid has the a allele at the MAT locus, whereas the a haploid has the (1 allele at the same locus. When haploids of opposite mating type meet, they will mate efficiently under the direction of the mating phermones, a-factor and o-factor, produced by a—haploid and a-haploid respectively. The mating phermones secreted by an a- or a-haploid can cause the cells of opposite mating type to arrest at the G1 stage of the cell cycle, to undergo morphological and physiological changes, to agglutinate, and then to fuse with the cells of opposite mating type to form a diploid zygote containing both a and a alleles at the mating type locus (Thorner, 1981). The a/a diploid formed can grow mitotically, undergo sporulation to produce two a-ascospores and two a-ascospores, but can no longer mate. Diploid cells that are homozygous at the mating type locus are asporogenous but they can still mate. Diploids homozygous and heterozygous at the MAT locus differ in their response to sporulation medium, providing substantial information about sporulation-specific events: a/a behaves as a, and 0/01 behaves as 0; hence the gene dosage of the mating type loci is unimportant. Therefore, even higher ploidy strains can. be constructed. ® 00““ ( Budding .J Haploids Germination Mating Figure 1.1: The life cycle of S. cerevisiac. 1.2 Events in sporulation Meiosis and spore formation in yeast are generally triggered by deprivation of a source of nitrogen and the presence of nonfermentable carbon source (Olempska- Beer, 1987). These processes are coordinately regulated and are accompanied by major biochemical and morphological changes. During sporulation, considerable macromolecular synthesis and degradation occur (Hopper et al., 1974; Illingworth et al., 1978; Kraig and Haber, 1980). Within several hours after transfer to sporulation medium, vegetatively growing cells will complete their mitotic cell cycle and arrest in G1 phase (Esposito and Esposito, 1974) in order to initiate meiosis and spore formation. 1.2.1 Three developmental stages in sporulating cells Three developmental stages have been described with respect to commitment to sporulation (Simchen et al., 1972). During the first stage, cells achieve “readiness” which is defined as the period when cells after exposure to sporulation medium are able to sporulate in water without further stimulus by the medium or revert to vegetative growth if transferred back to growth medium. Readiness begins 3 hours after exposure to sporulation medium and then lasts for about 4 hours. At this point, the cells are not yet irreversibly committed to sporulation. The second stage, “precommitment”, is very brief and is detectable after about 7 hours in sporulation medium. During precommitment, cells exhibit readiness but are thought to lose their ability either to return to vegetative growth or to sporulate in nutrient medim'n. The third stage, “commitment to sporulation”, begins at 7 to 8 hours incubation in sporulation medium. At this stage, cells sporulate even when challenged with growth medium. Irreversible commitment to sporulation occurs after premeiotic 5 DNA replication and initiation of commitment to genetic recombination, coincident with SPB (spindle pole body) separation at pachytene of meiosis. 1.2.2 Morphological development during sporulation After arresting at G1 phase, the cells gradually change their shapes from elon— gated to round, increase in size, and eventually form tetranucleate asci. During sporulation, the chromosomes are replicated and recombine, and two meiotic di- visions occur to yield four haploid nuclei. Toward the end of the second meiotic division, the nuclear membrane buds at each of the four poles, and a prospore wall initiates development at each nuclear lobe (Moens, 1971). The inner membrane of the prospore-wall becomes the spore plasmalemma, the outer membrane apparently contributing to the surface protein layer of the spore (Lynn and Magee, 1970; Illing— worth et al., 1973; Beckett et al., 1973). At prospore—wall closure, four ascospores with nucleus are formed. During sporulation, each cell contains only one mitochondrion,which is highly branched and extends throughout much of the cytoplasm. As prospore-wall forma- tion begins, the single mitochondrion, which surrounds the parental nucleus (Stevens, 1978), is distributed into the ascospores in development. A significant portion of the mitochondrion, however, is not incorporated into the ascospores and remains behind in the ascus cytoplasm. The distribution of the mitochondrion into the ascospores is apparently a nonrandom process. 6 1.2.3 Macromolecular synthesis and breakdown during sporulation DNA metabolism When a/oz diploids are transferred to sporulation medium, there is a small initial increase in incorporation of precursors into DNA during the first 2 hours as cells complete mitotic rounds of replication. The DNA content per sporulating cell then doubles during a discrete period beginning at 4—6 hours and ending at 8—10 hours (Roth and Lusnak 1970). In general, the increase in DNA content in a culture is proportional to the number of sporulating cells in the culture. The precursors for premeiotic replication may be derived primarily from the extensive turnover of RNA during sporulation (Simchen et al., 1972). Since there are events that occur in meiosis but not in mitosis, for example chromosome pairing and genetic exchange, it is possible that there are some unique gene functions for premeiotic replication that are nonessential for mitotic replication. From studies of cdc and spa mutants, it is suggested that mitotic and meiotic DNA replication are under separate genetic control but share some functions in common (Esposito and Esposito, 1978). In addition, the association of replication with other landmark events also differs in meiosis and mitosis. RNA metabolism There is a general increase in RNA synthesis immediately after induction of sporu- lation, and all classes of RNA are made during sporulation. Most of the synthesis occurs between 4 and 10 hours, reaching a maximum at 6 hours, regardless of the sporulation regimen (Hopper et al., 1974). The RNA polymerase activities have also been reported to change during sporulation (Magee, 1974). Extensive RNA 7 degradation was observed along with RNA synthesis (Croes, 1967; Hopper et al., 1974). A rise in the level of RNase within the first 8—12 hours after transfer to sporulation medium, specific for a/a sporulation-competent diploids, has also been reported (Tsuboi, 1976). Therefore, the total amount of RNA per cell appears to decline at the end of sporulation. In a/a diploids, approximately 50—70% of to- tal preexisting RNA is degraded, whereas in a/a and a/a cells, about half of that amount is broken down. These data suggest that at least a portion of the RNA degradation is sporulation-specific (Hopper et al., 1974). Protein metabolism Protein synthesis is necessary for the occurrence of most major events of sporu- lation (Magee and Hopper, 1974). There is a 10—35% increase in net amount of cellular protein during the first 6—10 hours of sporulation, and the net amount of cellular protein declines thereafter (Peterson et al., 1979). The protein patterns produced by one-dimensional and two-dimensional gel analyses showed differences between the vegetative state and the sporulating state of a/oz diploids. When the protein patterns of asporogenous a / a cells and a/oz cells were compared to a / cr cells in the same state, there was no detectable difference (Kraig and Haber ,1980). How— ever, several proteins have been detected which have differences in activities and in abundance between vegetative and sporulating states (Ballou et al., 1977; Colonna and Magee, 1978). A number of explanations have been proposed for the failure to find sporulation-specific proteins (Hopper and Hall, 1975; Peterson et al., 1979). All suggest that only a few crucial genes that require both MA Ta and o: for expression exist and that the products of these genes may be too few or too low in concen- tration to detect. Alternatively, such proteins may have poor solubility , extremely low isoelectric points, or low molecular weights, rendering them undetectable by the techniques used currently. One activity that may truly be unique to a/Oz sporulating cells is an a~1,4—glucosidase (Colonna and Magee, 1978). There is no activity in vegetative cells or in sporulating a/a cells prior to 8 hours and only trace levels of activity in 0/01 cells at any time during incubation in sporulation medium. The increase in protein content is not specific to a/a cells (Hopper et al., 1974). Extensive degradation of vegetative proteins occurs during sporulation (Betz and Weiser, 1976; Betz, 1977), and ranges from 25-70% in different studies. During sporulation, there is an increase in the activities of three proteinases: proteinase A, B, and C, and this is a/oz specific (Klar and Halvorson, 1975; Betz and Weiser, 1976). No new proteinase activities have been observed during sporulation (Klar and Halvorson, 1975). Diploids homozygous for a mutation that results in a low level of proteinase—A activity are unable to complete sporulation (Betz, 1975). Diploids defective at the structural gene of proteinase-B can complete sporulation but form asci that are abnormal in appearance (Zubenko et al., 1979; Zubenko and Jones, 1981). However, diploids defective at the PRC 1 -I locus have no proteinase-C activity but can sporulate normally (Wolf and Fink, 1975). The increase in proteinase C is not significant during sporulation (Betz and Weiser, 1976). Zubenko and Jones (1981) have shown that proteinases B and C are responsible for approximately half of the total amount of protein degradation during sporulation. Both proteinase and ribonuclease activities have been localized to the yeast vacuole (Hasilik et al., 1974), which undergoes extensive fragmentation both in a/cr and a/a cells ('l'flrew et al., 1979). Carbohydrate metabolism Sporulation is accompanied by the extensive accumulation of intracellular car- bohydrates, including trehalose and glycogen as reserve materials and mannan and glucan as the major carbohydrate components of yeast cell wall. The carbohydrates are synthesized to the same extent in both sporulating and non-sporulating cells af- ter transfer to sporulation medium and are considered to be a response to starvation conditions (Kane and Roth, 1974); however only sporulating cells exhibit a period of glycogen degradation coinciding with the appearance of mature spores. The enzyme which degrades glycogen, glucoamylase, appears specifically during sporulation. The accumulation of carbohydrates causes a dramatic increase (70%) in dry weight to occur early in sporulation (Croes, 1967; Esposito et al., 1969). Trehalose, which can be found in ascospores, is synthesized continuously during sporulation. rising to a level approximately tenfold higher than that in vegetative cells (Kane and Roth, 1974). At present, only trehalose accumulation has been shown to be required for ascus production, as mutants defective in its accumulation do not sporulate (Lillie and Pringle, 1980). Substantial degradation of glycogen specific to a/o- cells takes place late in sporulation, during the appearance of asci (Hopper et al., 1974; Kane and Roth, 1974). There are also other data that suggest that the activity responsible for glycogen degradation is sporulation-specific. For example, glycogen degradation depends upon new protein synthesis during sporulation (Magee and HOpper, 1974) and is inhibited by the addition of nitrogen (Opheim, 1979). During this same period in a/a cells, a tenfold increase in specific alkaline phosphatase activity occurs, which may be involved in the metabolism of phosphorylated sugars to glucose (Fonzi et al., 1979). These two events are thought to be responsible for the accumulation of glucose during the late stage of sporulation. 10 Lipid metabolism Cytological studies demonstrate the accumulation of lipid vesicles in the cyto- plasm during sporulation and they play a crucial role in spore-wall development. Biochemical studies have also shown that extensive lipid synthesis takes place during sporulation (Esposito et al., 1969; Illingworth et al., 1973). There are two different periods for lipid synthesis during sporulation: the first period occurs between 0— 10 hour and is not specific for a/a cells; the second period occurring after 20 hours in sporulation medium, lasting for 5—6 hours, coincides with the time of the first appearance of mature asci, and is specific to a/a cells and consists almost entirely of neutral lipid synthesis. 1.3 The regulation of sporulation 1.3.1 Control of sporulation In the yeast Saccharomyces cerevisiae, there are two parallel regulatory paths that govern entry into meiosis. They are the environmental signal, starvation, heterozy- gosity at MA T, which causes arrest of the mitotic cell division cycle at G1 phase, and the genetic signal, MATal and a2 (mating type locus), which activate the meiotic pathway (Mitchell, 1988). These two regulatory paths converge at some point to ac- tivate those sporulation-specific genes that are expressed only in a/a diploids during starvation. However, it should be noted that a very low, but detectable, fraction of a/a and 0/01 cells sporulates (Zakharov and Kozhira, 1967). There are several genes involved in nutritional control of sporulation; they are CYRI (adenylate cyclase [Matsumoto et al., 1983]), RASI and HAS? (stimulators of adenylate cyclase [Toda et al., 1985]), and genes encoding cAlV’IP-dependent protein kinase (Toda et al., 1987). CAMP-dependent protein kinase inhibits the entry of 11 cells into meiosis, but starvation can inhibit CAMP accumulation and thus inhibit the activity of the protein kinase (Tatchell, 1986). Glucose and other sugars repress sporulation by the suppression of gluconeogenic enzymes (Miyake et al., 1971). The regulation of sporulation by the MAT locus is mediated by the RME] (reg- ulator of meiosis) gene product. RMEI is expressed in haploid cell, its product inhibits meiosis. In a/a diploids, a1-a2 negatively regulates R/WEI expression and thus leads to derepression of meiosis and spore formation (Mitchell and Herskowitz, 1986). Two sporulation-specific genes, [MEI (inducer of meiosis) and IMEQ, are essential for meiosis and sporulation. MATa1-a2 may activate the expression of sporulation-specific genes through expression of [ME] (Kassir et al, 1988). Smith and Mitchell (1989) recently proposed that the main role of [ME] is to activate the expression of IMEQ, through which to induce the expression of other sporulation- specific genes. 1.3.2 Genetic control of meiosis and spore formation All of the cdc mutants, other than those defective in bud emergence and cytokine- sis, are also defective in meiosis. The rad mutants, particularly those involved in the repair of X-ray damage, are blocked in sporulation probably because of their involve,- ment in meiotic recombination and repair. The rationale is that meiosis and mitosis require common recombination functions. The cell types determined by the mating type locus also affect sporulation. The MAT mutants were examined because of the requirement for sporulation of heterozygosity at the mating-type locus. There are two codominant MAT alleles, MATa and MATa. The MA Ta allele produces two transcripts, but only a1 seems to carry out .MA Ta functions. .MA Ta is transcribed in opposite directions to give rise to two transcripts, a1 and Q2. The gene product of 12 MA Tal is thought to be a positive regulator of a-specific functions, while the gene product of MA Ta2 is thought to be a negative regulator of a-specific functions. Since neither the al nor the 0:2 transcript are synthesized in a-type haploids, a-specific genes are not turned on and a-specific genes are not repressed. Therefore, a MA Ta cell is constitutive for a-specific functions, is able to produce a-factor and is able to mate with an a-type cell. Both 01 and a2 transcripts are made in a-haploids, so the a-specific genes are expressed and the a-specific genes are silent in these cells. The a haploid can thus produce a factor and mate with an a-type cell. In MA Ta/ MA Ta diploids, the (12 product inhibits expression of the a-specific genes, while the a1 product inhibits the transcription of a1, preventing the turn on of transcription of the a-specific genes. These cells produce neither a factor nor a-factor and are un- able to mate. One reason that MATal- and MA Ta2-gene functions are necessary for sporulation is that they turn off RMEI (Mitchell and Herskowitz, 1986). A number of other nuclear genes are also required for sporulation. These include the numerous PET (petite) loci required for mitochondrial function, loci required for fatty acid metabolism (Keith et al., 1969), and loci required for the TCA cycle (Ogur et al., 1965). Mutations in any of these loci render the cells respiratory-deficient and, hence, incapable of sporulation. Several loci involved in amino acid biosynthesis are also required for sporulation (Lucchini et al., 1978). For example, met:9 and metIS’ mutations are methionine-auxotrophic and also result in an imbalance in different rRNA species and a lack of sporulation. These defects are reversible by the addition of methionine to the sporulation medium (Wejksnora and Haber, 1974). 13 1.4 Sporulation-specific genes and their mRNA and pro- teins 1.4.1 Sporulation-regulated genes It was established earlier that sporulation in S. cerevz'siae is dependent upon both sporulation-specific genes and certain vegetative genes. Genetic analyses have suggested that at least 50 loci code for functions indispensable to the process of meiosis and sporulation (Espsito et al., 1972). These loci contain genes affecting both early and late events in sporulation. Mills (1980) reported the first evidence for a sporulation-specific mRNA pop- ulation, about 7% of total mRNA. Recently, sporulation-specific genes have been isolated by selecting sequences that are preferentially transcribed during sporula- tion. They were named SPR genes (Clancy et al., 1983), SP5 genes (Percival-Smith and Segall., 1984), and SIT genes (Gottlin-Ninfa and Kaback., 1986) by different groups. The SPR genes were isolated from a Lambda Charon 28 library of yeast genomic DNA by using two cDNA probes; one complementary to polyA(+) RNA isolated from an a/a diploid incubated in sporulation medium, and the other com- plementary to poly(A)+ RNA of an a/a diploid under same situation. The Lambda plaques that hybridized to the a/a probe, but not the 0/0 probe were considered to contain sporulation-specific DNA. Twenty four different SPB genes were found to be represented by the 46 clones identified. In another differential hybridization screen, Percival-Smith and Segall isolated 15 different sequences, from a pBR322- yeast genomic DNA library, that represented 14 different sporulation-specific genes (SP5 genes). Gottlin-Ninfa and Kaback isolated genes, again from a gene—size insert- Lambda recombinant DNA library, which are complementary to a/a-dependent sporulation-induced transcripts. 14 The regulation of gene expression during sporulation could occur at the level of transcript accumulation or posttranscription. Each sporulation-specific expressed se- quence may have a function in sporulating cells, or these transcripts may be merely the result of a generalized relaxation of control in which many transcribable DNA sequences are expressed. The former case is similar to some other developing sys- tems characterized by the appearance of specific transcripts, e.g., globin transcripts in reticulocytes. In the latter case, specific transcripts would not always have dis- tinct or even definable functions, e.g., the lampbrush-chromosome stage found in oocytes of amphibians and other higher organisms(Davidson, 1976). In Aspergillus nidulans, some of the sporulation-specific RNAs are also present in hyphae but in a nonpolyadenylated form which later becomes polyadenylated during sporulation (Timberlake, 1987). These results suggest that posttranscriptional control mecha- nisms may play a role in the development regulation of gene expression. Kaback and Feldberg (1986) suggested the existence of a developmentally regu- lated program of gene expression for sporulation of S. cerevisiae. Various transcripts appeared and disappeared with different kinetics throughout this process. These pat- terns were not observed in asporogenous a- and a/a cells incubated in sporulation medium where all transcripts appeared to have similar kinetics. Those transcripts that became most abundant at 1 to 3 hours in the sporulating cells behaved almost identically in the asporogenous cells. Accordingly, this early behavior may not be part of the a/a-dependent sporulation-specific program. In contrast, only sporu- lating cells had the transcripts most abundant at later times. Notably, all cloned sporulation-specific transcribed sequences so far investigated also appear to be most highly expressed at these later times (Clancy et al., 1983; Percival—Smith and Segall, 1984). Consequently, the period after 3 hours would represent the predominant 15 period during which any developmental program would be operating. The accumulation of SPB transcripts in yeast cells during sporulation was ex- amined by Holaway et a1. (1987). They determined the transcription rates of three SPR genes, SPRI, SPRQ, and SPR3, using a filter binding assay. SPRI and SPB? mRNA abundance increased from less than 0.7 copies per cell in vegetative and early sporulating cells to 130 and 90 copies per cell respectively 8 hours after transfer to sporulation medium. Similarly, the level of fusion protein of SPR3—LacZ increased at least 700-fold. The accumulation of these SPR mRNAs may be due to the enhanced transcription of the corresponding genes. It is also possible that the SPR mRNAs ac- cumulate because of their increased stability. The average half-life of mRN A (20 min) and the proportion of polyA(+) RNA (1.3—1.4%) has been shown to be similar in vegetative and sporulating cells (Kraig and Haber, 1980). However, the results of Holaway et al. could not distinguish between these two possibilities. 1.4.2 Identification of sporulation-specific proteins One of the disadvantages of using yeast to study meiosis is the impermeability of sporulating cells to radiolabeled precursors. As cells progress in the sporulation pro- cess, they become more impermeable. In the past, attempts to elucidate the changes in sporulating cells at the protein level have not been very successful, although these earlier studies did identify changes in the electrophoretic pattern of proteins in both sporulating cells and asporogenous cells incubated in sporulation medium (Hopper et al., 1974; Peterson et al., 1979; Trew et al., 1979). If the changes in protein pattern observed are associated with changes in gene expression rather than modifi- cation of existing proteins, it should be possible to show that the mRNA content of the cells alters during meiosis and sporulation. Earlier studies could not distinguish 16 between those two possibilities. By using an in vitro translation system, one can prove that specific mRNAs can be translated at certain periods. VVeir-Thompson and Dawes (1984) first applied this system to study protein changes during sporula- tion in S. cerevisiae. Developmentally related changes do occur in the translatable mRNA population of S. cerevisiae during sporulation. Extensive degradation of total cellular RNA and a concomitant increase in RNase activity specific to sporulating cells is reflected in a specific decline in the functional mRN A population during the later stages of spore development. The availability of functional mRNA increased by about 25% during the first 4 hours after transfer of either sporulating or non- sporulating diploids to sporulation medium. Thereafter functional mRNA decreased such that in the a/a strain after 24 hours there was only about 50% of the amount in vegetative cells; a less marked decrease was observed in a/ a and a/a strains. A significant number of sporulation-specific changes in individual mRNA species was found to occur during meiosis and spore formation, although it is interesting that only four were found to involve completely new transcripts; the remainder were due to marked changes in the abundance of the mRN A species. Kurtz and Lindquist (1984) also observed changing patterns of gene expression during sporulation in yeast. They isolated RNAs from a/a sporulating cells and asporogenous a/ a and. a/a cells and determined their protein-coding capacities by translation in cell-free lysates. The ability of an RNA sample to stimulate incorpo- ration of labeled amino acids into protein in a cell-free translation system was taken to represent the availability of functional mRN A sequences in the sample. They also found that there is a rapid increase in the amount of RN As encoding a broad range of polypeptides 2 hours after transfer to sporulation medium in both a/a and a/ a cells. But two sets of abundant sporulation-specific mRNAs are induced in a strict 17 temporal order later in sporulation. The first appeared after 6 hours and encoded polypeptides of 17, 20, 25, 31, 38, 50, 65, and 68.5 kd. These RNAs were maxi- mally induced between 8 and 14 hour and then began to disappear. Their pattern of synthesis is similar to that of the four new transcripts seen by VVeir-Thompson and Dawes (1984). The second set of RNAs, encoding 21.5 kd and 34 kd polypeptides, was induced after 16 hours. These two RNAs continued to accumulate late into sporulation and were packaged into spores in translatable forms. These results are also in good agreement with the results of Clancy et al. (1983). These 10 sporulation- specific proteins were considered an absolute minimum, since this system can only detect mRNAs that accumulate in substantial quantities. Kurtz and Lindquist (1986) related the pattern of expression of those two sets of mRNAs to the morphological landmarks of sporulation to get some information about the functions encoded by these mRNAs. The induction of the first set of sporulation-specific mRNAs coincides with the initial events of nuclear budding and spore wall formation which occur in 8 hour sporulating cells. During the period when these messages are detected, spore walls increase dramatically in thickness (10 hours). By 16 hours, the spores have reached their mature size and the walls appear fully synthesized. At this point, the first set of sporulation-specific RN As is rapidly degraded. The first set of mRNAs was only found in ascal cytoplasm, and the later set of mRNAs was only found inside spores. The limitation of location of these two sets of mRNAs may be due to the degradation of RN As in the external com- partment when spore wall synthesis is complete. Thus, what appears to be selective degradation of these messages midway through spore development may be a simple consequence of their location in a compartment where degradation predominates. The first set of sporulation-specific RNAs encodes at least 8 polypeptides. The 18 authors suggest that these polypeptides are involved in spore wall assembly based on several observations: 0 The intensity of RNA translation in vitro indicates that the corresponding mes- sages are very abundant and that they most likely encode structural proteins. 0 These messages appear at the tetranucleate stage of meiosis, and they are degraded a few hours later. Their appearance is coincident with the initiation of spore wall synthesis. 0 Components of the wall must cross the membrane in order to be deposited within the double membrane. The translation behavior of the first set of mRNAs in vitro suggests that their translation is associated with membranes. The second set of sporulation-specific RNAs which is found only in spore compartment encodes two polypeptides. The authors suggested that the functions encoded by these RNAs may be involved in establishing the final stages of dormancy or may be stored for use during spore outgrowth. There are also some proteins with known functions that are sporulation—specific. The enzyme which degrades glycogen, glucoamylase, appears specifically in sporu- lating cells (Colonna and Magee, 1978). The expression of the sporulation-specific glucoamylase gene (SGA) is positively regulated by the MAT locus, both MA T211 and MA T02; and the control of SGA is exerted at the level of transcription. Expres- sion of SGA and the consequent degradation of glycogen to glucose in sporulating cells is not required for meiosis or sporulation, since a/a diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores. Diploid cells carrying the mutations in MATal and MATa2 could neither undergo sporulation 19 nor produce glucoamylase activity, while the mata2 and matal mutations affected neither sporulation nor enzyme activity. These results indicate that both a1 and 012 are essential for this enzyme production (Yamashita and Fukui, 1985). Another enzyme required for glucosamine synthesis, 2-amino-2-deoxy-D-glucose-6-phosphate keto-I-isomerase, is also sporulation-specific (Ballou et al., 1977). The isomerase enzyme activity is detected at the tetranucleate stage of meiosis. Mutation at this locus produces structurally abnormal, but viable, asci. 1.5 Phenotypes of sporulation-specific genes Aspergillus nidulans is another fungus that has been used as a model system to study development. Timberlake (1987) placed mutations that affect development in A. nidulans into two classes. The first class includes those mutants that prevent or alter the timing of initiation of conidiation. Genes in this class, regulating conversion from vegetative growth to conidiation, have been termed strategic by Clutterbuck (1969). The second class, termed auxiliary genes by Clutterbuck, are those whose mu- tations directly affect the formation of conidiophores or conidia, but mutant strains can initiate conidiophore development normally. These mutations can be further di- vided according to their functions. They include: genes that directly determine the structure and specialized physiological activities of conidiophores, genes that result in the formation of morphologically abnormal conidiophores or conidia, and genes that are expressed specifically during conidiation, but which have unknown physiological functions or little or no effect on the morphology or the Viability of conidiophores or spores. The number of genes that are expressed preferentially during conidiation in A. nidulans has been estimated directly by hybridization of an excess of polyA(+) RNA 20 from conidiating cultures with sporulation—specific and sporespecific cDNA probes (Zimmermann et al., 1980). The resulting data suggested that about 1200 putative mRNAs accumulate during conidiation and are absent from or present at low levels in hyphae. Of those 1200 mRNAs, approximately 1000 accumulate in conidiating cultures only. The remaining 200 accumulate specifically in mature conidia. It is likely that these RNAs encode enzymes or structural proteins that have specific functions in spore differentiation or spore germination. The same rules can be used to classify developmental genes of S. cerevisiac. Surprisingly, most sporulation-specific genes of A. nidulans and S. cerevisiae that have been cloned and characterized show no effect on sporulation and spore germination in strains containing these mutations. Segall’s group has worked on five SP5 genes, 5P5], SPSQ, SPSS’ (Percival-Smith and Segall, 1986), 5P54 (Garber and Segall, 1986), and SPSIOO (Law and Segall, 1988). Only two SP5 genes show alternate phenotypes when disrupted. SPSI is indispensable for completion of sporulation, and SPSIUO is required for spore wall formation and ascus maturation. Three SP5 genes, SPSI, 51352, and SPSS’, have been shown to be closely linked and they encode 1.9 kb, 1.6 kb, and 1.75 kb tran— scripts, respectively. Although SP52 expression is not essential for sporulation, Percival—Smith and Segall (1987) showed that increased copy number of the 5’ end of the SP5? sequence inhibits sporulation of S. cerevisiac. In other systems, the introduction of multiple copies of a DNA regulatory sequence into a cell may result in the in viva titration of transcriptional regulatory factors. Such studies can thus delineate control sequences and has demonstrated that common factors are involved in the expression of distinct genes. However, in the case of SPSQ, the reduction in ascus formation required the presence in the plasmid not only of the 5’ end sequences of the 51352? gene but also of 21 H the initial coding sequence of the gene. This observation suggests that the inhibition of sporulation by increased copy number of the 5’ end of the SP5? gene is not simply due to the competition for the regulatory factors. The authors concluded that the accumulation of a fusion protein containing the amino-terminal portion of the SP5? gene product, synthesized from the transcripts derived from the truncated 5P5? genes present in the high-copy-number plasmid, has an adverse affect on the progression of sporulation-specific events. The asporogenous phenotype is dependent on the synthesis of plasmid-derived SP5? transcripts, and hence requires that a promoter be present. There is not a stringent requiren‘ient for the SPS? promoter itself. The phenotype results from the overproduction of a protein containing the amino-terminal portion of the SPS? gene product. It is possible that this either leads to the accumulation of an aberrant sporulation-specific structure or prevents the assembly of a sporulation-specific structure, which in the former case inhibits and in the latter case is required for the continuation of the temporal progression of sporulation-specific events. Although the SP5? gene product may be dispensable, it nonetheless contributes to a sporulation-specific event. The cell can compensate for the absence of this gene product, but the putative overproduction of its amino-terminal portion is deleterious and prevents the completion of sporulation. The effect of overproduction of the entire protein has not been determined. The 5P54 gene encodes one of the prominent sporulation-specific proteins ob- served among the in vitro translation products of RNA from sporulating cells. From the nucleotide sequence of the SP54 gene, the putative product inferred is 38.6 kd with an isoelectric pH of 8.2 which is similar to the properties of one protein identified by Kurtz and Lindquist (1986). The gene product of 5PS4 is not essential for sporula— tion, since a/a diploids homozygous for a partial deletion of the SPS/, gene appeared 22 to be unaffected in their ability to sporulate and to form viable ascospores. The rela- tive ease with which plasmids containing the SP54 gene were identified suggested the possibility that this gene encodes a relatively abundant sporulation—specific mRNA. Alternatively, it may reflect the fact that the gene is present in several copies in the yeast genome. The 5P5100 gene (Law and Segall, 1988) is activated late in the sporulation process. It is first expressed at 12 hours of sporulation and remains unchanged from 15 to 35 hour. Several lines of evidence suggest that the gene product of SPSIUO may be a spore wall protein and have a protective function for the spores. The level of transcripts of the 5P5100 gene remains steady between 15 and 35 hours, a time coincident with spore wall maturation. The putative 34.2 kd protein encoded by 5P5100 contains a potential signal sequence and cleavage site. The resistance to ether treatment of spores of homozygous mutants of SPSIOO was delayed by 5 11 relative to the wild-type strain. Gottlin-Ninfa and Kaback (1986) also tried to determine the functions of three of the SIT genes, SIT?, SIT?, and SITI. All three genes proved to be dispensable for sporulation and spore germination based on a gene disruption experiment. One SIT? disruption contained a H153 insertion in one part of the gene and a second had a LEU? insertion into the site of a 0.3 kb deletion in another part of the gene. The authors reported the creation of a fusion transcript between a H153 RNA molecule and a 300bp long SIT? RNA molecule, giving a very low but finite possibility of SIT? activity. The other two genes, SI T3 and SIT4, were disrupted by simple inser- tion mutations, and thus might still possess some gene activity. Therefore, further experiments are required to create strains completely lacking SI T3 or SIT/,. message for phenotype exploration. 23 In this lab, we have worked on the SPRI and SPB? genes which are both abun- dantly transcribed late genes (Primerano et al., unpublished data). Disruption of SPRI and SPB? separately does not affect the sporulation process; the disruptants form four viable spores. The results from the DNA sequence of the SPRI gene and an SPRI-LacZ fusion suggested that the SPRI gene product may be a membrane protein and that the gene product is localized to a particulate fraction of the devel- oping spores. These data suggest that the SPRI gene product may have a role in spore wall maturation. The results obtained from three different groups suggest that not all sporulation- induced genes are essential for sporulation and spore germination. On the other hand, since examination of ascus formation and spore viability only monitors the most significant meiotic and morphological events of sporulation, a more careful analysis of spores and their formation may be required to determine the functions of these genes. 1.6 Focus of this study In the first part of this study, one sporulation-specific gene, SPRQ, was ana- lyzed molecularly and genetically. It was also compared to other previously studied sporulation-specific genes for a generalized view of all sporulation-specific genes. In the second part, the SPRQ gene was disrupted and the phenotypes of three SPR genes, SPRI, SPR?, and SPRQ, were analyzed from several different aspects. Two double mutants containing two defective sporulation-specific genes were also con- structed to gain insight into the relationship among these three genes. CHAPTER II Materials and Methods 2.1 Strains and media All strains used in this study and their genotypes are described in Table 2.1. All the media used are listed in Table 2.2. The media were prepared in either liquid form or in solid form. The solid form was made by adding 20 g Bactoagar per liter of media. For growing strains auxotrophic for different kinds of amino acids, media were supplemented with those amino acids required for growth at a final concentration of 40 mg per liter. 2.2 Culture conditions For cell growth and maintenance, the rich medium YEPD was normally used. For auxotrophic phenotype examination, a minimal medium (MIN) with or without various supplements was used. For testing respiratory—competence, YEPGlycerol was used. For regeneration of spheroplasts, solid MIN and YEPD both containing 1 M sorbitol were used. Sporulation in liquid medium was done at 30°C with vigorous aeration. Cells were inoculated into YEPD medium to grow overnight. The following day cells were transferred to presporulation medium (PSP2) supplemented with the auxotrophic 24 25 Table 2.1: List of strains. Strains Genotype Source W66—8A a/a (H0 adeZ-I leuI trp5-2 um3—1 met4-1 13132-1) Rothstein and Sher- man, 1980 W7 same as W66-8A, except spr9::URA3/SPR9 This study W7a same as W66-8A, except spr9::URA3/spr9::URA3 This study W7a-l same as W7a, except LEUI This study WSRBl same as W66-8A, except spr1-1::URA3/SPR1 This lab W61A same as W66-8A (sister spore to W61C) This lab W61C same as W66-8A, except This lab spr1-1::URA3/spr1 -1::URA3 SCMS7-1 a/a (H0 adeX 111294-519 leu?-3,112 This lab SPBl same as SCMS7-1, except spr1-?::LEU2/SPRI This lab 852A same as SCMS7—1, except spr1-2::LEU2/spr1- This lab 2::LEU? S52B same as SCMS7-1 (sister spore to S52A) This lab AKAS2 same as SCMS7-1, except spr2::LEU2/SPR2 This lab T4C same as SCMS7-1, except spr2::LEU?/spr?::LEU? This lab BSl a/a (H0 ADE?-1/ade?-I ADEX/adeX TRP5- This study 2/trp5-2 URAS—I/ura3-1 MET4-1/met4-1 LYS?- 1/lys?-1 HIS4-519/his4-519 LEU2-3,11?/leu2-3,11? spr1-?::LEU?/SPR1 spr9::URA3/SPR9) CS1 same as BSl This study 4-8-a a/a (H0 adeX met4-1 lys2-1 hid-519 spr1-?::LEU? This study spr9::URA3) CT2 same as BS1, except spr?::LEU?/SPR? This study CT3 same as CT2 This study 5-4-d a/a (HO 13132-1 spr2::LEU? spr9::URA3) This study APl a/a A3648. x a131-20 Hopper et al., 1974 APla/a derived from APl a/a Hopper et al., 1974 AP3a/a derived from APl a/a Hopper et al., 1974 YPH149 Hieter, unpub. 1989 26 requirements, to preadapt to respiratory metabolism. When the cell density reached 2—5 X 107 cells/ ml, cells were collected and washed twice in sterile, distilled water, and then transferred to sporulation medium (SP2). Cells began to sporulate after 12 hours of incubation in SP2 medium. For examining the ability to sporulate of a large number of samples, sporulation was performed on solid medium at 30°C. Cells were patched onto YEPD and grown overnight. Patches were then replicated onto PSP2 and incubated 48 hours. Each patch was then transferred to SP2 by toothpicks and incubated for three days. Heat shock was done as follows: Samples were grown in YEPD medium at 30°C until they reached log phase. Before harvesting, cells were transferred to 39°C for heat shock for 60 min. Cells were then used for further preparation. 2.3 Testing of free spores for thermoresistance and germi- nation For testing ascospore thermoresistance, strains were induced to sporulate in 10— 20 ml of SP2 medium as described before. For homozygous strains, equal numbers of asci (1 x 108 asci/ml) from Wild—type and mutant strains were mixed together. By mixing asci from different strains, we could minimize variations in treatment between wild-type and mutant strains that might occur during the preparation of free spores or during the experimental stress. For heterozygous strains, there were two wild-type and two mutant spores produced from one ascus; therefore no mixing between wild-type and mutant strains was needed. The asci were harvested at 16, 20, and 24 hour and washed three times with sterile, distilled water. Free spores were obtained by treating the ascus wall with fl-mercaptoethanol at 23°C for 30 min followed by glusulase (Endo Laboratories) treatment at 37°C until more than 90% 27 broken asci were observed in 5% SDS (Sherman et al., 1981). B-mercaptoethanol and glusulase were removed by washing three times in sterile, distilled water. The spores were resuspended in 10 ml of sterile water and then disrupted by three twenty second bursts of sonication at 150 mA. This converted 99% of all tetrad clusters to single spores. At this point, the numbers of total spores in each preparation were determined by plate count. Spore preparations were then distributed into individual vials and incubated at 4°C, 23°C, 30°C, 37°C, 42°C, and 45°C in various experiments. At several times (0, 24, 48, 72, 96, and 120 hour) spore viability was determined by plating diluted samples onto YEPD plates and incubating at 30°C for 48 hours. The colony counts from YEPD plates were used as the total survivors after heat treatment. Two hundred colonies were picked onto minimal plates with and without the auxotrophic supplements in order to determine the genotype of the spore colony and the percentages of mutant spores among the total survivors. Therefore, the number of wild-type and mutant survivors after heat treatment could be deduced by multiplying the number of total survivors by the percentage of wild-type or mutant spores (Figure 2.1). For testing the germination ability of mutant spores, free spores were prepared as described above. Instead of preincubation for heat treatment, diluted spore suspen- sions were plated immediately onto YEPD plates to germinate at different temper- atures. Plates were incubated at 23°C, 30°C, 37°C, and 40°C for 48 hours or longer depending on their growth conditions. The numbers of colonies formed when incu- bated at temperature T in the experiments were used as total spores. The genotypes of two hundred spore colonies were also determined as before and used to calculate the percentages of wild-type and mutant spores in the experiments. The number of total spores obtained by germinating at 30°C was considered as the starting 28 Sporulating cultures of Sporulating cultures of wild type strain were mutant strain were har— harvested at 16 h, 20 h, vested at 16 h, 20 h. and 24 h. and 24 h. Equal amounts of asci from wild type and mutant strains were mixed for free spore preparation. Glusulase treatment to remove the ascus wall Sonicate to disperse the spores Free spore suspension Heat resistance Germination Spore suspensions were Spore suspensions were preincubated at 4°C, 23°C, spread onto YEPD plates 30°C, and 42°C before after serial dilution and germination. germinated at 30°C. 37°C, ; and 40°C. Spore suspensions were Wild type and mutant spores diluted and spread onto were distinguished by their YEPD plates at the end of auxotrophic markers. The 0 h, 24 h, 48 h, 72 h, 96 h, number of spores germinated and 120 h of incubation at 30°C was considered as and germinated at 30°C. the total number of spores. The germination rate was # obtained by dividing the number of spores germinated at 37°C or 40°C by the total spores. Wild type and mutant spores were distinguished by their auxotmphic markers. Figure 2.1: Free spore preparation for thermotolerance and germination. 29 number of spores before treatment. The number of wild-type spores and mutant spores germinated at 30°C were considered to be the expected numbers, since we supposed that all spores could germinate at 30°C. Therefore, 0 The number of mutant spores germinated at temperature T = (the number of total spores at temperature T) times (% of mutant spores at temperature T) o The number of wild-type spores germinated at temperature T 2 (the number of total spores at temperature T) times (% of wild-type spores at temperature T) O % of germinated mutants at temperature T = (the number of mutant spores germinated at temperature T) / (the number of mutant spores germinated at 30°C) times 100% e % of germinated wild-types at temperature T = (the number of wild—type spores germinated at temperature T) / (the number of wild-type spores ger- minated at 30°C) times 100% 2.4 Construction of plasmids Restriction endonucleases and other DNA modifying enzymes were obtained from Bethesda Research Laboratories or Boehringer Mannheim and were used under the supplier’s conditions. A 5.8 kb ClaI fragment carrying the SPRQ gene from Asp104 was cloned into the ClaI site of pBR322 to give rise to p803. The 1.6 kb SalI-Xhol fragment of p803 was further removed to generate p803s/x and more unique restriction sites. For RNase protection, a 2.2 kb ClaI-SstI fragment containing the SPRQ gene was forced-cloned into a GEM3 and a GEM4 plasmid (Promega company) at Ach and 30 Sad sites to form G38 and G4S. All four plasmids constructed for this study are shown in Figure 2.2. To produce the mutant allele for one-step gene disruption, the 3.0 kb HindIII fragment containing the SPRQ gene of p803s/x was replaced by a 1.1 kb URAS—containing HindIII fragment as shown in Figure 4.2. 2.5 Yeast transformation Yeast transformation was done as described in Sherman et al (1987). Recipient strains were grown in 200—300 ml of YEPD until they reached 2—4 x 107 cells / ml. Cells were harvested by centrifugation in the GSA rotor for 5 min at 8 K. Cells were then resuspended in 20 ml SED (1 M sorbitol, 25 mM EDTA, pH 8.0; 50 mM dithiothreitol (DTT)) and incubated at 30°C for 10 min. After incubation, cells were washed once in 20 ml 1 M sorbitol and resuspended in 20 ml of SCE (1 M sorbitol, 0.1 M sodium citrate, pH 5.8; 10 mM EDTA). Then, 0.25—0.5 ml glusulase (Endo) were added to the suspension which was incubated at 30°C for 30-60 min with occasional shaking. Spheroplasting was assayed by checking the degree of lysis of cells in 5% sodium dodecyl sulfate under microscope. Because spheroplasts are fragile, cells were handled very carefully in subsequent steps. When Spheroplasting had reached 90%, the suspension was centrifuged and the pellet was washed twice in 1 M sorbitol and once in STC (1 M sorbitol, 10 mM calcium chloride, 10 mM Tris- HCl, pH 7.5). Cells were then resuspended in 1.0 ml of STC. Sheared salmon sperm DNA at a final concentration of 50 pg/ml was added to the spheroplast suspension as carrier. The suspension was then dispensed into 10 aliquots. DNA (0.1—5 pg in 1—10 pl) was then added and the tubes incubated at 23°C for 10 min. 1 ml of PEG (20% polyethylene glycol 4,000, 10 mM calcium chloride, 10 mM Tris—HCl, pH 7.5) was added to each tube. After 10 more min at 23°C, the cells were pelleted 31 Hindlll Hindlll G38 ClalIAccl . ISPG 5.1kb 77] Figure 2.2: Restriction maps of four plasmids. 32 carefully, resuspended in 150 #1 of SOS (10 ml 2 M sorbitol, 6.7 ml YEPD, 0.1 ml 1.2 M calcium chloride), and incubated at 30°C for 20 min. Cells were plated out by adding 6 ml of top agar to each tube and immediately pouring onto plates. The plates were incubated at 30°C until the transformants had appeared (usually two days). 2.6 Tetrad analysis and micromanipulation Yeast cells were incubated in SP2 medium until more than 30% of cells formed four-spore asci. The time required differs for different strains, but 16 hours is the usual minimum. The cells were harvested by centrifugation at 4000 rpm for 5 min, then washed with sterile distilled water three times. The samples were resuspended in 5 ml of sterile distilled water and 0.5 ml of glusulase (Endo) was added to the cell suspension. The suspension was incubated at 30°C until free ascospores were visible under the light microscope in a drop of 5% sodium dodecyl sulfate(SDS), usually 30 min. The glusulase was removed immediately by 3 washes in sterile water. The sample was resuspended in a small amount of sterile water, and this suspension was streaked along one edge of a slice of very thin YEPD agar. Individual intact asci containing four spores were picked, moved far from others, and dissected into four separate spores by micromanipulation as shown in Figure 2.3c. After they grew to form colonies, they were streaked onto a YEPD plate to give isolated colonies. They were subsequently checked for auxotrophic requirements. 33 a. Streak one spore suspension on the edge of one side of agar slice, and another suspension on the other side. .‘.'.'.'.'.’.'.'.’.'.'.'.'.'.‘.'.'.'.'.'.°.‘.'.'.°.°.'.'.’.'.'.’.'.'.'.'.'.'.'.'.'.‘.'.'.'.'.'.°.'.'.'. .......... b. "0V e on e four- Spore d a S CU S 6 to one spot far from all other asci with the needle .::::::t:::::::::::::::::::::::::::::::::::::::::::::::::' of m lcromanlpUlatOl". . . o c. Separate the four spores to a line on the 3 ar. e o 2 9 ~ 0 d. One four-spored ascus from o . O on on o . . g. a, the other sample was 0 o "b a. separated to four single 0 0 o co spores in the same way. a o. e. Two spores from different on do strains were pushed side 0' 0’ by side to mate. ~ on Figure 2.3: Micromanipulation of tetrads and spore-spore matings. 34 2.7 Nucleic acid preparation 2.7.1 Preparation of plasmid DNA Large amounts of plasmid DNA were prepared by a procedure described in Krieg and Melton (1984). The plasmids were transformed into appropriate E. coli strains. A single colony carrying the plasmid of interest was picked and inoculated into 10 ml of LB containing the appropriate antibiotic. The culture was incubated at 37°C overnight with vigorous shaking. One ml of the overnight culture was inoculated into 250 ml of the same medium and grown for 12 hours. The cells were harvested by centrifugation at 5,000 rpm, 4°C for 15 min. The pellet was resuspended in 6 ml of freshly prepared lysis solution (25 mM Tris-HCl, pH 7.5; 10 mM EDTA, 15% sucrose, 2 mg/ml lysozyme (Sigma)) by pipetting up and down with a 10 ml pipette. The suspension was incubated in ice water for 20 min. Then, 12 ml of 0.2 M NaOH, 1% SDS were added to the tube. The tube was carefully inverted several times to mix thoroughly, and incubated in ice water for 10 min. Then 7.5 ml of 3 M sodium acetate, pH 4.6 were added. The tube was carefully inverted several times until mixed thoroughly, and incubated in ice water for another 20 min. Then, the tube was centrifuged at 15,000 rpm for 15 min. The supernatant was removed to another tube, and 50 pl of RNase A (1 mg/ml)was added to remove cellular RNA. After 20 min of incubation with RNase A at 37°C, the supernatant was extracted twice with an equal volume of phenolzchloroform (1:1). Then, two volumes of 95% ethanol were added to precipitate the DNA. The DNA was pelleted by centrifugation at 10,000 rpm, 4°C for 15 min. The pellet was dissolved in 1.6 ml of sterile distilled water. 0.4 ml of 4 M NaCl were added to the tube and mixed, and followed by 2 ml of 13% polyethylene glycol. The tube was then incubated in ice water for 60 min. 35 The suspension was centrifuged at 10,000 rpm for 10 min after 60 min of incubation. The DNA pellet was washed with 70% ethanol, and then dissolved in an appropriate volume of sterile distilled water. 2.7.2 Preparation of yeast genomic DNA Yeast genomic DNA was prepared as described in Sherman et al. (1987). 10 ml of cells were grown overnight in YEPD. Cells were harvested at stationary phase and washed in 1 m1 of 1 M sorbitol. Cells were spheroplasted by resuspending in 1 ml of Spheroplasting buffer (1 M sorbitol pH 7.5, 50 mM potassium phosphate, 0.1% fi-mercaptoethanol, and 1 mg/ml Zymolase 20,000 (Kirin Brewery, J apan)) at 37°C. After 30 min of incubation, cells were centrifuged in an Eppendorf microfuge tube to remove the solution. The cells were then lysed in 0.5 ml of lysing buffer (50 mM N a EDTA, pH 8.5; 1 mg sodium dodecyl sulfate, 3 pl diethylpyrocarbonate (DEPC)) at 70°C for 30 min. 50 pl of 5 M potassium acetate were added at the end of incubation. After mixing the lysates and 50 pl of 5 M potassium acetate by rocking the tubes, they were incubated at 0°C for 30 min. The lysates were then centrifuged to get a clear suspension and the supernatant decanted into 1 ml of 95% ethanol. The tubes were gently rocked to mix the supernatant and ethanol. After centrifugation, the pellet was washed with 70% ethanol. Then the pellet was resuspended in 0.2 ml of TE and 0.1 pl of 10 mg/ml RNase A at 37°C for 30 min to remove RNA. The DNA was reprecipitated by adding 200 pl of isopropyl alcohol, centrifuged, washed with 70% ethanol, and dried. Finally, the pellet was resuspended in 50 pl of TE (10 mM Tris- Cl, 1 mM EDTA) and was ready for restriction enzyme digestion. 10 pg of enzyme digested genomic DNA of each strain was loaded onto a 0.7% agarose gel. Southern blotting was performed as described by Maniatis et a1. (1980). Radiolabeled probes 36 for filter hybridization were prepared by nick-translation of gel-purified restriction fragments (Maniatis et al., 1980). 2.7.3 Preparation of RNA The RNA was prepared in RNase-free glassware which had been baked at 250°C for 4 or more hours. Sterile, disposable plasticware was used without pretreatment for the preparation and storage of RNA. All solutions except those containing Tris were prepared by RNase-free techniques, treated with 0.1% diethylpyrocarbonate for at least 12 hours, and autoclaved. Total RNA was prepared from vegetative or sporulating yeast cells by the method described in Zitomer et al. (1981). 500 ml of cells grown in YEPD were harvested when the culture had reached 4 x 107 cells/ ml. For cells grown in SP2, they were harvested at different time points. After harvesting, the cells were washed once with sterile distilled water and the cell pellet resuspended in 0.5 ml of RNA lysis buffer (0.1 M Tris-Cl, pH 7.4; 0.1 M LiCl, 1 mM EDTA). One ml buffered-saturated phenol and 1/3 volume of glass beads (0.45 mm diameter) were added to the suspension. Cells were broken by vortexing 20 sec and chilling 20 sec for 10 cycles. The lysate was centrifuged to separate phases. The top, aqueous phase was transferred to a new tube. 0.5 ml lysis buffer, 0.5 ml buffer-saturated phenol, and 0.2 ml 10% Sarkosyl were added to the original tube. It was vortexed for another 3 cycles and centrifuged to separate phases. The aqueous phase was added to the original one. Usually four extractions were needed for completion of cell breakage and nucleic acid extraction. The pooled aqueous phases were then extracted two times with phenol:chloroform (1:1). The purified aqueous phase was precipitated by 1/ 10 volume of 3 M sodium acetate, pH 5.4 and 1.5 volumes of isopropanol at -—20°C for at least 1 hour. The 37 RNA was pelleted after precipitation and resuspended in 3.5 ml of DEPC-treated water for preparing an RNA/CsCl solution for CsCl density centrifugation. An equal volume of 10% Sarkosyl was added to the tube after the RNA had completely dissolved. Then, 0.25 g/ml of CsCl was added to the RNA solution, and finally 4 ml of 5.7 M CsCl, 1 mM EDTA solution was layered on top. The RNA/CsCl solution was centrifuged for 15 hours at 15°C in an SW41 rotor at 25000 rpm or an SW27 rotor at 20000 rpm. After centrifugation, the white RNA pellet could be seen at the bottom. The supernatant was removed with a Pasteur pipette. The RNA pellet was resuspended in DEPC-treated sterile water and its concentration was determined. PolyA(+) RNA was isolated by the method described in Harper et a1. (1980). Type II oligo(dT) cellulose (Collaborative Research, Inc.), which bound 1.2 mg to 4 mg of polyA(+) RNA per gram of resin (dry weight), was used to enrich for the polyadenylated fraction of total RNA. There is usually 2-4% polyA(+) RNA in total RNA of yeast, therefore 1 gram of oligo(dT) resin is sufficient to process at least 30 mg total RNA. Total RNA was dissolved in DEPC-treated water, and KCl and Tris-HCl were then added to final concentrations of 0.5 M and 0.01 M, respectively. The RNA solution was heat-denatured at 50°C for 10 min. Then, the RNA solution was applied to the oligo(dT) column with A buffer (0.5 M KCl, 0.01 M Tris). The polyA(+) RNA was allowed to adsorb to the resin for 15 min, after which the column was washed with 10 volumes of A buffer. The polyA(+) RNA was then eluted with DEPC-treated water, and the first 1 ml was collected. The polyA(+) RNA was precipitated with ethanol in the presence of 0.3 M sodium acetate, pH 5.4, pelleted at 10,000 rpm in a microfuge, and resuspended in DEPC—treated water for measuring its concentration. For Northern blots, 25 pg of either total or polyA(+) RNA was fractionated on 38 1% denaturing agarose gels (2.2 M formaldehyde), blotted onto nitrocellulose filters, baked at 80°C for 2 hours, and finally hybridized to radiolabeled probes according to procedures described by Thomas (1980). After hybridization, filters were washed twice with 5 x SSC (0.75 M NaCl, 0.25 M sodium citrate), 0.1% SDS for 15 min at 23°C, and twice with 0.2 X SSC (0.03 M NaCl, 0.01 M sodium citrate), 0.1% SDS for 15 min at 45°C. Filters were then air-dried and exposed to Kodak X-ray films for a period of time according to the intensity of the signals. For slot blot analysis, 5 to 10 pg of polyA(+) RNA was dissolved in 100 pl of DEPC-treated water followed by adding 60 p1 of 6 X SSC and 40 pl of formaldehyde. The RNA solution was denatured at 65°C for 15 min. At the end of denaturation, the tubes were placed in ice and 100 pl of ice-cold 5 M NaCl was added. The RNA samples were then loaded onto nitrocellulose filters. The filters were then handled as for Northern blot analysis. 2.8 Contour-clamped homogeneous electric field gel elec- trophoresis Intact yeast chromosomes were prepared from strain YPH149 by the agarose embedding method of Carle and Olson (1985). Cells were grown in 50 ml YEPD until late log phase. The cells were then centrifuged and washed twice with 50 mM EDTA, pH 7.5; and one time with 10 ml of SCE (1 M sorbitol, 0.1 M sodium citrate, pH 5.8; 0.01 M EDTA, pH 7.5). Cells were then resuspended in 3.25 ml of SCE and 3.0 ml of the cell suspension was mixed with 1 ml SCE, 50 pl fl-mercaptoethanol, 2.0 mg Zymolase 20,000 (Kirin Brewery, Japan), and 5 ml of 1% low melting agarose in 0.125 M EDTA, pH 7.5 at 42°C. The agarose mixture was poured into a 6 cm petri plate immediately and allowed to gel at 23°C. After solidification, 5 ml of 0.45 M 39 EDTA (pH 9.0), 0.01 M Tris-Cl (pH 8.0), and 7.5% fl-mercaptoethanol were poured on the gel. The petri plate was then sealed in a bag and incubated at 37°C overnight. After incubation, the solution was replaced by 0.45 M EDTA (pH 9.0), 0.01 M Tris- Cl (pH 8.0), 1% Sarkosyl, and 1—5 mg proteinase K. The plate was sealed in a bag again and incubated at 50°C overnight or until the agarose was clear. The solution was then replaced with 5 ml of 0.5 M EDTA, pH 9.0. At this point, the agarose was ready to be used for electrophoresis or stored at 4°C. Yeast chromosomes were resolved by contour-clamped homogeneous electric fields (CHEF) gel electrophoresis for 20 hours in 1.2% agarose gels at 300 Volts with a switching time of sixty seconds (Chu et al., 1986). Chromosomes were then trans- ferred to nitrocellulose filters according to Carle and Olson (1985). Hybridization with radiolabeled probes and autoradiography were performed as for Southern blot— ting. 2.9 RNase protection Plasmids containing the gene of interest were linearized at a restriction site within the gene or close to the end of the gene that was far from the phage promoter used for transcription. After linearization of the plasmid, 5 pl of 20 pg/ pl proteinase K was added to the reaction mixtures which were then incubated at 37°C for 1 hour to remove restriction enzymes and RN ases. Phenolzchloroform (1:1) purification was performed and DNA was precipitated with 1 / 10 volume of 3 M sodium acetate and 2.5 volumes of ethanol. These linearized plasmids were used as templates to prepare radioactive RNA probes. 1 pg of plasmid DNA was resuspended in 1 pl of DEPC- treated sterile water. 5 pl of 5X reaction buffer, 0.5 pl of 1 M DTT, 0.5 pl of 40 units/p1 RNasin (Promega), 2.5 pl of a 1 mM/ml solution of all four nucleotides, 40 10 pl of sterile distilled water, 0.5 pl of T7 or SP6 polymerase, and 5 pl of 32P-UTP were added and incubated at 37°C for 1 hour. After the RNA probe was synthesized, 225 pl of DNase buffer (40 mM Tris—HCl, pH 7.9, 10 mM NaCl, 6 mM magnesium chloride) and 1 pl of RNase—free DNase (1 mg/ml) were added and incubated at 37°C for 15 min to remove the template DNA. Phenolzchloroform (1:1) extraction and ethanol precipitations were performed, and the ethanol suspension was left at -—20°C for 1 hour. The probe (100,000 cpm) was then resuspended in 30 pl of sterile distilled water. The hybridization procedure was performed by coprecipitation of 20 pg of isolated yeast RNA and 1 pl of probe. The RNA mixture was resuspended in 30 p1 of hybridization buffer (80% formamide, 0.4 M NaCl, 0.04 M PIPES) and incubated at 90°C for denaturation and then transferred to 55°C overnight. After overnight incubation, 300 pl of TE (pH 8.0), 1.2 pl of 10 mg/ml RNaseA, and 0.6 pl of 1.1 mg/ ml of RNaseT1 were added and the solution was incubated at 37°C for 1 hour to remove unhybridized and single—stranded RNA. The enzymes were then removed by treatment with 2.5 pl of proteinase K (20 mg/ml) and 20 pl of 10% SDS at 37° for 15 min. The suspension was then phenol:chloroform (1:1)—extracted and ethanol-precipitated. The RNA pellet was collected and washed with 70% ethanol. The pellet was then resuspended in 3 pl of loading buffer (90% formamide, 10 mM EDTA, dye). The suspension was denatured at 90°C for 2 min and loaded onto a 6% urea-polyacrylamide gel immediately. The gel was run at 30 W for 4 hours, then autoradiographed. 2.10 DNA sequencing The region of the SPR9 locus between Clal and SstI was sequenced by the dideoxy method of Sanger et al. (1977). Three strategies were used to generate the subclones 41 for sequencing. First, several well-characterized restriction fragments were subcloned into replicative forms of M13mp18 and M13mp19 (Boehringer Mannheim). Second, the HindIII-Sstl fragment was digested with Sau3A and subcloning the generated fragments into both phage vectors at appropriate sites. Third, specific oligomers were synthesized and used as primers to fill in the remaining gaps in the sequence. Sin- gle stranded templates were prepared from insert containing M13mp18 or Ml3mp19 phage vectors by the procedure of Messing and Vieira (1982). The sequencing reac- tions were prepared by using the Sequenase 2.0 version kit (United States Biochemical Corp.) under the supplier’s conditions. 42 Table 2.2: List of media. Media Ingredients Reference YEPD 1% yeast extract, 2% peptone, 1% dextrose Fink, 1970 MIN 1% dextrose, 0.67% yeast nitrogen base w/o Sherman et al., 1981 amino acids YEPGlycerol 1% yeast extract, 2% peptone, 1% Glycerol Sherman et al., 1981 PSP2 1% KOAc, 0.67% yeast nitrogen base w/o Roth and Halvorson, amino acids, 5% phthalic acid, pH 5.5 1969 SP2 1% KOAc, 0.2% raffinose Hopper et al., 1974 LB 1% peptone, 0.5% yeast extract, 1% NaCl Maniatis et al., 1982 CHAPTER III Analysis of the Sporulation-Specific Gene, SPR9 3.1 Cloning and mapping of SPR9 The SPR genes were isolated from a genomic library of Saccharomyces cerevisiae by Clancy et a1. (1983). These SPR genes were identified by their ability to be transcribed preferentially during sporulation. The Lambda phage clone 104 carries the SPR9 sequence whose transcript lies within a 5.7 kb Clal fragment. This Clal fragment was cloned into a pBR322 vector to give plasmid p803 (Figure 2.2) which was then mapped with several restriction enzymes (Figure 3.1). 3.2 Developmental regulation of SPR9 Some genes may be turned on during sporulation as a physiological response to the change in conditions that are needed to induce sporulation, and they may not be involved in meiosis or spore formation. These genes would be transcribed in nonsporulating cells in the same changing conditions. On the other hand, there are. other genes that are essential for sporulation to occur, and they may be sporulation- specific or nonsporulation—specific genes. For example, many genes that control the cell division cycle, mating type, radiation sensitivity, etc. have been shown to be essential for sporulation (Dawes,1983). It is important to distinguish genes expressed 43 Sstl Xhol Hindlll 44 Xbal Ncol Sau3AI Seu3Al 93" BstEl ""°' //’ Indiu Pstl H al HP" EcoRl p 3" a IH 5““ LHlndlll Sstl Haelll ' EcoRV Clal III E (flel N§§f“" °°R' SPRQ to Mb . p803 p8038/X 638 or G48 L0 kb L Figure 3.1: The restriction map of the SPR9 locus. The 5.8 kb Clal fragment was cloned into pBR322 to produce plasmid p803. p803 was then digested with enzymes XhoI and SalI to produce p803s/x (SalI site is on pBR322). The arrow represents the transcript of SPR9 gene. 45 during sporulation from those induced by sporulation conditions or by shock such as the heat shock gene, HSP?6 (Petko and Lindquist, 1986). To prove that SPR9 gene is not turned on solely due to environmental stress but only during sporulation, we used the following approach. First, polyA(+) RNA was prepared for slot blot analysis from strain W66-8A (a/a) grown at 23°C in rich medium with and without shifting to 39°C for one hour of heat shock. PolyA(+) RNA from vegetative cells (grown in PSP2) and 10 hour sporulating cells (grown in SP2) was also prepared for slot blot analysis to show that HSP?6' was turned on by sporulation. The same filter was probed sequentially with three different radiolabeled plasmids: p803 for detecting SPR9 message, pHSP26 for detecting HSP?6 message, and pGAP for detecting the message of glyceraldehyde-3-phosphate dehydrogenase gene as a positive control. The result shows that both the SPR9 and HSP?6 genes are turned on by sporulation as predicted, and trace SPR9 transcripts are detected after heat shock treatment (Figure 3.2a). To induce sporulation, a nitrogen-source is limiting in the medium. Therefore, genes that are turned on by nitrogen-starvation are also expressed in sporulation medium (Primerano, unpublished data). Since these genes will also be expressed in asporogenous diploid strains grown in sporulation medium, polyA(+) RNA was prepared from both vegetatively grown and sporulation-induced cells of strains AP1 a/a, and AP3 a/a for slot blot analysis. Radiolabeled p803 and pGAP were used as probes. No SPR9 message was detected from those two strains under vegetative growth or sporulation induction (Figure 3.2b). Therefore, we conclude that the SPRQ gene is not responding solely to environment changes or nitrogen starvation but is a developmentally regulated gene. woo-8A AP 1 Wet .AP39G W7a veg, veg veg. W66-8A V99- Room temp W66-8A spo. 39°C Probe SPR9 veg. GAP 46 SPR9 Probe HSP26 III! IIIIIl web-8A AP1 oC/ac GAP spo. spo. spo. Probe SPR9 GAP ._ i Q Figure 3.2: Slot blot analysis of the SPR9 transcripts. a. The response of the SPR9 gene to heat shock at 39°C. A diploid strain W66-8A (a/a) was grown at 23°C in YEPD until stationary phase. For heat shock treatment, part of the culture at stationary phase was grown at 39°C for another hour. PolyA(+) RNA was prepared from both cultures for slot blot analysis. PolyA(+) RNA was also prepared from the same strain grown in presporulation medium (veg.) and sporulation medium (spo.). Radiolabeled p803, pHSP26, and pGAP were used as probes. b. The response of the SPR9 gene to nitrogen starvation. PolyA(+) RNA was prepared from three diploid strains W66-8A (a/a), AP1(a/a), and AP3 (a/a) grown in presporulation (veg) and sporulation medium (spo.) for dot blot analysis. Radiolabeled p803 and pGAP were used as probes. W7a (a/a) is a diploid strain which is defective at both SPR9 alleles. 47 3.3 Kinetics of the transcription of SPR9 The dot blot analysis of Holaway et al. (1985) suggests that SPR9 is a middle gene which is turned on at about the time of DNA synthesis, maximally transcribed at meiosis II, and the mRNA present for at least the next 3 h. Since it is impossible to tell by dot blotting if the message is intact or degraded, we analyzed the RNA by hybridization of Northern blots. In this study, polyA(+) RNA was prepared from vegetative cells (0 h) and sporulating cells of an a/a diploid strain, W66-8A, at different times (2 h, 4 h, 6 h, 8 h, 10 h, 15 h, and 20 h cells in sporulation medium). In order to be certain that the extracted RNA was from sporulating cells, the percentage of asci in the cultures was determined at the time of extraction by counting 200 cells under a light microscope. Before RNA extraction, a 5 ml culture of each time point was saved and further incubated to determine the sporulation efficiency after 24 h incubation in sporulation medium (Figure 3.3). The RNA and polyA(+) RNA were prepared as described in Materials and Methods. Both the total RNA and polyA(+) RNA were loaded onto a 1% denaturing gel. The filters were first hybridized to a 0.9 kb EcoRI fragment containing part of the SPRg coding sequence to show the pattern of transcripts (Figure 3.3). A 1.0 kb transcript could be detected by this probe. Transcripts of the SPR9 gene begin to appear 4 h after transfer to sporulation medium; they increase in abundance as sporulation proceeds. They remain abundant after 20 h in sporulation medium. The same filter was then probed with pGAP as a control to show that equal amounts of RNA sample were loaded in each lane of the gel (Figure 3.3). A Northern blot of total RNA was prepared using RNA extracted from the same cultures used for preparing polyA(+) RNA. This blot was hybridized to the radiolabeled 0.9 kb EcoRI fragment. There 48 was no detectable difference in the patterns of transcript abundance whether total RNA or polyA(+) RNA was used (data not shown). 3.4 Chromosomal location of SPR9 The chromosomal location of the SPR9 gene was determined by CHEF (Chu et al., 1986). The chromosomes of strain YPH149 were separated and blotted as de— scribed in Materials and Methods. The filter was then cut into three strips, and these strips were hybridized to different probes: radiolabeled plasmid YIp333 which car- ries a chromosome II marker, LY5?; a radiolabeled 0.9 kb EcoRI fragment containing part of the SPR9, and both LYS? and SPR9. The result is depicted in Figure 3.4; the picture of the CHEF gel is at the top of the figure and the picture of the filter is at the bottom. Both probes hybridized to chromosome 11 despite the fact that YIp333 also hybridized to two other chromosomal bands, 11a' and 11a". To make YPH149 useful for CHEF, chromosome VII was separated into two fragments (chromosomal bands 11a’ and 11a") (Hieter, 1989, unpublished data) by attaching telomere sequences to the broken ends of chromosome fragments, which resolve from chromosome XV (chromosome band 11b). Both chromosome fragments also contained part of the pBR322 sequences from the plasmid carrying the telomere sequence during the con- struction process, so when YIp333, a pBR322 derivative, was used as a probe both bands hybridized. 3.5 Determination of the sense strand of SPR9 The orientation of the transcription of the SPR9 gene was determined by RNase protection by hybridizing the polyA(+) RNA from vegetative cells (grown in PSP2 medium) and sporulating cells (10 hours in SP2 medium) of strain \v’V66-8A to two 49 g of sporulation at 24h 67% 67%65% 65%66%62%66% 65% 0% 0% 0% 1% 5% 33% 42% 58% 0h 211 4b 6h 8h 10h 15h 20b 1 2 3 4 5 6 7 8 SPR9 ' ' "" —l.0kb Figure 3.3: The kinetics of the appearance of SPR9. PolyA(+) RNA was prepared from vegetative cells (0 11) and sporulating cells of an a/a diploid strain, W66-8A, at different times (2 h, 4 h, 6 h, 8 h, 10 h, 15 h, and '20 h cells in sporulation medium) for Northern blot analysis. Sporulation efficiency of cultures used to prepare RNA was examined at the time of extraction and 24 11 after transfer to sporulation medium. The filter was first hybridized to a radiolabeled 0.9 kb EcoRI fragment containing part of the SPR!) coding sequence, then to radiolabeled pGAP. Probe LYS2 SPR9+ L S2 SPR9 Chromosome -—‘VI| ———Vl| ‘. a. Figure 3.4: The chromosomal location of the SPR9 locus. The chromosomes of strain YPH149 were separated by CHEF (contour-clamped homoge- neous electric field gel electrophoresis) and then blotted onto a filter. The picture of the CHEF gel is at the top of the figure. The filter was cut into three strips which in turn were hybridized to different radiolabeled probes: YIp333, mixtures of YIp333 and 0.9 kb EcoRI fragment, and 0.9 kb EcoRI fragment alone. 51 riboprobes. The two riboprobes were made from the two complementary strands of a region encoding the SPR9 transcript. A 2.2 kb ClaI-Sstl fragment was force-cloned into a GEM3 or a GEM4 plasmid (from Promega company) at Ach and Sstl sites. The GEM plasmids contain two viral promoters, T7 and SP6, that can produce two complementary run-off transcripts from the DNA fragment inserted. The resulting G33 plasmid was linearized at the HindIII site for the T7 promoter and at the Sstl site for the SP6 promoter. The transcripts from the T7 and SP6 promoters are 1800 and 2200 bases long, respectively. The template region of G38 and the transcripts from the SP6 and the T7 promoters are shown in Figure 3.5a. After hybridization, the RNA-RNA hybrids were treated with RNaseA and RNaseH to remove all single stranded RNA. The samples were analyzed on a 6% urea-polyacrylamide gel following RN ase treatment. The result is shown in Figure 3.5b. The marker DNA was at lane 1. Lanes 2 and 3, which showed no protection of SPR9 transcripts, were hybridized to the riboprobe from the T7 promoter. Lanes 4 and 5 were hybridized to SP6 transcripts, and only the RNA from sporulating cells but not vegetative cells was protected from RNase digestion. The protected band is about 1.0 kl) in size, and the RNA sample shows some degradation. This result indicates that the strand used for SPR9 transcription is the one complementary to the transcript made from SP6 promoter. The data also confirm that the SPR9 transcripts are made only during sporulation. 3.6 DNA sequencing of SPR9 locus The SPR9 gene was sequenced by Ml3 dideoxy-sequencing to gain information about its protein product. The strategies used are illustrated in Figure 3.6. For the first strategy, plasmid 803 was digested with EcoRI restriction enzyme to generate 52 a 0 Eco!!! Bo Ell Hum ' I F"" c al El —l§| span see "map! ( 2.2 kb) T7 transcript ( 1.3 m) i; ,__ [,0 n b . 1.0 kb _ Lone]. Marker 2. T7 9 Veg. RNA 3. SP6 9 Veg. RNA 4. T7 0 Spa. RNA 5. SP6 ’ Spo. RNA Figure 3.5: Determination of the sense strand of SPR9. a. The template region of G38 and the transcripts made from the SP6 and the T7 pro- moters. b. The result of RNase protection analysis of SPR9 transcript on a 6% urea.- polyacrylamide gel. Lane 1 is the radiolabeled marker DNA. Lanes ‘2 and 3 are the hy- bridization mixture of vegetative RNA from W66-8A (a/a) and RNA transcripts from T7 and SP6, respectively. Lanes 4 and 5 are the sporulating RNA of W66-8A hybridized to the RNA transcripts from T7 and SP6, respectively. 53 four fragments. They are 0.4 kb, 0.9 kb, 1.0 kb, together with a 8.0 kb fragment containing pBR322 sequences. The 0.4 kb, 0.9 kb, and 1.0 kb fragments were lig- ated to both M13mp18 and M13mp19 linearized at the EcoRI site. For the second strategy, a 3.0 kb HindIII fragment containing the SPR9 gene from plasmid 13803 was isolated and further digested with Sstl restriction enzyme. The resulting frag- ments were either ligated to the HindIII-Sstl linearized Ml3mp18 and Ml3mplQ or further digested with Sau3A, and then cloned into the two Ml3 vectors. For the gap that could not be sequenced by the strategies described above, two 17-mers (~TGGAGTCCTTCTTCCCC—) and (-GCAAGAGCCTTCCCCCA—), were synthe- sized and used as primers to obtain the entire sequence of the SPR9 gene. The nucleotide sequence and the deduced open reading frame (ORF) are shown in Figure 3.7. Within this 2.5 kb sequence, one long ORF from position +1 to +435, encoding a 16.8 kD protein is found. Two possible TATA boxes (TATAAA) are found 93 bp and 174 bp upstream from the putative AUG initiation site, and there is another degenerate possible TATA box, TATAAC (5/ 6 matches) at position ~45. When the upstream sequence was compared to that of other spornlation-specific genes, no unique consensus sequence was found for all the SPR genes sequenced. However, pairwise homology in different upstream regions between SPR] and SPRQ, and SPR2 and SPRQ was observed (Figure 3.8). There also has been no significant homology found to any DNA sequence in the GENBANK library (release 54-) or to any protein sequence in the PIR15 library (release 15). The sequence, AATAAA or AATATA, suggested by Fitzgerald and Shenk (1981) for the addition of polyadenylic acid tracts appeared twice after the termination codon. Both of them, +563 to +568 and +681 to +686, would produce transcripts that were much shorter than 1.0 kl). The predicted protein is composed mostly of hydrophilic amino acids. 54 EcoRV EcoRl EcoRl Sstl Rsal BstEll Hlndlll places. EcoR' T... Halolll W ( c... Sequoncod region +t%* I* ’> -—--—D> “——- ID t= 5' 1 4 4 e t k Sequencing stratogy 4——” :D> 3‘ ‘fir .1: at ++€3 Figure 3.6: The sequencing strategies for the SPR9 locus and flanking regions. ORF is the potential open reading frame for SPR9 protein. The open bars are the synthetic primers. -688 -638 -588 ~538 -488 -438 -388 -338 -288 -238 -188 -138 -88 -38 1 1 46 16 91 31 136 46 181 61 226 76 271 91 316 106 361 121 406 136 437 487 537 587 637 687 737 787 837 887 937 987 GCCCTCAAAG GGTTCAAGAT CAAAGAACCC TACATTCAGC GGATTTTCAA GCCATGACTA TGTAAACCAA ATATCGCAGT TTCGCTCATG CCGCCCTTCC AAACTAATGG CACACGTTTT ATCAAAAAGC TAACAATTCG CCTTATAAGA ATCGCTGAGC ATTATAGAAG AGATTTATGC GTACCCTAAA CTTCGTGGAA ATACAAGTTT AGCACTCTCG TTTCTTCGAG CCACACACAC aaragggggg TTAATTTATT CCAAGCCTCA AAATCTCTAA 55 AGAACCCTTT TCAGAATCTA AGTATAGTGT GAACTTCTAA GAACAAATGG AACACTGGGT AGAGCATAAC CAGGGGAGTG GGGCGCATAT AAAAAAAAAA CAAATAAGTA TTTTTTTTTA AACAAGAGTC GCTCGCTTAT CATGAAGTTT TCTCTACAAC AGCGGCTAGT GAAACAGTGT CTCGAACAGT TTATAGACAA GTTCCGATCA GCGACTCATA TGTGACGCTA GGTTAAGGAT AGTAGATACT CATATCTTAA TTATCACTCC AGTTGACA TTTGAAATTG AATTCGTATA ATATACCGTT CATAACCAGT TTCTTAGCCA GTGGTATGAT TCAGACGTCA ATTGCGTGAA ATATTATGTA AAAGATAAGT GTAAATAGAA TTTAATATAA_ ATTWA ATG Met TTT Phe GAA Glu GAC Asp ATC Ile AAG Lys AAG Lys ACA Thr GAT Asp AGA Arg ACC Thr AAA Lys AAC Asn GAT Asp CTG Leu AAA Lys GGC Gly CGG Arg AGG Arg AGG Arg ATA Ile AAG Lys AAT Asn AGC Ser ACT Thr GGC Gly CTT Leu CAC His TTC Phe AGC Ser TTT Phe CAA Gln GGT Gly ATG Met AGA Arg GTT Val GTC Val CAG Gln GAT Asp TAC Tyr TCT Ser GAG Glu TTC Phe GAG Glu AAC Asn Asn GCC Ala AAC Asn GAT Asp TTG Leu AGG Arg CCT Pro ATA Ile GCG Ala AGA Arg AAT Asn AAT Asn ACA Thr AAA Lys AGA Arg TTC Phe TCC Ser AAC Asn GAC Asp AAC Asn CAT His GAA Glu TAT Tyr CAA Gln TAC Tyr TCA Ser CCC Pro ATA Ile ATT Ile AAT Asn TGC Cys CAC His TCA Ser AGC Ser AAG Lys TAT Tyr ATT Ile Lys TCA Ser TTG Leu ATA Ile Lys CCA Pro AGA Arg AAA Lys TTT Phe GAA Glu TCC Ser GAT Asp GGT Gly GAT Asp TTG Leu GAG Glu ATA Ile TAA *** GAT Asp ATA Ile TTA Leu AGA Arg AAG Lys TTG Leu GAT Asp GAT Asp CTA Leu AGT Ser ATC Ile GAA Glu GAA Glu GTT Val AAT Asn AAC Asn TCC Ser Lys CTA Leu TAT Tyr AGC Ser ATG Met GCC Ala GAG Glu GAT Asp GTG Val TGT Cys TTC Phe TGC Cys CCC Pro GCT Ala ATT Ile CTG Leu AAC Asn GAA Glu AGC Ser TCC Ser AAT Asn ACT Thr ACA Thr GAT Asp Lys TCA Ser TTT Phe ACT Thr GACAATTGAG GTCAGAAAGA AAAAAGTAAT GATTAGTTCT CCTCAGGAAG GCGAGGAACA TAGAAGAAGC TTTGAAGCAA TGTCATTCCT AGAATTATCT AACATTCCGA TGAAAAATTG CTGCTTCATT GTGTTCTCTA ATCATATACT ATTAATTACT AAGGGGTTCT ATGGATGGGA CAAAAAAAAA TTGCGTGTCT CAATTCTTCC TCCGCAAIIA. AGCCACATAT CAAGATAAAA ATATATCTTG TGTTTTTGTG ATTAATAATA ATTGACACAC TAGGAAAATT ACAAAACCAT AAAAACAGAA CGGTTTACGC AGGAAGCCTT AACGACGATG TCCTTACTTC TCAAAGTTGA ATATAATCAC CTTCCATAAA AAAGTTATAC TTTGAATTCC TTTTCTTTTT GTTCAGCCTT ACGCCAACCA TACCAATTTA TCAACAAGAA ATAGCTCTGC ATGAAACCAC CGATTTGAAT CTTTCTTATC TATATAGTTC TGAATATATT CTAAAIIAAA TAATATAAAA CAACAATTGG TGCACTTTTC GCTCTGCCTA GAGCCAATTG CGTTGCTGCC ATGTAGAAAG GCTACTGCTT Figure 3.7: The nucleotide and deduced amino acid sequence of the SPR9 locus. Underlined are the potential TATA sequences. is the termination codon for translation. are possible polyadenylation sites. *** 56 SPRl - 6 7 1 AC TTTCTTGGGACATACCACAATTAC ***** ** ***** **** **** -333 ACTTTGTTAATACATATCACAGTTAC SP R2 SPR9 -158 TTTTTTTTTACATATCTTAATTTAATATAA *** ** ******** * ** **** * -331 TTTGTTAATACATATCACAGTTACATATTA SPR2 Figure 3.8: The homologous sequences in the upstream regions of the SPR genes. The homologous regions are found in the upstream regions between the SPR] and SPR9 genes and between the SPR? and SPR9 genes. * represents the matching nucleotides for the two sequences. CHAPTER IV Phenotypic Analysis of Three SPR Genes, SPR], SPR2, and SPR9 4.1 Disruptions of SPRI, 2, and 9 In order to get insight into the functions of the SPR genes during sporulation, the SPR genes were mutated by one-step gene disruption which replaces part of the gene of interest with a marker gene (Rothstein, 1983). In this technique, the DNA fragment containing the disrupted gene is transformed into a diploid H O yeast strain deficient for the marker gene. Since the DNA fragments can recombine with homol- ogous sequences in the genome, one copy of the wild-type chromosomal sequence of the gene of interest can be replaced by the disrupted sequence and the transfor- mants can be selected by complementation of their auxotrophic phenotype by the marker gene (Figure 4.1). The transformants thus produced are heterozygous at the disrupted locus and can sporulate to produce two wild-type spores and two spores mutant at that locus. Each spore will diploidize due to the presence of the HO gene (Herskowitz and Oshima, 1981), and the ability of the resulting a/a homozygous spr mutant strains to sporulate can be tested. If they do sporulate, their spores can be isolated for further examination. The SPR] and SPR2 loci were mutated by Primerano et al. (1990 and 58 marker gene 2 marker gene 9909 2' Figure 4.1: One step gene disruption. The cloned fragment containing Gene Z+ is digested with a restriction enzyme that cleaves within the coding region of the gene 2. A fragment containing a selectable yeast gene (marker gene) is cloned into the site. The fragment containing the disrupted gene Z is liberated from the plasmid, and the liberated fragment retains homology to the Gene Z region on both sides of the insert. Transformation of the yeast cells with the linear fragment. results in the substitution of the linear disrupted sequence for the resident chromosomal sequence. (Rothstein, 1983) 59 unpublished data), and these transplacements are shown in Figure 4.2. For the SPR9 gene replacement, the 3.0 kb region between two Hindlll sites was replaced by a 1.1 kb HindIII fragment carrying the URA3gene to create the spr9 null mutant allele. The 3.5 kb Clal fragment with the missing SPR9 locus was then introduced into W66- 8A, an H 0 arm? diploid strain (Figure 4.2c). Transformants carrying the 3127‘sz URA3 allele were selected on minimal medium containing all the required supplements except uracil. Southern blot analysis of their genomic DNA confirmed that the gene was replaced by the marker gene. The heterozygous SPR9 /spr9 transformant was sporulated and the asci dissected into individual spores by micromanipulation. The URA3 spore grew into homozygous sprg diploids. The genotype of homozygous sp'r9 diploids was also confirmed by Southern blot analysis. Figure 4.3a shows the genomic DNA patterns of the SPR9 locus in the wild—type (W66-8A), the heterozygous (W7), and homozygous mutant strains (W7a). The wild-type SPR9 locus gives rise to a 5.8 kb Clal fragment, while the spr9zzURA3 mutant Clal fragment is 3.5 kb when probed with a nick-translated 0.9 kb EcoRI fragment containing the SPR9 gene. The gene replacement for SPR] and SPR2 genes was done by Primerano et al. (1990, and unpublished data). For the SPR] locus, the genomic DNA was digested with Xhol restriction enzyme and detected by nick-translated pCG33 probe (carrying the wild-type SPR] and flanking sequence). The wild-type SPR] locus lies on a 4.4 kb Xhol fragment, while the disrupted sprl-szLEUQ allele gives a 6.6 kb Xhol fragment. Therefore, the wild-type strain SCMS-7 should show only the 4.4 kb band, the mutant strain (S52B) should show a 6.6 kb band, and the l‘ieterozygous strain (SPBl) should show both bands (Figure 4.3b). For the SPR2? locus, the genomic DNA pattern was generated by HindIII restriction enzyme and probed with nick-translated pACI (carrying the wild-type SPR2 and flanking sequences). The 60 BamHI EcoRl Sstl XhOI 5""7 I rein] l XhOI / . Sstl SPF" BamHl 1.5 ltb “I“ San/Xhol 58" xj' LEUZ (3.0 kb) Wlld type Xhol fragment: 4.4 kb 1.0 u, Mutant Xhol fragment: 6.6 Itb M EcoRl Pvull Seal Scal Xhol Sma Neel P tl Hlndll l I 1 I, firmndm Hlndlll Patl Hlndlll ECORI PVUII spnz Scal Xhol LEU2 (3.7 kb) Wlld type Hlndlll fragment: 2.7 kb l9") kbl Mutant Hlndlll fragment: 5.8 kb Hlndlll Hlndlll Hlndlll Clal EcoRl EcoRl l/E¢:oRl Xhol Clal 3.0 ltb $539k!» Hlndlll Xhol Clal CI l L URA3 l J 1.1 kb 1.0 kb Wild type Clal fragment: 5.8 kb I'— Mutant Clal fragment: 3.5 kb Figure 4.2: Disruption of the three SPR genes. The restriction maps of wild-type loci and mutant loci for: a. SPR] gene; b. SPR2 gene; and c. SPR9 gene. The arrows represent the transcripts of the SPR genes. 61 wild-type SPR2 gene gives a 2.7 kb HindIII fragment, a 5.8 kb fragment for the spr2zzLEU2 mutant strain (T4C), and both for the heterozygous strain (AKAS'Z) (Figure 4.3c). 4.2 Detection of SPR messages in single mutants To make sure that there are no SPR transcripts made in these spr mutants, total RNA was prepared from the spr strains. It was proven previously by Primerano et al. (1990 and unpublished data) that there are no SPR] and SPR2 messages in S528 (sprI-Q/sprI-Q) and T4C (spr2/spr2) strains, respectively. For W7a (sprQ/sprg), the poly A(+) RNA was prepared and hybridized with the radiolabeled 0.9 kb EcoRI fragment carrying a part of the SPR9 gene as probe. As shown in Figure 32b, SPR9 transcripts can be detected in the sporulating wild-type strain, while SPRQ transcripts are seen in neither the W7a vegetative nor sporulating cells. The behavior of cells without one or more of the SPR gene product was then examined. 4.3 Phenotype detection of SPR9 To determine the function of the gene product of SPR9, the behavior of the ho- mozygous spr9 mutant strain during vegetative growth and during and after sporu— lation was examined. We first examined vegetative growth of wild-type and the homozygous spr9 mutant strains. The growth rate of W 7a (sprg/sprg) at 23°C, 30°C, and 34°C was compared to its isogenic parent, W66-8A. There was no obvious difference in growth rate at any of the temperatures tested, as shown in Figure 4.4. This result suggests that the SPR9 function is not needed for vegetative growth. We then asked whether or not spore formation was affected by the 8])1‘9 mutation at 30°C. Not only were four-spore asci formed with about the same kinetics as 62 Clal digest of SPR9 locus I 2 3 t t - W.T. —- - Mut. '— Probe= 5.8 kb Clal —5.8 kb ~-- -—3.5|(b 7 fragment c. Xhol di est of Hindlll digest of SPR] ocus SPR2 locus + 1- - f - l 2 3 I 2 Mut. —. ‘ — 5.8 kb Mut.-—~ - . —6.6 kb W.T. — . _ 2.7 kb W.T. —.C —4.4 kb ' Probe: pCG33 Probe: pACl Figure 4.3: Southern blot analysis of the three SPR loci. a. The genomic DNA from three strains W66-8A (lane 1), W7 (lane ‘2), and W7a (lane 3) was digested by Clal enzyme for Southern blot analysis. The filter was hybridized to a radiolabeled 5.8 kb ClaI fragment for the detection of the SPR9 locus. b. shows the detection of the SPR] locus in strain SCMS (lane 1), SPBl (lane ‘2), and 852B (lane 3). Radiolabeled pCG33 was used as probe. The genomic DNA was digested by Xhol enzyme. c. shows the detection of the SPR2 locus in strain SCMS (lane 1), AKA82 (lane '2), and T4C (lane 3). The genomic DNA was digested by HindIII enzyme. Radiolabeled pACI was used as probe. +: wild—type band, -: mutant band. 63 wild-type, but also the efficiency of sporulation did not change because of the missing SPR9 function (Figure 4.5). Both samples of sporulating cultures were also stained with DAPI at different stages for examining nuclei of the cells and examined under the phase-contrast microscope, no difference was seen among them (data not shown). These observations indicated that the SPR9 gene may not be involved directly in the formation of spores. A similar situation has also been found in bacterial sporula— tion (Dion and Mandelstam, 1980; Moir, 1981). In B. subtilis some events occurring during sporulation are not essential for spore formation, but are essential for the subsequent germination or outgrowth steps during which the spores return to the vegetative state. To investigate whether the SPR9 gene is involved in events after spore maturation (such as germination) or in maturation of the spore itself (such as resistance to heat and chemicals), spores at different stages (16, 20, and 24 hours in sporulation medium) were isolated for tests. In some experiments, only spores isolated 16 hour after transfer to sporulation medium were used for examinations. Sixteen hour spores of the strains used in this study are at that stage most sensitive to heat according to our experiments. The thermotolerance of the spr9 mutant spores of strain W 7a after 16 hours in sporulation medium was compared to that of VVG6-8A (wild—type strain). In this experiment, spore suspensions were incubated at various temperatures (4°C, 23°C, 30°C, and 42°C) for various times. To reduce the plating error and differences in the treatment of each sample, equal amounts of asci from wild—type and mutant strains (isogenic except at the SPR9zz URA3 locus) were mixed after harvesting and the mixed cultures used for all further treatment. The survivors were distinguished by scoring for the uracil requirement. At all temperatures tested there is no obvious 64 3 a. a I 3: c: 2" :3 U . u 0 H s 1 - g —I— W66-8A '4 —0- W7a-1 0 fi ' I ' I v 0 10 20 30 3 b. 3 I :1 r: 2'- :3 U 1 u 0 H E 1 g‘ + W66-8A O I 1 I ' I ' O 10 20 30 3 C. 3 II p 1: :1 2 u u d 0 .-r :‘5 1- 8‘ —I— W66-8A A —9_ W7a-l O | I ' I ' O 10 20 30 Figure 4.4: The growth curve of the wild-type and sp1'9 mutant strains. Both strains were pregrown in YEPD at 30°C overnight then transferred to fresh YEPD and grown at a. 23°C, b. 30°C, c. 34°C. W88-8A: wild-type strain, W7a: 519129 mutant strain. 65 go. 1 I 8 fl *1 50' u ‘U .. H 3 4o 0 O. (I) 1 ~¢ o~ 20., 9" NEG-8A —’_ W7a-1 ""l'l"l 0 10 20 30 40 50 Time (Hours) Figure 4.5: The sporulation of the wild-type and spr9 mutant strains at 30°C. W88-8A: wild-type strain, W7a: spr9 mutant strain. 66 difference in the heat tolerance of the 16 hour spores between wild-type and nmtant strains (data not shown). Actually, spores from both strains can survive a prolonged time at 4°C in water (data not shown). To test the germination ability of mutant spores, the same procedure for prepar- ing free spores was used and these spores were spread onto YEPD plates (without preincubation in water) and incubated at four different temperatures. Since yeast cells can barely grow when the temperature reaches 42°C and above, germination temperatures used in this study were 23°C, 30°C, 37°C, and 40°C. For homozygous ads? strains, the cells will accumulate red pigments due to incapable of turning over the intermediates produced in adenine synthetic pathway. However, some of the germinated colonies turned white when the temperature was 37°C despite their ads? genotypes (Figure 4.6a). At 40°C, all colonies germinated were white (Figure 46b). At 23°C or at 30°C, mutant spores germinated as efficiently as the wild—type spores for all four stage spores tested, however when germinated at higher tempera- tures (37°C and 40°C), mutant spores showed lower germination efficiency than the wild-type spores (Table 4.1 and Figure 4.7 ). After 12 hours in sporulation medium at 30°C, about one third of the cells formed four-spore asci in both strains examined, and no difference in germination efficiency between the wild-type and mutant spores was observed at any temperature. In 16 h sporulating cultures, the number of four-spored asci increased to more than 40% at 30°C. When placed at 37°C, only 50% of the mutant spores germinated compared to those at 30°C, whereas the parental spores germinated as well as at 30°C. At 40°C, the germination of mutant spores dropped to 30% while the wild-type retained the same germination efficiency. Small colonies appeared on germination plates at tem- peratures higher than 37°C(Table 4.1) for 16 h spores. 87.5% of small colonies arose 67 Figure 4.6: The formation of small colonies during germination at temperature 2 O a. The plate was incubated at 37°C for germination. b. The plate was incubated at 40°C for germination. All colonies germinated at 40°C turned white despite their udc2 genotypes. 68 from mutant spores, while of the regular sized colonies, only 24% were mutants. The progeny of small colonies grew to form small colonies even at 30°C. They cannot grow in medium which contains glycerol as the sole carbon source. All these observations suggest that these small colonies are petites. The appearance of petites which may be induced by the heat during germination. The sporulation rate of 20 h cultures reached 60% at 30°C for both strains. The germination efficiency at 37°C was slightly decreased to 46% for 20 h mutant spores, while at 40°C the lowest germination of mutant spores were observed, 14%. The parental spores germinated at 37°C and 40°C with 100% and 90% efficiency, respectively. There were lower percentages of small colonies formed when a 20 h sporulating culture was germinated at either 37°C or 40°C compared to a 16 h sporulating culture at the same temperatures. For a 24 h sporulating culture, the sporulation rate was the highest at 30°C. The germina- tion efficiency of mutant spores was also the highest among the four stages of spores examined, 68% at 37°C and 45.5% at 40°C. The 24 h wild—type spores germinated equally well at all three temperatures tested. There were very few small colonies formed for 24 h spores at either 37°C or 40°C. About equal numbers of mutant and wild-type spores gave rise to both small and large colonies at 37°C, and 47% and 37% of mutants in small and large colonies, respectively, at 40°C. The results of heat tolerance and germination suggest that the function of the SPR9 gene product is important to spore germination. The occurrence of the small colonies may be related to the spore stage rather than the mutation, since the per- centages of small colonies decreased at both 37°C and 40°C as the spores got more mature. When germinating at temperatures higher than 37°C, both wild-type and mutant 20 h spores were most affected by heat. A much more severe deficiency of mutant spores in germination was observed at 40°C for all three stages of spores. It 69 est. 3?. <2 3% 2 <2 33 Osww <2 8828 =25 .88 8 s 35 3% <2 0% .2. <2 38 was <2 8828 was. .88 8 cs 78 Ta 3.88 T». 2;. 28m in 2;. Team assoiaaw News: s2 s2 s2 e2 $2: .32 $2: $2 32 828588 a. .3 8 cs 2 x as 2 x E. 2 x ms 2 x as 2 x m 2 x 3 2 x 2 2 x 3 2 x 3 seam H .3 .8 a 38 was as: 38 38 new” 35 $88 was” 88% a. .3 8 3 3.8. was we: as: $9. 022 so” 3% s2 messes» eases 8 s 2 x 3 2 x as 2 x 3 2 x e 2 x m 2 x 2. 2 x 3 2 x 3 2 x 2 88% ass: 8 a was” ea... Has... 28 $9. $8 $3 ass” same ”28% 3888 8 cs 2 x a. 2 x m 2 x as 2 x n he x n 2 x a 2 x A...“ 2 x a. 2 x a. 88% 38 .8 a use. can can use. oat“ 02 02. can 02. 852888 8:22:80 3.8% : vw 8.8% s cm 88% a .2 6.20% mmlm. 23:8 28 war: 23 me :OSaEEEU ”Has $23. % germination 8 I 201—.— spr9-16h 10: —O— SPR9-16h 0 r-I'I'I'r'l'l 28 30 32 34 36 38 40 42 % germination 8 I 20" —"'— spr9-20h 10 ' —*— SPR9-20h ' T ' l ' l ' l ' l ' l ' I 0 23 30 32 34 36 3B 40 42 is germination 8 r] 20- —¢—' spr9-24h 10- —0- SPR9-24h Temperature (°C) Figure 4.7: Germination efficiency of wild-type and 511129 mutant spores. a. 16 h spores, b. 20 h spores, c. 24 h spores 71 is difficult at this moment to draw any further conclusions about the SPR9 function during germination. 4.4 Detection of the SPR] phenotype The function of the SPR] gene was also investigated in this study. The sprl mutant strain can sporulate as efficiently as its wild-type parent (Primerano, 1990). The ability to germinate and the heat tolerance of the SPR] spores were examined by the same procedures used for SPR9 strains. Two different homozygous sprl strains, W61A (sprI-Izz URA3/spr1-1:: URAS’) and S52B (sprl-Q::LELI’Q/sprl-Q::LELII'Q), were tested for thermotolerance to show that the heat-sensitivity of sprl mutant spores observed in this study was not strain specific (Figure 4.9 and Figure 4.10). To test the germination ability of the sprl mutant spores, strains S52B (sprI/sp-rl) and S52A (SPRI/SPRI) were used. For 16 h sporulating cultures, the germination efficiency of mutant spores declined as temperature increased and so did the wild- type spores. At all temperatures, the germination efficiency of mutant Spores was only slightly lower than that of wild-type spores. At 37°C, 79.3% of mutant spores germinated compared to those germinated at 30°C, while 87.8% of wild-type spores germinated under the same conditions. At 40°C, the germination efficiency for mu— tant spores was 62.5% compared to 71.0% for wild-type spores. For a 32 h sporulating culture, no difference was observed between wild-type and mutant spores in their ger- mination ability on YEPD plates at all temperatures tested. At 37°C and 40°C, 80% of both wild-type and mutant spores germinated compared to at 30°C (Table 4.2 and Figure 4.8). The thermotolerance of sprl mutant spores was slightly lower than their isogenic wild-type spores under some conditions tested (Figure 4.9 and Figure 4.10). When 72 a. 100 90 C .2 80 g 70 r: 80 a” E 50 g, 40 up 30 20 —fi_ sprl-lGh 10 —0—' SPR1-16h OW ' I ' I ' I ' I ' I fI ' I 28 3O 32 34 36 38 4O 42 Temperature (°C) b- ‘00 -\___‘ g germination —°_ epr1-32h .—0—- SPRl-32h o I ' I ‘ I ' I ' I ' I ‘ I ' I 28 30 32 34 36 38 40 42 Temperature (°C) 388888388 Figure 4.8: Germination efficiency of wild type and spr! mutant spores. a. 16 h spores, b. 32 h spores 73 incubated at 4°C, 23°C or 30°C, the sprl mutant spores harvested at all four times survived as well as wild-type spores (data not shown). When the temperature of incubation was 42°C, the mutant spores were killed at a higher rate than the wild- type spores. This heat sensitivity of mutant spores was first apparent in 16 hour sporulating cultures. The difference in cell death between the mutant and the wild- type spores began to appear after 48 to 72 hours of incubation. After 120 h of incubation, the mutant spores showed 1% survival to the wild—type spores, while the wild-type spores had 3.3 x 10’5 survival. For 20 hour sporulating cultures, the differential killing effect of heat was less severe than to the 16 hour ones, although a 10 fold decrease in relative survival of the sprl mutant spores was seen at 120 h. The 24 hour mutant spores were killed by heat at the same rate as wild—type ones. This result suggests that the more mature the spores are, the less the SPR] function is needed. The spores from the SPRI/spr] heterozygous strain (SPBI) were also tested for heat sensitivity (Figure 4.11). This strain produces 2 wild—type spores and 2 mutant spores in one ascus. Therefore, it was expected that mutant survival would be less than the wild-type survival. Surprisingly, equal numbers of wild-type and mutant spores survived at all temperatures for all four stages. This may suggest that either the SPR] gene product functions before the formation of the individual spore wall, or that it can diffuse through spore wall and membrane. 4.5 Phenotype detection of SPR2 The phenotype of the SPR2 gene was also examined. The spr2 mutant strain did not show any difference from wild-type strains in all tests applied. They sporulated and germinated as efficiently as wild-type (Table 4.3 and Figure 4.12), and their 74 a. 10° 3 7 g 10 D 8‘ 10° 1-1 5 5 ,3 1o 2‘ 3 10‘ —I— 3523 o —o— SSZA U 3 10 F'jv'l""l"'rr""l"'rl o 24 4e 72 es 120 8 10 b. 0) ti 7 g 10 0" 5 a. 10° H O '44 >1 q 105 3 —.— $523 8 —-0- SSZA 4 10 I ' UV " t V V V V I V V f. I V V. U ' I I I I' o 24 48 72 96 120 10° C. to {J '3 :1 107 O 5. S 10° 0 ’44 >1 3 105 H —9— $528 8 —v- $52A 10“.r.........3........,....j 24 4a 72 93 120 Hours Figure 4.9: Thermotolerance of the wild—type and sprl mutant spores in SCMS-7 derivatives. The wild-type spores of strain 852A and the sprI mutant spores of strain 852B were preincubated at 42°C for various time and then grown on YEPD plates. Spores were from a. 16 h, b. 20 h, and c. 24 h sporulating culture. Colony forming units 3 (I °1 M h b O V N O Q Colony forming units 3 0| O N ‘ ‘ O N N 0 OJ Colony forming units Figure 4.10: Thermotolerance of the wild-type and sprl mutant spores in WGG-SA derivatives. The wild-type spores of strain W61C and the spr] mutant spores of strain “761A were preincubated at 42°C for various time and then grown on YEPD plates. Spores were from a. 16 h, b. '20 h, and c. '24 h sporulating culture. 76 a . 107 O) 4.) '3 a 5 10 m 5 5 105 o u 2‘ 10‘ '3 + Mutant ,3 -0— Wild type 103 ' V V V V I T V V V I V V V V I V V V V I V V V fl 0 24 48 72 96 120 b. C) 4) H r: :1 or c: E o u 2' g —"l— Mutant 8 4 —'9_ Wild type ‘0 I""l"“I""I""l"“j O 24 48 72 96 120 8 c. 10 3 «H 7 c: 10 S 2‘ 10° vi 5 5 3 1O ‘5‘ g 104 —9_ Mutant 8 —0— Wild type 103 ' V 1 I I I V V V V I V V V V I V V V V ' V V V V 0 24 48 72 96 120 Hours Figure 4.11: Thermotolerance of the wild-type and sprl mutant spores of SPB]. Strain SPBl (SPRI/sprl) will produce two mutant spores and two wild-type spores in one ascus. The spore suspensions were preincubated at 42°C for various time and then grown on YEPD plates. Spores were from a. 16 h, b. ‘20 h. and c. ‘24 h sporulating culture. 77 resistance to heat was the same as wild-type. We have detected no phenotype for the SPR2 gene; we do not know whether the SPR2 gene product has an important function in the events during and after sporulation. Further studies should be done in order to identify the role of the SPR? product during sporulation. 78 Table 4.2: Germination of wild-type and 3177‘] mutant spores. 16 h spores 32 h spores Germination temperature 30°C 37°C 40°C 30°C 37°C 40°C # of total spores 3.0 x 10" 2.5 x 107 2.0 x 107 2.6 x 107 2.0 x 107 2.0 x 107 % of mutant spores 52% 51% 50% 50% 50% 50% # of mutant spores 1.6 x 107 1.3 x 107 1.0 x 107 1.3 x 107 1.0 x 107 1.0 x 107 % of mutants germinating 100% 79% 63% 100% 80% 80% % of W. T. spores 48% 49% 50% 50% 50% 50% # of W. T. spores 1.4 x 107 1.2 x 107 1.0 x 107 1.3 x 106 1.0 x 107 1.0 x 107 % of W. T. germinating 100% 88% 71% 100% 80% 80% Table 4.3: Germination of wild-type and sprQ mutant spores. 16 h spores 32 h spores Germination temperature 30°C 37°C 40°C 30°C 37°C 40°C ' # of total spores 2.0 x 107 1.5 x 107 4.0 x 106 1.5 x 107 1.2 x 107 8.0 x 10‘3 % of mutant spores 58% 63% 50% 54% 62.5% 63% i # of mutant spores 1.2 x 107 9.4 x 106 2.0 x 106 8.1 x 106 7.4 x 106 5.0 x 106 ‘70 of mutants germinating 100% 81% 17% 100% 91% 61% % of W. T. spores 42% 37% 50% 46% 37.5% 37% # of W. T. spores 8.8 x 106 5.6 x 106 2.0 x 106 6.9 x 106 4.6 x 106 3.0 x 106 % of W. T. germinating 100% 64% 23% 100% 67% 43% 79 a. 100 90 5 00 '13 70 ”E 60 a: if. U‘ a, 30 20 —9'- spr2-16h 10 —0— SPR-16h OjVI'I‘I'I'I—' I ' I 28 30 32 34 36 30 40 42 Temperature (°C) b. 100 90 g 00 13 70 c: 00 '3 50 t: a, 40 30 w . 20 —u— spr2-32h 10 —9— 3232-321: OI—' I ' I 1 I ' I ' I ' I ' I 28 30 32 34 36 38 40 42 Temperature (°C) Figure 4.12: Germination emciency of wild type and spr2 mutant spores. a. 16 h spores, b. 32 h spores CHAPTER V Construction and Analysis of Double Mutants 5.1 Construction of double mutants defective at two SPR loci There were two reasons for constructing mutants defective at more than one gene; one was to determine the epistasis of these SPR genes during the sporulation process if they are in the same pathway; the other was to examine whether proteins of duplicated function are produced during sporulation. By mating two haploid H O spr single mutants, one can get a diploid that is heterozygous at both SPR loci. This heterozygous strain will produce spores of four different combinations of SPR genotypes after sporulation. They are wild-type, mutant at either SPR locus, and mutant at both SPR loci. Each spore will then diploidize and become homozygous for all loci except the mating type locus. In this study, all the strain constructions made were between derivatives of two strains, W66-8A and SCMS7-1 (Figure 5.1). SCMS7—1 is auxotrophic for leucine, being defective at leu2—3,112. For the derivatives of SCMS7—1, the LE U2 gene was inserted into the wild-type SPR gene and prototrophy for leucine was used to detect cells that contained spr mutations. W66-8A is also auxotrophic for leucine, at the Zen] locus. In order not to confuse the Zen] and Zen? genotypes, we decided to isolate 80 81 LE U1 revertants of W66-8A derivatives before mating with SCMS7-l derivatives, so that the auxotrophic phenotype for leucine observed in their offspring will be from [6272 only. The LE U1 revertants of W66-8A derivatives were either UV-induced or sponta- neous revertants. The spontaneous reversion rate for the Zen] strain is 10‘9, cal- culated from this study, while the UV-induced rate is 10’7 (data not shown). Ten revertants from both treatments were checked for their genotypes and they all showed the same markers as the untreated parents except at the [en] locus (data not shown). Retention of the SPR9 disruption (UR/13) was also confirmed by Southern blot anal- ysis of their genomic DNA; they all showed the correct construction at the SPRQ locus (data not shown). Since these revertants are heterozygous at the LE U1 locus, they were sporulated and diploidized to produce colonies that were homozygous at all loci except the mating type locus. The LE U1 cells were then retested for their homozygosity at the SPR9 locus by Southern blot analysis (data not shown). This strain was named W7a-1 and used for further constructions of double mutants. Spore-to-spore mating (Figure 2.3d and e) was used to construct sprl/Q double mutants. Spores from S52B (sprl-szLEUQ/sprl-QxLEUQ) and spores from W7a-1 (spr9zz URA 3)/sp7‘9:: URA3) were placed side by side by micromanipulation on a piece of thin YEPD agar to facilitate their mating. The diploid hybrids formed by mating should be both H0 and heterozygous for all the auxotrophic markers including the two SPR loci, since the two parent strains have completely different auxotrophic markers except that both are auxotrophic for adenine. Therefore, these diploids could grow on minimal medium supplemented with adenine while the cells from unmated and self-mated spores could not. The resulting diploid products carried one wild-type allele and one mutant allele for both the SPR] and SPR9 loci as shown by 82 Southern blot analysis (Figure 5.2a). All five hybrids examined are heterozygous at both loci. These strains were then sporulated and the asci were dissected. All the colonies grown from single spores were tested for their genotypes on minimal medium with different combinations of supplements (Figure 5.1). Those colonies grown after dissection were further examined for their SPR] and SPR9 loci by Southern blot analysis (Figure 5.3). For the construction of the SPRQ/ 9 double mutant, a similar approach was ap- plied, except that the single mutant strains used as parents for spore to spore mating were T4C (spr2zzLEU2/spr2zzLEU2) and W7a-l. The genomic DNA of cells with URA3 and LEUQ genotypes were examined for their SPR2 and SPR9 loci (Fig- ure 52b). All hybrids were heterozygous at both loci except the CT1 strain, which was defective only at SPR2 but not at the SPR9 locus and therefore was not used for further experiments. The reason for observing differences in relative intensity of the wild-type and mutant bands of the Southern blot analysis is not known at this moment. The genomic DNA of colonies grown after dissection was examined for their SPR2 and SPR9 loci by Southern blot analysis (Figure 5.3b). 5.2 Phenotypes of the double mutants The disruption of both loci of the two double mutants, 4-8-a (sprI/Q) and 5-4-d (sprQ/Q), was checked by Southern blot analysis (Figure 5.3). Their growth rates, like the single mutants, were not affected by mutations at two SPR loci (data not shown). The two double mutants can also sporulate when tested, but only about 40% of the population form four-spore asci at 30°C compared to 70% of the wild- type and the heterozygote (Figure 5.4). Figure 5.4a shows that 4-8-a sporulated 20% to 30% less than W66-8A (wild-type) and BS1 (heterozygous at both the SPR] 83 W7a a/ (HO ade2-1 leul trpS-2 met 4-1 lysZ-l ura3-l spr9::URA3) spontaneous reversion at leul locus W7a-1 a/ (HO ade2-l LEUl/leul trp5-2 met4-l lysz-l ura3-l spr9::URA3) sporulation 2 LEUl spores diploidization and sporulation 2 leul spores : 2 H0 adeZ-l trpS-Z mew-l lysz-l urn3-l spr9::URA3 3 H0 adeZ-l trpS-Z met4-1 lysz-l un3-1 spr9::URA3 HO sde2-1 trp5-2 met4-1 1ys2-1 un3-l spr9::URA3 HO sdeZ-l trpS-Z met4-l lys2-l unS-l spr9::URA3 k 8528 a/ (HO adex his4-Sl9 leu2-3,112 spr1::LEU2) 0r T4C a/ (HO adex his4-519 leu2-3,112 spr2::LEU2) sporulation H0 adex 11184-519 lad-3,112 spr1(or 2)::LEU2 H0 adex 11184-519 leul-3,112 spr1(or 2)::LEU2 0 HO sdex 11134-519 leu2-3,112 sprl(or 2)::LEU2 3 H0 sdex 11134-519 lend-3,112 sprl(or 2)::LEU2 .4 spore to spore mating a/ (HO ADE2-l/ade2-l ADEX/adex HIS4-Sl9/his4-Sl9 LEU2/leu2-3,112 LYS2-l/lysz-1 MET4-1/met4-l URA3-l/ura3-l SPR1(2)/spr1(2)::LEU2 SPR9/spr9::URA3) sporulation All different genotypes of spores were identified by examining the auxotrophic markers and Southern blot analysis. Figure 5.1: Construction of mutants defective at two SPR loci. 84 a. C18! digest of b. SPR9 locus 1 2 3 4 5 1 2 3 4 5 CD '1'. W.T. — O O O O O Mut. — I 35 .. -/ . Probe=5 Bkb Clal fragment Xhol digest of Hindlll digest of SPR] locus SPR2 locus Mut. __ 6.6 Mut.—‘. . - - g —5.8 WT — _44 WT. _ i . ... .7 Probe: pCG33 ”"0139”,“:I Figure 5.2: Southern blot analysis of the heterozygous strains. The genomic DNA of cells which are prototrophic for uracil and leucine were prepared for Southern blot analysis. a. The genomic DNA of strains BSl, BS2, C51, C82, and D81 was digested with Clal enzyme for detection of the SPR9 locus and XhoI for detection of the SPR] locus. b.The genomic DNA of strains BT1, BT2,CT1, CT2, and CT3 were digested with ClaI enzyme for detection of the SPR9 locus and HindIII for detection of the SPR? locus. +: wild-type band, —: mutant band. 85 a. Clal digest of Xhol digest of SPR9 locus SPR] locus t - - - _ ’1' I I- w.r. _’ 4' 6.6 kb MUL— Lo . . 4.4 kb 3 . ' , i 7 7 b. Clal digest of Hindlll digest of SPR9 locus O i‘ 2 3 4 ' W.T. ' * . —5-8 kbMut.— ' . — 5.8 kb ' — 2.7 kb .~~ - - —3.5 kbw'T' = _ SPR2 locus _ , , .. Mut. — Figure 5.3: Southern blot analysis of the spr double mutants. a. Southern blot analysis for the detection of spr1/9 double mutants. The genomic DNA from 5 spore colonies was digested by Clal enzyme for the detection of SPR9 locus and by XhoI for SPR] locus. b. Southern blot analysis for the detection of spr2/9 double mutants. The genomic DNA from 5 spore colonies was digested by Clal enzyme for the detection of SPR9 locus and by HindIII for SPR] locus. +: wild-type band, -: mutant band. 86 and SPR9 loci). However, the kinetics of sporulation of 4-8-a are similar to that of the wild-type and heterozygous strains. Figure 5.4b shows the sporulation kinetics of W66-8A, CT3 (heterozygous at both the SPR2 and SPR9 loci), and 5—4-d. The result is similar to that of 4-8-a. The thermotolerance of double mutant spores was also tested under the same conditions as single mutants. The result was that spr1/9 double mutant spores were very sensitive to heat, while the spr2/9 double mutant spores had about the same thermotolerance as spr9 single mutant spores. Table 5.1 shows the spore viability of 4-8-a and 5-4-d after incubation at five different temperatures compared to two wild-type strains, W66-8A and SCMS-7. After incubating 3 days at 42°C, spore viability of 4-8-a dropped 105 and those of 5-4-d dropped 20 fold while the wild-type spores retained the same viability. At 30°C, the viability of both double mutant spores improved, a 500 fold decreases in spore viability for 4-8-a and wild-type level of viability for 5-4-d. At 4°C, the spores of wild-type and both double mutants had the same viability after 6 days of incubation. The same experiment was repeated by using micromanipulation to produce single spores, which were incubated at 42°C, and a similar result was observed (Table 5.2). As the period of incubation increased, the number of intact four-spore asci decreased for all four strains tested. However, the number of viable spores decreased much more for 4-8-a. By the third day, no viable spores were detected by plate count. Furthermore, the spores of 4-8-a were so fragile that they broke when touched with the micromanipulator needle, which was not true of the spores of the other three strains. 87 a. . 80's a 1 .2 so- ‘3 —I '3 H 40-1 0 o. «n 3% —9— "66-85 ""'l"" 4-8-a 0 - . ' . ' ' 0 10 20 30 4O 50 Time (Hours) b. a c: #3 0 or. u t“ ‘—-I H a u o D: 01 .\° ass-BA CT3 5-4-d I ‘ I 40 50 Time (Hours) Figure 5.4: Sporulation efficiency of double mutants. The sporulation efficiency of three strains W66-8A (wild-type), BSl (heterozygous at both SPR] and SPR9 loci), and 4-8-a (homozygous spr1/9 at 30°C was in Figure a. Figure b shows the sporulation efficiency of strains W66-8A, CT3 (heterozygous at both SPR2 and SPR9 loci), and 5-4—d (homozygous spr2/9 at 30°C. 88 5.3 Multiple heat effect on germination To test if preincubation at high temperature and germination at high ten'iperature have an additive effect on spore germination, spores of 7 strains were preincubated at 4°C, 30°C, or 42°C for 48 h and then germinated at 30°C or 40°C. The free spore suspension of each strain was divided equally into three tubes for preincubation at three different temperatures, 4°C, 30°C, and 42°C. After incubating for 48 hours, each spore suspension was spread onto two YEPD plates for germination. One plate was incubated at 30°C and the other one at 40°C. The result is shown in Table 5.4. The numbers of cells preincubated at 4°C and then germinated at 30°C were taken as the numbers of starting cells, since spores do not lose viability when incubated at 4°C. The three single mutants, S52B, T4C, and W-7-a, showed a similar heat tolerance to the wild-type strains, since the heat-sensitive phenotype of S52B mutant spores does not become evident until 72 hours. Comparing the ability of the spores to germinate at high temperature, W-7-a was sensitive to heat during germination but the other two single mutant strains were not. For the double mutant strain, 4-8-a, the absence of two SPR gene products was detrimental when high temperature was applied both to preincubation and germination. The number of surviving mutant cells dropped more than lOO-fold when compared to wild—type spores under same conditions. For 5- 4-d, the effect of the absent SPR9 gene product was observed in the lower germination efficiency when high temperature was applied to both preincubation and germination. 5.4 Linkage between SPR9 and LY52 The results of CHEF analysis indicated that the SPR9 gene lies on chromosome 11 where the LY52 gene is also located. The linkage between these two genes can be determined by recombination analysis. The first step was to construct a strain that is 89 heterozygous at both the SPR9 and the LY52 loci. The scheme used to generate the heterozygotes is the same as that for constructing double mutants (Figure 5.1). The heterozygotes from the cross of W7a-1 to S52B and the cross of W 7a-1 to T4C were then sporulated and dissected for analyzing the recombination frequency between the two loci. There were 89 and 62 asci dissected from the cross involving T4 C and S52B, and the percentages of four viable-spore asci were 57% and 68%, respectively. Only 92 of them were complete tetrads and were examined for the genotype of each spore. Among those 92 asci, only tetrads that were 2 URA-l- : 2 URA— and 2 LYS+ : 2 LYS- were used for calculating the map distance. The reason was that there were 4 URAS’ and 2 mm? alleles in the heterozygotes instead of the normal 2 URA3 and 2 171113 distribution. Two of the URAS’ alleles were from S52B or T4C, and the other two URA3 were 319129.: URA3 alleles from W7a-1. Only the asci with the 2 URA+ : 2 URA— pattern were certain to be 2 SPR9: 2 sprg, since both the normal URAS’ and the sprgzz URA3 alleles were segregated into the same spore. Some other asci were confirmed by Southern blot analysis for the 2 SPR9 : 2 sprQ pattern(data not shown). The numbers of parental ditype, tetratype, and nonparental ditype asci for SPR9 and LYS? loci and map distance are shown in Table 5.1. Let (I be the map distance in cM between two markers (Esposito and Klapholz, 1981), then d =100(TT + 6NPD)/2(PD + TT + NPD), where PD is parental ditype ascus, TT is tetratype ascus, and NPD is nonparental ditype ascus. The map distance obtained from these data (about 67 CM) suggested that the SPR9 locus and the LY52 locus are not linked. Thus, there are more than 50 cM between the SPR9 locus and the LYSQ locus. The centromere linkage of the SPR9 locus was also examined. Since the map 90 distance between the centromere and a gene is about one half the percentage of second division segregation (SDS), there is a formula for determining the distance between a gene and its centromere: For two heterozygous sites, each on different chromosomes, and with SDS fre- quencies X and Y, respectively, let F be the frequency of tetratype asci for the two markers (Esposito and Klapholz, 1981). F = X +Y—3/2(XY) In this case, the other centromere linked gene used as reference is H154 which is 20.8 cM from its centromere (Mortimer and Schild, 1980). Therefore, the percentage of SDS for H154 is twice the number of the centimorgans between H154 and its cen- tromere, which is 0.40. The frequency of tetratype asci for H154 and SPR9 obtained from tetrad analysis of heterozygotes of both loci is 0.58. The data are shown in Ta- ble 5.4. The calculated distance between centromere II and SPR9 is about 22.3 CM. Since the distance between centromere II and LY52 is 54 cM (Mortimer and Schild, 1980), these data indicate that SPR9 should be on the opposite arm from LYSZ? (which is on the shorter arm). However, further experiments have to be done for precisely mapping the position of SPR9 locus on chromosome 11. 5.5 Linkages among SPR], SPR2, and ADE2 Primerano et al. showed that both the SPR] and SPR2 genes lie on chromosome XV by OFAGE analysis (1990 and unpublished data). Their linkage to the .4 DE? locus (chromosome XV) could be obtained from the same heterozygotes used for determining SPR9 and LYSQ linkage. The only difference was that there were two ADE loci involved in this cross: ADE? and ADEX. The ads}? mutants are not only auxotrophic for adenine but accumulate a red pigment which turns the colonies red. 91 The adeX mutants are auxotrophic for adenine, and are white. The adCB/ader double mutants are also white. There would be three different types of asci produced by the heterozygotes containing the ode? and adex loci. In only two of them could the genotypes at these loci of all four spores be distinguished (Table 5.5), the parental ditype and nonparental ditype asci, and only those asci were used to determine the linkage to the SPR] and SPR? loci. In a type I ascus, the genotype at the ADE? locus of the two auxotrophic white ascospore colonies cannot be determined from the information obtained. The map distances determined from the data in (Table 5.4) were 59 cM between SPR! and ADE? and 65 cM between SPR? and ADE? Both of them are probably not linked to the ADE? locus. The centromere linkage of these two loci was also examined by using the H154 locus as a reference to determine the frequency of second division segregation. The map distance from centromere XV to SPR] was 23.5 cM, and to SPR? 4.5 cM. The map distance between ADE? and the centromere was 65 CM (Mortimer and Schild, 1980). Further experiments are needed to establish the precise map relationship among SPR], 5PR?, and ADE? loci. 92 Table 5.1: Thermotolerance of double mutants. W66-8A SCMS-7 4-8—a 5-4--d SPR genotype (1+)(2+)(9+) (1+)(2+)(9+) (1—)(2+)(9-) (1+)(2—)(9—) 42°C Day 0 6.5 X 107 3.4 X 107 1.2 X 107 1.5 X 107 Day 3 1.5 x107 2.3 x107 <1.0 x102 5.5 x 105 30°C Day 0 1.5 X 108 1.5 X 107 5.0 X 106 1.3 X 107 Day 3 1.0 x107 1.0 x107 1.0 x104 8.0 x 106 4°C Day 0 5.0 X 107 2.2 X 107 2.5 X 107 3.0 X 107 Day 6 4.0 x 107 1.0 x 107 3.0 x 106 3.0 x 107 93 Table 5.2: Thermotolerance of double mutants by micromanipulation followed by incubation at 42°C. W66-8A SCMS07 4-8-a 5-4-d Genotype (1+)(2+)(9+) (1+)(2+)(9+) (1—)(2+><9-) (1+)(2—)<9—> 0 h Total spores 44 49 40 36 Total tetrads 11 1O 11 9 Viable spores 42 48 29 35 24 h Total spores 42 15 24 52 Total tetrads 6 4 1 13 Viable spores 39 12 4 46 48 h Total spores 36 40 a 36 Total tetrads 9 10 ~~~ 9 Viable spores 30 24 — 25 72 h Total spores 41 32 --- 40 Total tetrads 10 4 —— 10 Viable spores 28 16 —— 23 a. Spores broke when touched with needle. The plate count showed no viable spores after 72 h incubation at 42°C. 94 .2 x 3 v ”2 x 3 v .2 x 3 v .2 x q... .2 x S. .2 x 2: .2 x 3 0.2. T 2 2; 0.2. .2 x 3. .2 x as .2 x 3 .2 x as .2 x as .2 x 3 .2 x 3 0.2 T 2 as 0.2. .2 x 3 v .2 x 3 v .2 x 3 v .2 x A: .2 x 3. .2 x on .2 x S 0.2. T E 2; 0.2 .2 x 3 .2 x as .2 x 2 .2 x f .2 x as .2 x 3 .2 x 3 0.2.. T 2 2; 0.8 .2 x 3 .2 x 3 v .2 x as .2 x as .2 x as .2 x S .2 x em 02. T 2 22 o... .2 x as .2 x S .2 x 3 .2 x 3 .2 x o.” .2 x as .2 x o.” 0.2 T 3 22 o... .58 82235.2 $242+: $232-: Ta2+~2+3 $232+: $2222-: 2+a2+~2+s 2+a2+~2+3 8:88 2% 2.-.-.. is. 3-3 0.9 £2 328 8.83 .ooSeEEuow :o flute ousaeuomEou 23:52 ”m6 oEeE 95 Table 5.4: Linkage analysis of three SPR genes. Number of Asci PDQ TTb NPDc Freq. of SDSd Map distance SPR 9-L Ysz. SPR 9-11154 SPR 1 -A DE‘Z , 51931-11154 SPR2-ADE? SPR2-H154 5 23 6 6 18 7 3 7 1 4 13 5 2 7 1 1 8 9 0.58 0.59 0.44 unhnked unlinked unlinked unlinked unlinked unlinked Q G- c. NPD is nonparental ditype ascus. Q. Q . PD is parental ditype ascus. . TT is tetratype ascus. . SDS is second division segregation. . The distance between centromere II and LYS? is 54 CM. f. The distance between centromere XV and ADE? is 65 CM. 96 . I a ll pm: I L. 353 I .3 . ”moEo—oo 20 .228 BE. .22? 2:32: H .I. .32? .8 .>> M .+. .o .cosficofioaasm octave 252.23 82a 7:2 e co 38w 3 35% 23. .v .258 unzip Eacoaeaco: mm sz . .258 earn—253 mm BB U .288 093% Eacouea mm mm A .d a + I I a l I n a I u u e .H + I I 3 T .. I .2 + I I m a .. + - a + + + a - + - m a .. + .. a + + + a + + + 2 .8_8 2.3.. 852 25a .28 «max 252 £35 :28 .23.. .25.. .232» 22s 3an E 25. 25x: 2 25 2.2.: 2 25. .Eesm 82e\k®Q< moue\mmm~v. Eod poneuocom «one 20 89$ 8:3 .23. ”mm oEeH. CHAPTER VI Discussion In this study, the SPR9 locus was analyzed molecularly in terms of transcription, regulation, and nucleotide sequence. The phenotypes of three SPR genes, SPR], SPR2, and SPR9, were also studied in terms of thermotolerance during spore forma- tion and germination. Two double mutants, sprI/Q and spr2/9, were constructed for investigation of the relationships among these three genes during sporulation. The results of this study show that the three SPR genes are not involved in the process of sporulation, but the gene products of SPR] and SPR9 affect the spore thermotoler- ance and germination under high temperature. Despite the fact that the biochemical functions of these SPR genes and hence the reasons for the mutants showing the phe- notypes are not known, some evidence suggests that some of the sporulation-specific gene products such as SPR] and SPSIOO are associated with the spore membrane (Primerano et al., 1990; Law and Segall, 1988). Additional sporulation-specific genes may be involved in the spore membrane assembly and functions. Sporulation is an intricate process which switches yeast cells from one state to another. Most of the sporulation—specific genes studied so far (Percival-Smith and Segall, 1986; Garber and Segall, 1986; Law and Segall, 1988; Gottlin-Ninfa and Kaback, 1986; Primerano et al. unpublished data) do not block sporulation. This 97 98 observation may be explained by the existence of more than one sporulation pathway in yeast cells to ensure sporulation. Alternatively, the genes involved in sporulation may be functionally redundant, or it may be that some of the gene products are not essential for sporulation and the absence of them only creates an undetectable defect in sporulation. However, few of the sporulation-specific genes have been examined for the behavior of the spores produced by homozygous mutant strains. Little is known about the process of spore development and germination, although they are obviously important parts of the developmental process. Mutations at SP5 100 (Law and Segall, 1988), SPB], and SPR9 (This work) are found to affect spore resistance to stress and germination competence. There may be more sporulation-specific genes that do not affect sporulation, but are nonetheless involved in these processes. More studies are needed to relate those gene functions to the biochemical events in spore development. The protein predicted from the SPR] nucleotide sequence has a putative transit sequence, and the fusion protein of SPRl-LacZ was found in the membrane fraction in yeast cells (Primerano et a1. 1990). Holaway et al. (1985) detected abundant transcripts of the SPR] gene in sporulating cells and the accumulation of the tran- scripts lasted at least 20 h after transfer to sporulation medium. In this study, we found that mutant spores missing the SPR] gene product are more sensitive to heat than are the wild-type spores. All these results suggest that the gene product of SPR] may be one of the components of the spore membrane. The mutant spores, however, regain heat resistance as sporulation progresses, and they finally reach the wild-type level of heat resistance after 24 h of incubation in sporulation medium. The delay in the heat resistance may suggest a delay in spore maturation. In the heterozygous strain, SPBl, both mutant and wild—type spores show the wild-type 99 level of thermotolerance. This result indicates that the SPR] gene is maternally acting. The possibilities for the maternal action of the SPR] gene product are that SPR] functions before the formation of the spore wall or before the closure of as- cospores, or that the SPRI protein can travel through the spore membrane freely. The nucleotide sequence of SPRI is not homologous to any sequence in Genebank, nor is the predicted protein homologous to any protein sequence in the protein bank (Primerano et al. 1990). Therefore, it is important in future to isolate the SPR] protein for biochemical analysis in order to reveal its function in vivo and its role in spore formation. The SPSIUO gene isolated by Law and Segall (1988), which is expressed 12 h after transfer to sporulation medium, is also involved in spore maturation. The ether resistance of the 3123100 mutant spores is delayed when compared to that of wild- type spores. The nucleotide sequence of SPSIOO also predicts a transit sequence and shows many potential glycosylation sites for this protein. Their results also suggest that the SP5100 protein is associated with the spore membrane. The ether- resistance of the spr] mutant spores was also examined in this study; the mutant spores exhibit the same level of resistance to ether as the wild-type spores (data not shown). Although different strains were used as recipients for constructing the 3123100 and sprl mutant strains, the kinetics of sporulation of the strains were similar. Both mutant spores became resistant to stress conditions at the same time (‘24 h after transfer to sporulation medium). These observations suggest that SPSIOO and SPR] function in protecting the spores from different kinds of stress. The reason for different gene products causing the same pattern of delay in spore maturation when missing is not clear. The heat-sensitivity of sprl mutant spores and the ether-sensitivity of 312.3100 100 mutant spores only exist during a few hours of development. Both mutant spores regained resistance in later stages of spore development. The reason that the gene functions of SPR] and SPSIOO are not absolutely required is not known. There may be proteins which appear later that can replace the functions of SPR] and SP5 100. We may get the answer when the biochemical functions of these genes are known. One mutation of B.subtilis, ger E36, also shows a conditional phenotype. The endospores formed at 42°C were phase-gray, while when sporulated at lower temperature the spores became phase-bright (Moir, 1981). The basis for the conditional phenotype of the ger E36 is not known, either. The homozygous mutant strain spr2/spr2 did not show any difference from the wild-type in any of the tests used in this study, including sporulation efficiency, ther- motolerance, germination at high temperature, and resistance to ether (data not shown). The lack of phenotype of the spr2 mutants may be due to the fact that only a limited number of criteria for testing mutant phenotypes during sporulation have been used, there may be other proteins that can replace the function of SPR2, or it may be simply that the function of the SPR2 gene is nonessential. In B. megaterium, there are highly related acid-soluble polypeptides that are degraded to provide a source of amino acids during germination. These proteins may be redun- dant in function and the absence of any particular one would be without significant phenotype (Dignam and Setlow, 1980). Obviously, more experiments have to be done in order to draw any conclusions about the role of the SPR2 gene during sporulation. The SPRQ gene is expressed after 4 h incubation in sporulation medium and the transcripts remain in abundance after at least 20 h more of incubation. The protein predicted from the nucleotide sequence of the SPR9 locus is 16.8 kD, which is close to the size of one of the early sporulation-specific proteins identified by Kurtz and 101 Lindquist (1984). The predicted SPR9 protein consists mostly of hydrophilic amino acids. No transit peptide sequence is present. No homologous sequences have been found for either the SPR9 nucleotide sequence or the predicted protein sequence. The results of CHEF also revealed that there is no closely homologous sequence on any other chromosome of S. cerevisiae. At temperatures higher than 37°C, the germination efficiency of the sprQ mutant spores was found to be lower than the wild-type spores, for spores isolated from 16 h, 20 h, 24 h, and even 32 h sporulating cultures (data not shown for 32 h spores). Al- though reduced germination efficiency appeared in spores at all four times, the 20 h spores showed the most severe defect when germinated at high temperature. The decrease in germination efficiency did not result from a delay of response to germi- nation signals, since prolonged germination time did not improve the germination efficiency of mutant spores (data not shown). It seems that either the maturation of mutant spores was not affected by the sprQ defect, or the spore maturation was delayed even after 32 h of incubation in sporulation medium. If the first case is true, it suggests that the SPR9 gene exerts its function after spore formation, or spore germination and formation are functionally independent. To test the second case, spores incubated longer than 32 h in sporulation medium should be used for the same kind of experiments. However, the heat-resistance of the spr9 mutant spores was normal. This result suggests that the defective SPR9 gene affects only the ger- mination process. From the facts described above, we conclude that not only are the functions of SPR] and SPR9 totally different, but they also act at different stages of spore formation. The formation of small colonies was observed when the spores of strains W 66-8A and derivatives were germinated at temperatures above 37°C. The appearance of 102 the small colonies was most likely due to heat-induced induction of mitochondrial mutations. These mitochondrial mutants are called petites, they are defective in respiration and can not utilize glycerol as a carbon source. Petite ade] or rider? colonies are white and small. These small colonies not only are white, they also can not grow in YEPG medium. The result with germinating wild—type and spr9 mutant spores suggests that the appearance of small colonies may be a consequence of the extent of maturation of spores germinated. This is supported by the observation that the more mature the spores are, the fewer small colonies are formed. Therefore, the fact that most small colonies are germinated from mutant spores is because that mutation at spr9 locus makes spores more sensitive to heat during germination. Multiple mutants, lacking more than one gene from a specific developmental pathway, are required for resolving the relationships among the genes in that pathway. If the genes involved are functioning in a sequential order, a damaged gene which functions at an early stage will block the expression of the genes functioning later. Thus, the multiple mutant will show the same phenotype as a. single mutant blocked at the earliest gene. If genes disrupted are functioning independently, the multiple mutant will show a combination of mutant phenotypes in the same cell. If single mutants show no phenotype due to gene function duplication, the multiple mutants may show a defective phenotype. In this study, two double mutants were constructed to demonstrate the interrelationship among the SPR], SPR2, and SPR9 genes. For spr1/9 double mutant spores, the heat-sensitivity is much increased over the sprl single mutant spores. The number of viable spores of 16 h sprI/9 spores dropped 105 after 72 h of incubation at 42°C, while sprl spores at the same stage only dropped lO-fold under the same conditions. The 16 h double mutant spores also showed a lower germination efficiency than the spr9 single mutant spores at high temperature. 103 The above observations suggest that spores missing the two SPR gene products show greater sensitivity to heat both in viability and in germination efficiency than do the single mutants; i.e., the phenotype of a spore missing these two SPR gene functions is greater than the sum of the phenotypes of the two single mutants missing one SPR gene function. Therefore, we conclude that SPR] and SPR9 genes do not function in sequence, nor do they have duplicated functions. For spr2/9 double mutants, the mutant spores showed increased thermosensitiv- ity in addition to the predicted defect in germination. In addition, the efficiency of germination at high temperatures of double mutant spores was also lower than that of sprQ single mutant spores. These results may suggest that the sp'rQ defect, nonobservable in a single mutant, is revealed in a double mutant. From these result, we can also conclude that SPR2 and SPR9 genes do not function sequentially, nor have the same functions. The physiology and the structure of endospores and spore germination of Bacil- lus have been studied in depth. Although the formation of endospores of bacteria is quite different from sporulation in S. cerevisiae, the knowledge obtained from bac- terial spore formation can still serve as a model system for studying yeast spore formation. The spore coat of B. subtilis consists of twelve or more different peptides ranging in size from 8000 to 65,000 Mr, and the major component is a peptide of Mr 12,000. Four structural genes of spore coat, cotA, cotB, cotC’, and cotD have been studied, which encode high abundance proteins of Mr 65,000, 59,000, 12,000, and 11,000, respectively. None of the cot mutations studied so far block the sporulation process. But the cotD mutant was found to germinate somewhat more slowly than the wild-type spores, and the COM is responsible for sporulation-associated pigment production (Donovan et al., 1987). James and Mandelstam (1985) suggested that 104 the spore coat of B. subtilis was acting as a protective shell, which participated in response to germination signals. Future work should identify the biochemical roles of sporulation-specific genes during sporulation and later events. The regulation of expression of these genes is also an important and interesting question to be resolved. By comparing the sequence of the regulatory region of sporulation-specific genes, we may find some interesting sequences that control their developmentally regulated expression. Although such a sequence has not yet been found among these three genes, it may be that different sequences are used at different stages. Further sequencing should be performed on more sporulation—specific genes to identify such sequences, and these sequences can be used to isolate the factors, which react with them. By this process, the mechanism of regulating the expression of sporulation-specific genes may eventually be understood. BIBLIOGRAPHY BIBLIOGRAPHY [1] Ballou, C., S. Maitra, J. Walker, and W. Whelan. 1977. Developmental defects associated with glucoseamine auxotrophy in Saccharomyces cerevz'sz'ae. Proc. Natl. Acad. Sci. USA 74: 4351-4355. [2] Baker, B. S., A. T. C. Carpenter, M. S. Esposito, R. E. Esposito, and L. Sandler. 1976. The genetic control of meiosis. Annu. Rev. Genet. 10: 53-90. [3] Beckett, A., R. F. Illingworth, and A. H. Rose. 1973. Ascospore wall development in Sacchammyces cemvisiae. J. Bacteriol. 113: 1054- 1057. [4] Betz, H. 1977. Protein degradation and sporulation in yeast. In Cell differenti— ation in microorganisms, plants and animals (ed. L. Nover and K. Mothes), p. 243. Gustav Fischer, Jena. [5] Betz, H., and U. Weiser. 1976. Protein degradation and proteinases during yeast sporulation. Eur. J. Biochem. 62: 65-72. [6] Carle, G. F., and M. V. Olson. 1985. An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA 82: 3756-3760. [7] Casey, J., and N. Davidson. 1977. Rates of formation and thermal stabilities of RNAzDNA and DNAzDNA duplexes at high concentrations of formamide. Nucleic Acids Res. 4: 1539-1595. [8] Chu, G.,D, Vollrath, and R. W. Davis. 1986. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234: 1582-1585. [9] Clancy, M. J., B. Buten-Magee, D. J. Straight, A. L. Kennedy, R. M. Parn- vidge, and P. T. Magee. 1983. Isolation of genes expressed preferentially during sporulation in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 80: 3000-3004. [10] Clutterbuck, A. J. 1969. A mutational analysis of conidial development in As- pergz'llus nidulins. Genetics 63: 317-327. [11] Colonna, W. J., and P. T. Magee. 1978. Glycogenolytic enzymes in sporulating yeast. J. Bacteriol. 134: 844-853. [12] Croes, A. F. 1967a. Induction of meiosis in yeast. I. Timing of cytological and biochemical events. Planta 76: 209-226. 105 106 [13] Croes, A. F. 1967b. Induction of meiosis in yeast. 11. Metabolic factors leading to meiosis. Planta 76: 227-237. [14] Dawes, I. W. 1975. Study of cell development using derepressed mutations. Nature 255: 707-708. [15] Dawes, I. W. 1983. Genetic control and gene expression during meiosis and sporulation in Sacchammyces cerevisiae. In Yeast Genetics Fundamental and Applied Aspects. p29-58. Springer-Verlag New York Inc., New York. [16] Dignam, S. S., and P. Setlow. 1980. In vivo and in vitro synthesis of the spore specific proteins A and C of Bacillus megaterium. J. Biol. Chem. 255: 8417-8426. [17] Dion, P. and J. Mandelstam. 1980. Germination properties as marker events characterizing later stages of Bacillus subtilis spore formation. J. Bacteriol. 143: 786-792. [18] Donovan, W., L. Zheng, K. Sandman, and R. Losick. 1987. Gene encoding spore coat polypeptides from Bacillus subtilis. J. Mol. Biol. 196: 1-10. [19] Esposito, R. E., and S. Klapholz. 1981. Meiosis and ascospore development. In The Molecular Biology of the Yeast Sacchammyces. vol. 1.: Life Cycle and Inheritance, p211-287. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. [20] Esposito, M. S., and R. E. Esposito. 1969. The genetic control of sporulation in Saccharomyces. I. The isolation of temperature-sensitive sporulation-deficient mutants. Genetics 61: 79-89. [21] Esposito, M. S., and R. E. Esposito. 1974. Genes controlling meiosis and spore formation in yeast. Genetics 78: 215-225. [22] Esposito, M. S., and R. E. Esposito. 1975. Mutants of meiosis and ascospore formation. Methods Cell Biol. 11: 303-326. [23] Esposito, M. S., and R. E. Esposito. 1978. Aspects of the genetic control of meiosis and ascospore development inferred from the study of spa (sporulation- deficient) mutants of Saccharomyces cerevisiae. Biol. Cell. 33: 93-103. [24] Esposito, M. S., R. E. Esposito, M. Arnaud, and H. O. Halvorson. 1969. Ac- etate utilization and macromolecular synthesis during sporulation of yeast. J. Bacteriol. 100: 180-186. [25] Fitzgerald, M., and T. Shenk. 1981. The sequence 5’-AAUAAA-3’ forms part of the recognition site for polyadenylation of late SV40 mRN As. Cell 24: 251-260. [26] Fonzi, W. A., M. Shanley, and D. J. Opheim. 1979. Relationship of glycolytic intermediates, glycolytic enzymes and ammonia to glycogen metabolism during sporulation in the yeast Saccharomyces cerevisiae. J. Bacteriol. 137: 285-294. [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] 107 Fowell, R. R. 1975. Ascospores of yeast. In Spores(ed. P. Gerhart et al.), vol. 6, p.124. American Society for Microbiology, Washington. Garber, A. T., and J. Segall. 1986. The SP5] gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA. Mol. Cell. Biol. 6: 4478-4485. Gottlin-Ninfa, E., and D. B. Kaback. 1986. Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae. Mol. Cell. Biol. 6: 2185-2197. Haber, J. E., M. S. Esposito, P. T. Magee, and R. E. Esposito. 1975. Current trends in genetic and biochemical study of yeast sporulation. In Spores (ed. P. Gerhart et al.) , vol. 6, p.132. American Society for Microbiology, Washington. Hahn, S., E. T. Hoar, and L. Guarente. 1985. Each of three “TATA” elements specifies a subset of the transcription initiation sites at the C YC-I promoter of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 82: 8562-8566. Harper, J. F ., M. J. Clancy, and P. T. Magee. 1980. Properties of polyadenylate- associated RNA from Saccharomyces cerevisiae ascospores. J. Bacteriol. 143: 958-965. Hasilik, A., H. Muller, and H. Holzer. 1974. Compartmentation of the tryptophan-synthase-proteolyzing system in Saccharomyces cerevisiae. Eur. J. Biochem. 48: 111-117. Herskowitz, I., and Y. Oshima. 1981. Control of cell type in Saccharomyces cere- visiac: Mating type and mating-type interconversion. In The Molecular Biology of the Yeast Saccharomyces. vol. 1.: Life Cycle and Inheritance, p145-210. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. Holaway, B. L., G. Kao, M. C. Finn, and M. J. Clancy. 1987. Transcriptional regulation of sporulation genes in yeast. Mol. Gen. Genet. 210: 449-459. Holaway, B. L., D. J. Leman, D. A. Primerano, P. T. Magee, and M. J. Clancy. 1985. Sporulation-regulated genes of Saccharomyces cerevisiae. Curr. Genet. 10: 163-169. Hopper, A. K, and B. D. Hall. 1975. Mating type and sporulation in yeast. 1. Mutations which alter mating-type control over sporulation. Genetics 80: 41-59. Hopper, A. K., P. T. Magee, S. K. Welch, M. Friedman, and B. D. Hall. 1974. Macromolecular synthesis and breakdown in relation to sporulation and meiosis in yeast. J. Bacteriol 119: 619-629. [39] Horesh, O., G. Simchen, and A. Friedmann. 1979. Morphogenesis of the synap- ton during yeast meiosis. Chromosoma 75: 101-115. [40] Illingworth, R. F., A. H. Rose, and A. Beckett. 1973. Changes in the lipid com- position and fine structure of Saccharomyces cerevisiae during ascus formation. J. Bacteriol. 113: 373-386. 108 [41] James, W. and J. Mandelstam. 1985. spo VIC, a new sporulation locus in Bacillus subtilis affecting spore coats, germination and the rate of sporulation. J. Gen. Microbiol. 131: 2409-2419. [42] Kaback, D. B., and L. Feldberg. 1985. Saccharomyces cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation. Mol. Cell. Biol. 5: 751-761. [43] Kane, S., and R. Roth. 1974. Carbohydrate metabolism during ascospore devel- opment in yeast. J. Bacteriol. 118: 8-14. [44] Kassir, Y., D. Granot, and G. Simchen. 1988. [ME], a positive regulator gene of meiosis in S. cerevisiae. Cell 52: 853-862. [45] Keith, A. D., M. Resnick, and A. B. Haley. 1969. Fatty acid desaturase mutants of Saccharomyces cerevisiae. J. Bacteriol. 98: 415-420. [46] Klar, A. J. S., and H. O. Halvorson. 1975. Proteinase activities of Saccharomyces cerevisiae during sporulation. J. Bacteriol. 124: 863-869. [47] Kraig, E., and J. E. Haber. 1980. Messenger ribonucleic acid and protein metabolism during sporulation of Saccharomyces cerevisiae. J. Bacteriol. 144: 1098-1112. [48] Kuenzi, M. T., and R. Roth. 1974. Timing of mitochondria DNA synthesis during meiosis in Saccharomyccs ccrcvisiae. Exp. Cell Res. 85: 377- 382. [49] Kurtz, S., and S. Lindquist. 1984. Changing patterns of gene expression during sporulation in yeast. Proc. Natl. Acad. Sci. USA 81: 7323-7327. [50] Kurtz, S., and S. Lindquist. 1986. Subcellular differentiation in sporulating yeast cells. Cell 45: 771-779. [51] Law, D. T. S., and J. Segall. 1988. The SPSIOO gene of Saccharomyces cere- visiae is activated late in the sporulation process and contributes to spore wall maturation. Mol. Cell. Biol. 8: 912-922. [52] Lillie, S. H., and J. R. Pringle. 1980. Reserve carbohydrate metabolism in Sac- chammyces cerevisiae: Responses to nutrient limitation. J. Bacteriol. 143: 1384- 1394. [53] Lucchini, G., A. Biraghi, M. L. Carbone, A. DeScrilli, and G. E. Magni. 1978. Effect of mutation in the aromatic amino acid pathway on sporulation of Sac- charomyces cerevisiae. J. Bacteriol. 136: 55-62. [54] Lynn, R. R., and RT. Magee. 1970. Development of the spore wall during ascospore formation in Saccharomyces cerevisiae. J. Cell Biol. 44: 688-692. [55] Magee, P. T. 1974. Changes in DNA dependent RNA polymrerase in sporulating yeast. Mol. Biol. Rep. 1: 275-281. 109 [56] Magee, P. T., and A. K. Hopper. 1974. Protein synthesis in relation to sporula- tion and meiosis in yeast. J. Bacteriol. 119: 952-960. [57] Maniatis T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A labora- tory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. [58] Matsumoto, K., I. Uno, and T. Ishikawa. 1983. Initiation of meiosis in yeast mutants defective in adenylate cyclase and CAMP-dependent protein kinase. Cell 32: 417-423. [59] McCusker, J. H., and J. E. Haber. 1977. Efficient sporulation of yeast in media buffered near pH6. J. Bacteriol. 132: 180-185. [60] Messing J ., and J. Vieira. 1982. A new pair of M13 vectors for selecting either strand of double digest restriction fragment. Gene 19: 269-276. [61] Miller, J. 1972. Experiments in molecular genetics. Cold Spring Harbor Labo- ratory, Cold Spring Harbor, New York. [62] Mills, D. 1980. Quantitative and qualitative analysis of polyadenylated RNA sequences during meiosis of Saccharomyces cerevisiae. In Tenth International Conference on Yeast Genetics and Molecular Biology, Louvain-la—Neuve, Bel- gium. p.55. [63] Mitchell, A. P. 1988. Two switches govern entry into meiosis in yeast, p. 47- 66. In F. Haseltine and N. First (ed.), Meiotic inhibition: molecular control of meiosis. Alan R. Liss, Inc., New York. [64] Mitchell, A. P., and I. Herskowitz. 1986. Activation of meiosis and sporulation by repression of the RMEI product in yeast. Nature (London) 319: 738-742. [65] Miyake, S., N. Sando, and S. Sato. 1971. Biochemical changes in yeast during sporulation. II. Acetate metabolism. Dev. Growth Differ. 12: 285-296. [66] Moens, P. B. 1971. Fine structure of ascospore development in yeast Saccha- romyces cerevisiae. Can. J. Microbiol. 17: 507-515. [67] Moir, A. 1981. Germination properties of a spore coat-defective mutant of Bacil- lus substilis. J. Bacteriol. 146: 1106-1116. [68] Mortimer, R. K., and D. Schild. 1980. Genetic map of Saccharomyces cerevisiae. Microbiol. Rev. 44: 519-576. [69] Nagawa, F ., and G. R. Fink. 1985. The relationship between the “TATA” se- quence and transcription initiation sites at the H154 gene of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 82: 8557—8561. [70] Ogur, M., A. Roshanmanesh, and S. Ogur. 1965. Tricarboxylic acid cycle mu- tants in Saccharomyces: Comparison of independently derived mutants. Science 147: 1590. 110 [71] Olempska-Beer, Z. 1987. Current methods for Saccharomyces cerevisiae. II. Sporulation. Anal. Biochem. 164: 278-286. [72] Opheim, D. J. 1979. Effects of ammonium ions on activity of hydrolytic enzymes during sporulation of yeast. J. Bacteriol. 138: 1022-1025. [73] Percival-Smith, A., and J. Segall. 1984. Isolation of DNA sequences preferen- tially expressed during sporulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 4: 142-150. [74] Percival-Smith, A., and J. Segall. 1986. Characterization and mutational anal- ysis of a cluster of three genes expressed preferentially during sporulation in Saccharomyces cerevisiac. Mol. Cell. Biol. 6: 2443-2451. [75] Percival-Smith, A., and J. Segall. 1987. Increased copy number of the 5’ end of the SP5? gene inhibits sporulation of Saccharomyces cerevisiae. Mol. Cell. Biol. 7: 2484-2490. [76] Petersen, J. G., L. Olson, and D. Zickler. 1978. Synchronous sporulation of Saccharomyces cerevisiae at high cell concentrations. Carlsberg Res. Commun. 43: 241-253. [77] Petersen, J. G., M. C. Kielland-Brandt, and T. Nilsson-Tillgren. 1979. Protein patterns of yeast during sporulation. Carlsberg Res. Commun. 44: 149-162. [78] Petko, L., and S. Lindquist. 1986. HSP26 is not required for growth at high temperature, nor for thermotolerance, spore development, or germination. Cell 45: 885-894. [79] Primerano, D. A., G. Muthukumar, S.-H. Suhng, and P. T. Magee. 1989. A sporulation-regulated gene, SPR], contributes to ascospore thermoresistance in Saccharomyccs cerevisiae. (In press). [80] Ramirez, C., and J. J. Miller. 1964. The metabolism of yeast sporulation. VI. Changes in amino acid content during sporogenesis. Can. J. Microbiol. 10: 623- 631. [81] Rose, A.H., and J. S. Harrison. 1969. The biology of yeasts In Yeasts, Vol.1, p.303. Academic Press, New York. [82] Rothstein, R. 1983. One-step gene disruption in yeast. Methods Enzymol. 101: 670-676. [83] Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467. [84] Sherman, F., G. R. Fink, and J. B. Hicks. 1987. Chapter XXII in Methods in yeast genetics: A Laboratory Manual, p106-p110. Cold Spring Harbor Labora- tory, Cold Spring Harbor, New York. 111 [85] Shilo, V., G. Simchen, and B. Shilo. 1978. Initiation of meiosis in cell-cycle initiation mutants of Saccharomyces ccrevisiae. Exp. Cell Res. 112: 241-248. [86] Simchen, G., R. Pinon, and Y. Salts. 1972. Sporulation in Saccharomyces cere- visiae: Premeiotic DNA synthesis, readiness and commitment. Exp. Cell Res. 75: 207-218. [87] Smith, H. E., and A. P. Mitchell. 1989. A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae. Mol. Cell. Biol. 9: 2142-2152. [88] Stevens, B. J. 1978. Behavior of mitochondria during sporulation in yeast. In Electron microscopy (ed. J. M. Sturgess), vol. 2, p. 406. Microscopical Society of Canada, Toronto. [89] Struhl, K. 1985. Naturally occurring poly(dA-dT) sequences are upstream pro- moter elements for constitutive transcription in yeast. Proc. Natl. Acad. Sci. USA 82: 8419-8423. [90] Tatchell, K. 1986. RAS genes and growth control in Saccharomyces cerevisiae. J. Bacteriol. 166: 364-367. [91] Thomas, P. S. 1980. Hybridization of denatured RNA and DNA fragments trans- ferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77: 5201-5205. [92] Timberlake, W. E. 1987. Molecular genetic analysis of development in As- pergillus nidulans In Genetic regulation of development, p63-p82. Alan R. Liss, Inc., New York. [93] Toda, T., S. Cameron, P. Sass, M. Zoller, and M. Wigler. 1987. Three different genes in S. cerevisiae encode the catalytic subunits of the CAMP-dependent protein kinase. Cell 50: 277-287. [94] Toda, T., U. Isao, T. Ishikawa, S. Powers, T. Kataoka, D. Broek, S. Cameron, J. Broach, K. Matsumoto, and M. Wigler. 1985. In yeast, HAS proteins are controlling elements of adenylate cyclase. Cell 40: 27-36. [95] Trew, B. J., J. D. Friesen, and P. B. Moens. 1979. Two-dimensional protein pat- terns during growth and sporulation in Saccharomyces cerevisiae. J. Bacteriol. 138: 60-69. [96] Tsuboi, M. 1976. Correlation among turnover of nucleic acids, ribonuclease ac- tivity, and sporulation ability of Saccharomyces cerevisiae. Arch. Microbiol. 111: 13-19. [97] Wang, H.-T., S. Frackman, J. Kowalisyn, R. E. Esposito, and R. E. Elder. 1987. Developmental regulation of SP013, a gene required for separation of homologous chromosomes at meiosis I. Mol. Cell. Biol. 7: 1425-1435. [98] Weir-Thompson, E. M., and I. W. Dawes. 1984. Developmental changes in trans- latable RNA species associated with meiosis and spore formation in Saccha- romyces cerevisiae. Mol. Cell. Biol. 4: 695-702. 112 [99] Wejksnora, P. J., and J. E. Haber. 1974. Methionine-dependent synthesis of ribosomal ribonucleic acid during sporulation and vegetative growth of Saccha- romyces cercvisiae. J. Bacteriol. 120: 1344-1355. [100] Wickner, W. T., and H. F. Lodish. 1985. Multiple mechanisms of protein in- sertion into and across membranes. Science 230: 400-407. [101] Wolf, D., and G. Fink. 1975. Proteinase C (carboxypeptidase Y) mutant of yeast. J. Bacteriol. 123: 1150-1156. [102] Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103-119. [103] Yamashita, I., and S. Fukui. 1985. Transcriptional control of the sporulation- specific glucoamylase gene in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 5: 3069-3073. [104] Zakharov, I. A. and T. N. Kozhina. 1967. The genetics of mating in yeast. Genetika 11: 110-115. [105] Zimmermann, C. R., W. C. Orr, R. F. LeClerc, E. C. Bernard, and W. E. Tim- berlake. 1980. Molecular cloning and selection of genes regulated in Aspergillus development. Cell 21: 709-715. [106] Zitomer, R. S., D. L. Montgomery, D. L. Nicholas, and B. D. Hall. 1979. Transcriptional regulation of the yeast cytochrome c gene. Proc. Natl. Acad. Sci. USA 76: 3627-3631. [107] Zubenko, G. S., and E. W. Jones. 1981. Protein degradation, meiosis, and sporulation in proteinase-deficient mutants of Saccharomyces cerevisiae. Genet- ics 97: 45-64. [108] Zubenko, G. S., A. P. Mitchell, and E. W. Jones. 1979. Septum formation cell division and sporulation in mutants of yeast deficient in proteinase B. Proc. Natl. Acad. Sci. USA. 76: 2395-2399. "IIIIIIIIIIIIII“