.' .vl vvvv ... ... yummflw... a... «Em 21%.”; .-.. . . ‘. . , a4 2.2.....:.w...ri&v . . ”...“...T? ‘ . . 243.7. .. if; [‘0 b l' I' ‘ ‘ Eli... 13””5‘161 9— LIERAE’Y H H “NH fl!IIUIHIIIIHUHIMil/H111W! Michigan State University I III 3 1293 00611 7877 This is to certify that the dissertation entitled ISOLATION OF SP017, AN ESSENTIAL EARLY SPORULATION GENE IN SACCHAROMYCES CEREVISIAE presented by ALLAN L . KENNEDY has been accepted towards fulfillment of the requirements for Ph. D. degree in Microbiology and Public Health V 17qu Major professor Date 5‘////!”)7 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 m“?— ‘ ‘ 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 MSU Is An Affirmative ActiorVEquel Opportunity Inditmion ISOLATION OF SP017, AN ESSENTIAL EARLY SPORULATION GENE IN SACCHAROMYCES CEREVISIAE BY Allan L. Kennedy 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 1989 ABSTRACT ISOLATION OF SP017, AN ESSENTIAL EARL! SPORULATION GENE IN SACCHAROMYGES CEREVISIAE BY Allan L. Kennedy Sporulation in the yeast Saccharomyces cerevisiae involves the fundamental process of meiosis. Entry into the pathway is known to require the conditions of starvation and diploidy, but the lack of understanding of regulatory mechanisms is due, in part, to the dearth of characterized genes involved in the early events of sporulation. One such early sporulation gene has been isolated because it complements strains that are blocked early in meiosis by a mutation, spol7, first identified in the strain SL484. A spol7 recipient strain, R92W, was constructed by crossing SL484 with the ade2 strain W66-8A and screening the progeny for certain selectable markers and for heterozygosity at the ADE2 locus. After transformation of R92W with a YCpSO yeast genomic library, transformants were screened for sporulation-competence by assaying for the production of red papillae on white colonies; in a homothallic ADEZ/adeZ background red papillae can be produced when cells sporulate and produce progeny that have undergone diploidization and are adeZ/ade2. A complementing plasmid, p1912, was found to bear SP017 on a 5.8 kb yeast insert. When the coding region (identified by deletion mapping) was disrupted by in vitro mutagenesis and transformed into wild-type strains, the cells lost sporulation-competence. Northern analysis demonstrated the presence of at least two bands, 2.7 and 2.1 kb, in wild-type cells under both sporulation and vegetative conditions, but these signals are replaced by smaller messages in sporulation-deficient disruptants. To determine the influence of SP017 on RMEI, a gene that exerts its effects by repressing the meiotic inducer gene IMEI, the spol7/spol7 rmel/rmel double mutant strain 221a3 was constructed. 221a3 is unable to sporulate under any conditions. Thus it was concluded that SP017 does not exercise its effect by regulating RMEI. The activity of the SP017 product in the sporulation process may lie downstream of the activity of RMEI or on a pathway independent of the RMEl-IMEI cascade. TABLE OF CONTENTS List of tables . List of figures Introduction . Chapter I. Chapter II. Chapter III. Appendix A: Appendix B: Appendix C: Review of Literature Overview of early events in sporulation Regulation of meiosis and spore formation ARTICLE: Isolation of SP017, an essential early sporulation gene in S. cerevisiae Abstract Introduction . . Materials and methods . Results Strategy for cloning the SP017 gene Mapping of SP017 . . . . . . Function of SP017 Discussion References Discussion (ARTICLE) Isolation of genes expressed preferentially in the yeast Saccharomyces cerevisiae . . . . . Disruption of the sporulation-specific gene SPRZ (ARTICLE) Identification and characterization of mutations affecting sporulation in Appendix D: Saccharomyces cerevisiae . SP017, additional data . List of References . iv vi 28 29 31 36 39 40 43 47 64 69 74 77 86 95 Table LIST OF TABLES Yeast strains . Plasmids used and their sources . Complementation of spol7 mutants by plasmids Determination of the extent of the spol7 gene . RAD6 does not complement the spol7 mutation . Sporulation of the rmel::LacZ spol7::LEU2 double mutant . . . . . . . . . . . Page 52 53 54 55 56 57 Figure UIFWNH bWNH LIST OF FIGURES CHAPTER I A general model of sporulation . Biochemical events during sporulation cAMP- -dependent regulation of meiosis . Regulation of meiosis by IMEI Position of IMEI on the sporulation pathway CHAPTER II Plasmid constructs . OFAGE analysis . Southern analysis Northern analysis APPENDIX B Disruption of SPRZ Location of the STE4 transcript APPENDIX D Growth of experimental strains after exposure to sporulation conditions at 30° C . . Growth of experimental strains after exposure to sporulation conditions at 34°C . RMEI expression in a spol7 haploid and a spol7 homozygous diploid . . . . . . . . . . . vi Page ll 13 22 23 24 58 6O 61 62 75 76 89 91 93 INTRODUCTION Sporulation in the yeast Saccharyomyces cerevisiae is a fundamental process which involves meiosis. One way to study such basic cellular processes in microorganisms is by generating and characterizing mutant strains that can be used in genetic and molecular studies. Although many sporulation deficient mutants have been generated, few of the affected genes have been isolated. Moreover, most genes that have been characterized are involved in the later events of sporulation, and their products are not likely to be regulators of early sporulation events. Genes expressed early in the sporulation pathway are of particular interest because they are likely to be involved in the regulation of meiosis. This work was undertaken to isolate and characterize genes involved in sporulation. Initially, it was part of a larger effort (reviewed in Appendix A) to develop a library of sporulation-specific genes, based on the differential expression of those genes during sporulation. Appendix B is a brief survey of data developed from work on SPRZ, a gene from the sporulation-specific library. SPRZ, although expressed at significantly higher levels under sporulation conditions than in vegetative media, is not essential for sporulation. In vitro mutagenesis of SPRZ by both insertion and deletion methods, followed by transformation and diploidization of wild-type strains, did not seem to 2 affect sporulation. This result and the work of others suggested that genes isolated by methods that rely on differential expression are likely not to be essential for sporulation and meiosis. Though they are interesting, their functions are probably quite varied and may involve spore structural development or fine-tuning of the processes. Some workers have even suggested that sporulation may include a period of general deregulation wherein many messages, including those of genes not required for sporulation, are indiscriminately expressed (Gottlin- Ninfa and Kaback, 1986); the genes for such messages might easily be included in a screen based on differential expression. The work of Smith (1985) attempted to address the problem by the more conventional approach of UV mutagenesis. She developed an assay for an enzyme, sporulation amyloglucosidase (SAG), which is expressed at high levels in sporulating cultures. After UV mutagenesis of haploid spores from the homothallic strain SCMS7-l, she screened surviving diploid colonies for SAG activity and identified two strains that are sporulation-deficient, SL484 and SL641. The mutations in these strains have been given the designations spol7 and sp018, respectively. Her work can be reviewed in Appendix C. The work reported in this thesis is based upon and extends those results. Because of the rising interest in the characterization of early sporulation genes, I sought to isolate the SP017 gene, which would complement strain SL484. This was done by introducing the spol7 mutation into a red ade2 strain which was made heterozygous at the ADEZ locus by plating on adenine-deficient media and selecting a white revertant colony. The new spol7/spol7 ADEZ/adeZ strain was transformed with a yeast genomic library and the transformants were subjected to 3 sporulation conditions. Following transfer to rich growth media, colonies were screened for sporulation-competence by scoring for the appearance of multiple red papillae, and plasmids isolated from such strains were found to bear the complementing gene, SP017. To prove that SP017 is essential to sporulation, several experiments were performed, including an in vitro mutagenesis analysis. Spores from transformed mutagenized strains segregated 2:2 for the ability to sporulate. Though initial entry by cells into the sporulation pathway is not well understood, it has been shown to depend upon at least two influences, available nutrition and the mating type locus, MAT. Interactions between environmental conditions, the MAT locus, and the meiotic regulator genes IMEI and RMEI have been demonstrated by studies using double mutants. Work with the rmel imel strain D320 (Kassir et a1. 1988) showed that IMEI, which induces sporulation, is negatively regulated by RMEI as well as by nutritional signals. The rmel mutation permits cells to enter the meiotic pathway in strains that ordinarily do not, such as a/a diploids, a/o diploids or haploids. Thus, IMEI may represent a key point in the pathway where different influences on sporulation converge. Can SP017 be positioned on the pathway? In an attempt to answer this question, the spol7 mutation was introduced into an rmel strain, thereby creating a double mutant, and the sporulation—competence of the new strain was assessed. The results suggest that SP017 does not interact directly with RMEI; the strain does not sporulate in spite of the rmel mutation. Furthermore, the SP017 transcript is expressed normally in the rmel/rmel strain. On the other hand, the expression of 4 RMEI does not seem to be affected in spol7/spol7 cells. Thus, the position of the SP017 product in the sporulation pathway probably lies downstream of RMEl or on a parallel pathway. Chapter I of this thesis is a review of the current literature. Chapter II is a prepared manuscript that has been submitted to Molecular and Cellular Biology. This work was carried out by me under the direction of Dr. Magee and is being submitted under joint authorship. Chapter III is a larger discussion of the results reported in Chapter II and other unpublished data. Appendix A is the publication which resulted from the initial work done by Clancy et a1. (1983) using the differential hybridization approach. My contribution to this work was screening the lambda library and identifying clones that contain sporulation-specific yeast DNA sequences. A brief overview of the work done on SPRZ is contained in Appendix B. Appendix C is the work of Smith et al., in which the spol7 mutant SL484 was isolated. The acceptance of this work for publication was dependent upon the proof that the mutation in SL484 was different than that in SL641, a second-sporulation mutant strain also reported in the paper. This could not be done until I had isolated the plasmid complementing SL484 and showed that it could not complement SL641. Additional data produced during the isolation of SP017 but not included in the manuscript (Chapter II) are in Appendix D. REVIEW OF LITERATURE Because of their fundamental nature, eukaryotic developmental pathways have become the focus of many investigators. Sporulation in yeast has provided a major access to the study of basic processes such as genetic recombination and chromosome segregation - events involved in meiosis - because of the well-developed genetics and molecular biology in this organism. The ascomycete Saccharyomyces cerevisiae has many distinctly eukaryotic features (for review, see Struhl 1983), including a well- defined nucleus and chromosomes which contain a centromere, two telomeres and multiple DNA replication sites. Transcription and translation characteristics include three distinct RNA polymerases, 5'capped-3' poly(A) mRNAs, translation beginning at the first AUG codon, lack of operons, and occasional splicing of mRNAs. The yeast cell cycle involves two haploid cell types, a and a, which will mate to form diploid zygotes; these zygotes will grow vegetatively as diploids. The latter undergo meiosis and sporulation under specific conditions. Cellular organelles are similar to those in higher eukaryotes: lysosymes, Golgi apparatus, mitochondria, 80s ribosomes and nuclear membranes are all present. Although some have argued that S. cerevisiae is not truly representative of higher eukaryotes (Beggs et a1. 1980; Langford et al. 1983; Kaufer et al. 1985), this yeast offers an extremely powerful approach to the study of eukaryotic gene expression, DNA replication, 6 recombination, transposition, chromosome segregation, chromatin structure, secretion, cell cycle and control of cell type (Struhl 1983). Many distinctly eukaryotic proteins, such as histones and tubulin, are present in yeast. Studies of the yeast RAS proteins and their effector system have contributed greatly to the development of a model for this important eukaryotic system (Field at al. 1988). A variety of genetic manipulations are possible because of the yeast mating system. Mating is controlled by the NAT locus on chromosome III, where either of two genetic configurations, designated as mating type a or mating type a, can exist. The stability of these configurations is under the control of another locus, the H0 gene, whose activity confers homothallism: germination of haploid spores of homothallic strains is followed by a mating type switch by certain daughter cells, so that a "self" mating between daughter cells is ensured, thus producing a new homozygous diploid strain. Heterothallic (ho) strains, on the other hand, are stable at NAT, and in the absence of haploid cells of the opposite mating type, switch their MAT locus, mate, and diploidize only rarely. These features of yeast genetics have permitted the development of techniques to study many biochemical systems. For example, heterozygous diploids in homothallic backgrounds can be sporulated and their tetrads dissected to isolate spores; after germination these strains diploidize, giving rise to homozygotes. In this way the effects of a recessive mutation can be evaluated. Other studies such as mapping, linkage, and cross-over frequency analysis are easily performed by mating various haploid mutants and analyzing phenotypes of colonies grown from their dissected spores. Molecular techniques have further enhanced yeast genetics for 7 metabolic studies. The well-developed transformation system (Struhl 1983; Rothstein 1983) permits introduction of metabolic or regulatory genes in several ways; an intact or disrupted gene may be integrated at its normal chromosomal location (Shortle et a1. 1982), it may be carried on a multicopy plasmid, or it may exist as a single copy on an artificially constructed chromosome. Gene disruptions effected in homothallic strains may, upon sporulation and tetrad dissection, be rendered homozygous at the mutant locus. Furthermore, haploids may be made temporarily homothallic by transformation with a plasmid-borne H0 followed by curing. Mutant haploids may be generated either by dissection of spores from mutagenized heterothallic diploid strains or by direct mutagenesis. In addition to their strongly eukaryotic features, yeast have many characteristics analogous to those of prokaryotes; these may also be exploited in the laboratory. Gene isolation, which is accomplished with relative ease by complementation, may be followed by studies on regulation and gene dosage effects. Other advantages include the generation time (only about 90 minutes), the ease of growing and culturing the organism under a variety of experimental conditions, and its non-pathogenicity. Thus, the ease of genetic, biochemical and molecular manipulations, as well as the extensive characterization of many of the biochemical and physiological pathways, make yeast an attractive system for eukaryotic studies. The process of meiosis in yeast is an integral part of sporulation and is easily induced. Although cultures often sporulate at less than 75% under optimum conditions and sporulating cells may exhibit asynchrony, qualitative analysis of sporulation can be accomplished 8 with ease (for review, see Esposito and Klapholz 1981). Most studies of meiosis in the yeast system have been directed toward the morphology, biochemistry, and genetics of wild-type cells, and have involved altered nutritional conditions and the generation of sporulation mutants. More recently, molecular approaches have been incorporated, whereby the involved genes themselves have been isolated by complementation of mutants blocked in the process. Many sporulation-specific genes of unknown function have also been isolated and are now being analyzed for their contribution to this developmental pathway (Clancy et a1. 1983; Percival-Smith and Segall 1984). Molecular approaches have identified several genes that have products essential to the successful completion of the pathway, including those that permit sporulation to occur under non-sporulation conditions (Hopper et a1. 1975; Kassir and Simchen 1976; Mitchell and Herskowitz 1986). 0v ew h vent S u n Meiosis in yeast is closely related to mitosis, in that many of the mitotic processes are essential for meiosis, including spindle pole body duplication and segregation, DNA replication, and chromosome segregation. Almost all genes known to be involved in regulating the nuclear events of mitosis are required also for meiosis (Pringle and Hartwell 1981). General conditions for entry into the wild type meiotic pathway are the presence of both MAT; and MATo information, nitrogen deprivation, and a nonfermentable carbon source such as acetate. The optimum temperature is approximately 30°C with ascus formation greatly reduced below 25°C and above 35°C. Glucose and some other sugars inhibit sporulation by a variety of mechanisms, including increased levels of cAMP (Matsumoto et a1. 1983), repression of the TCA cycle enzymes which are required for acetate utilization, and repression (Miyake et a1. 1971) of gluconeogenic enzymes (which are involved in the synthesis of storage carbohydrates). Nitrogen repression of developmental pathways in microorganisms is usually meiosis-related and is a phenomenon which has been generally observed though not well understood; ammonium ions, glutamine, and methylamine (an ammonium analog) have been shown to inhibit yeast ascus development. Ammonium ions have also been observed to block a variety of developmental processes in other organisms, including gametogenesis, heterocyst formation and sporulation (Heywood and Magee 1976; Esposito and Klapholz 1981). Sporulation requires aerobic conditions, and though entry into the pathway can be enhanced by pregrowth in acetate 10 medium before shifting to nitrogen starvation, acetate exposure is probably not continuously required. Maximum sporulation is achieved when cells are grown to about 5x107 cells/ml in complete acetate medium, then transferred to acetate medium lacking a nitrogen source. The events of sporulation proceed in a general temporal order as follows (Esposito and Esposito 1978): l. Premeiotic DNA synthesis 2. Commitment to recombination 3. Polycomplex body formation 4. Synaptonemal complex formation 5. Commitment to sporulation 6. First spindle pole body duplication 7. Nuclear elongation and Meiosis I 8. a) Second SPB duplication b) Initiation of spore wall synthesis 9. a) Nuclear budding and Meiosis II b) Spore wall growth 10. a) Nuclear separation b) Spore wall closure 11. Ascospore maturation Spore wall formation is dependent upon, and begins at about the same time as, the second spindle pole body duplication and appears to exist on a parallel pathway. These events were subsequently arranged and modeled (Esposito 1987) to reflect the existence of the separate pathways (Figure l). ll rstO Synaptonemal Complex Formation Dependent Meiotic , Recombination Recombination 39611 Enzymes 8.6. Independent —{——DNA Synthosit cool cch cdc21 cdc28 RMEi cdc2 cdc1 cdoiio sppi 3 spot 2 r I —. IM E1 Meiosis I MAT - cdo5 me" “'91 4 mei2 . J Meiosis ll mei3 spot . ’ Spore Well “:03 Development spo4 spos Figure l. A general model of sporulation (Esposito 1987), modified to include spol7 and spol8. After introduction to starvation medium budded cells complete cell division, and are arrested in the G1 phase of the cell cycle. The fraction of budded cells falls from 30% to 2% within 48 hours. A cell failing to complete the cycle is thought to be unable to sporulate, remaining as a mother with an anucleate bud. Thus, cells enter meiosis from the GI phase (Hartwell 1974). After about 7 hours in sporulation medium, cells may be returned to growth medium and mitotic growth will resume; however, at about 5 hours commitment to both intragenic and intergenic recombination has 12 occurred. This feature permits a brief window for the analysis of recombination events and characterization of mutants, since cells committed to meiotic recombination are not committed to sporulation, and the return to rich medium permits cells to resume vegetative growth after meiotic events have occurred. Landmark biochemical events that take place during sporulation are shown on a time course in Figure 2. Of these processes, glycogen degradation (Hopper et al. 1974; Colonna and Magee 1978) is of particular interest because of its utility as an assay for sporulation. The two sporulation mutants isolated by Smith (1988), SL484 (spol7) and SL641 (spol8) were isolated because of their inability to produce the glycogen degradation enzyme, sporulation amyloglucosidase (SAG), although the enzyme itself is not required for completion of sporulation (Yamashita and Fukui 1985). From about 5 hours after introduction into sporulation medium until about 9 hours, the DNA content of the sporulating cell doubles (Simchen et a1. 1972). Inhibitors of DNA synthesis such as hydroxyurea and 8-hydroxyquinoline when applied during the first 8 hours of sporulation prevent ascus formation, but the cells generally remain viable if plated on rich medium (Simchen et al. 1976). RNA synthesis also increases during sporulation between 4 and 10 hours, with a peak at about 6 hours (Hopper et a1. 1974), but declines by the time of spore formation due to an extensive degradative (RNase) activity which may be sporulation specific (Hopper et al. 1974). The appearance of sporulation-specific messages has permitted the construction of cDNA probes (Mills 1980); about 7% of the cDNA made PHYSIfiOGICAL PARAMETER: Respiration pH DNA synthesis RNA synthesis RNA degradation Protein synthesis Protein degradation Neutral lipids Phospnolipids Trenolose Monnon Glycogen Glycogen degradation Dry weight a LIGHT MIQROSCOPW Binocleote cells Tetronacleote cells Spores .1 V 13 HOURS IN SPORULATION MEDIUM as s 9 '9 I; L4 M 2.02.225 L__.L L t----- L _j_ s_ L L k L P L L L L L——_ L £—-L— L Figure 2. Biochemical events during sporulation (Esposito and Klapholz 1981). 14 from the messages of sporulating cells fails to hybridize to vegetative RNA. Sporulation-specific probes have been constructed and used to isolate sporulation-specific genes (Clancy et a1. 1983; Percival-Smith and Segall 1984; Kaback and Feldberg 1985), but many of these genes, while exhibiting strong expression during sporulation, are not required for the completion of spore formation. Later events of the sporulation pathway make it hard to define other parameters. A gradual increase in the pH of the medium inhibits the uptake of labeled precursors. Spore wall formation hinders cell disruption. Assays for macromolecular synthesis are difficult because of the extensive RNA and protein degradation and recycling. Nonetheless, for studies of early meiotic events S. cerevisiae remains the system of choice. The later major temporal events lie outside the scope of this discussion, and have been reviewed (Esposito and Klapholz 1981). Two general approaches have been employed in the characterization of sporulation genes. The first, which relies on the generation of spa mutants, followed by the isolation of the genes themselves, has led to the development of the general model (Esposito and Klapholz 1981; Esposito 1987). The second approach has relied on the differential hybridization screen (St. John and Davis 1975) to identify clones from a yeast genomic library that bear genes which hybridize specifically to cDNA probes made from RNA of sporulating cells and do not hybridize to probes from non-sporulating cells (Clancy et a1. 1983; Percival-Smith and Segall 1984). Although a large number of genes has been isolated by the second method, they have added, as yet, little to the understanding of developmental regulation. Hybridization-screened 15 genes disrupted in vitro have given either no visible phenotypes (Kaback and Feldberg 1985; Percival-Smith and Segall 1986; Primerano et al. 1988b; Clancy, personal communication), or phenotypes which exhibit only a slight interference with successful spore formation (Law and Segall 1988; Primerano et al. 1988a). Only one gene isolated by this technique, the SPSl gene (Percival-Smith and Segall 1986), has been shown to be absolutely required for sporulation. The apparent lack of requirement for many if not most of the genes isolated by the differential hybridization method has led to much speculation. Kaback and Feldberg (1985) have proposed that a generalized relaxation of transcriptional control during sporulation could result in expression of sequences that do not have specific functions in the process. While this may be true, there are two points to bear in mind. First, some important sporulation-required genes may have been missed by the screen because they are expressed in undetectable quantities or because they are also expressed during vegetative growth; their absence from the pool of characterized genes distorts the view we have of sporulation. Secondly, later evidence for two genes, SPRl and SPSIOO has shown that their mutant phenotypes are obvious only after spores have been subjected to a battery of experimental conditions. Thus many sporulation-specific genes seem dispensable for spore formation, but they may make subtle and highly regulated contributions to the process. Four temporally distinct classes of genes have been observed in sporulation. The early, middle and late designations (Holaway et a1. 1985) are based on the time of transcript appearance in the rapidly sporulating strain SKl. Transcripts of early genes appear soon after l6 transfer to sporulation medium. Those for the middle genes appear at about the time of DNA synthesis, and late gene transcripts make their appearance at about the time of meiosis I. A fourth class, the very late genes, appears even later (discussed below). SP013 (Klapholz and Esposito 1980a; Klapholz and Esposito 1980b) is a prototype early gene. Its expression, which begins shortly after transfer to sporulation medium, gives a product which functions in chromosome separation during meiosis I. The preceding events, DNA synthesis, genetic recombination, and spindle formation, proceed normally in spol3 mutants, and after the failure of chromosome disjunction, cells proceed through meiosis II and give rise to two diploid spores. SP013 is developmentally regulated by MAT, but initial induction is also triggered by starvation, regardless of the MAT phenotype (Wang et a1. 1987). The late gene SPRl (Primerano et al. 1988a) has been shown to play a role in spore viability; spores produced from cells homozygous for the sprl mutation exhibit increased sensitivity to elevated temperatures. Moreover, the gene product of SPRI bears a hydrophobic leader sequence and is thought to accumulate in the spore wall (Primerano et al. 1988a). The late gene SPRZ however, has no apparent essential function in sporulation (Primerano et al. 1988b). Among a cluster of three genes, SPSI, SPSZ, and SPS3, only one, SPSl, gives an asporogenous phenotype upon disruption. The 5' region of SPSZ, which is first expressed at about 15 hours after introduction into starvation medium, inhibits sporulation when introduced into cells on a high copy number plasmid, but disruption of SPSZ itself does not inhibit sporulation (Percival-Smith and Segall 1986; Percival-Smith and 17 Segall 1987). The very late genes, whose transcripts do not appear until 12 hours after transfer to sporulation medium and continue to be expressed steadily until 35 hours, are represented by SPSlOO and SPSlOl (Law and Segall 1988). SPSIOO is involved in spore wall development; resistance to ether (Dawes and Hardie 1974) is delayed in mutants by about 5 hours. The interest in early genes has been enhanced by the characterization of the later genes. Whether attempts to identify a mutant phenotype were successful (SPRI, SPSI, and SPSlOO) or unsuccessful (SPSZ, SPS3, SPRZ), these genes have provided little information about meiosis or its regulation. Though they may eventually provide avenues for the isolation of other regulatory genes, their functional products may act primarily at the end of the pathway to spore formation and thus be representative of processes quite specific to spore-forming organisms. To date, attention continues to focus on the variety of early mutants as a means to isolate the early genes. Several mutants that are blocked in premeiotic but not mitotic, DNA synthesis have been isolated; these include spo7, 8, 9 and 11 (Esposito et al., 1972), spol7 and 18 (Smith 1988), meil, me12 and mei3 (Roth 1973), and the dominant SP098 (Esposito et al, 1972). Two mutants, cch and cdc7, undergo premeiotic DNA replication, yet fail to undergo mitotic DNA synthesis (Esposito and Klapholz 1981) and some mutants, cdc4, cdc8 and cchl, are blocked in both processes (Simchen 1974; Game 1976). The relationship between mitosis and meiosis thus seems to be one of intricate and complex regulation. Re u atio o e s s d re t n After the completion of DNA synthesis, the process of sporulation is separated into several distinctly regulated pathways, each of which involves a separate commitment. A similar observation has been made for bacterial sporulation (Mandelstam 1971). Commitments to recombination and chromosomal segregation, which have been shown to occur separately (Klapholz and Esposito 1980) in spol mutants (see Figure l), are followed by separate commitments to other downstream events. For example, cells that are unable to undergo Meiosis I (spolZ or spol3 mutants) can nonetheless be committed to and complete all other processes of sporulation. In addition to the apparent separation of commitments, arrest of spore development and resumption of mitotic growth occurs more readily when cells are returned to rich rather than poor medium, suggesting that some stages of commitment are sensitive to the medium composition. Thus, single events in the pathway of sporulation appear to be dependent on a series of conditions, and commitment to those events is part of a complex multistep process (Esposito and Klapholz 1981). Mutational analysis has provided a useful, though limited, understanding of the regulation of sporulation. Structural mutations are not easily distinguished from regulatory mutations, because little is known yet of the enzyme activities of the process. The discovery of the separation of the events, however, has permitted the construction of the model in Figure 1. Regulation by the mating type locus is fundamental to DNA 18 19 replication (Roth and Lusnak 1970), genetic recombination (Friis and Roman 1968; Roth and Fogel 1971), nuclear division, and spore formation (Roman and Sands 1953). Although upon transfer to sporulation medium many of the initial responses of a/a strains resemble those of a/g and a/a diploids, only cells heterozygous at the mating type locus will undergo meiosis and sporulation - diploid homozygotes a/g and a/a do not carry out premeiotic DNA synthesis (Roth and Lusnak 1970). The products of MATgl and MAToZ (reviewed by Herskowitz and Oshima 1981; Nasmyth 1982) are thought to form a negative regulator. In sporulating wild type strains, heterozygosity at the MAT locus is brought about by mating. Products of the haploids MAT; (a-factor) and MATo (a-factor) are secreted and arrest cells of the opposite mating type in the 61 phase of the cell cycle so that mating may occur (see Thorner, 1981, for review). Strains which have a stable mating type are designated as heterothallic and cannot undergo a switch to the opposite type except at very low frequencies. Homothallic strains, however, undergo a switch that is catalyzed by the product of the H0 gene: transposable information of the opposite mating type is inserted into an expression locus within the mating type region. In a cell line arising from a germinated spore, a mating type switch is engineered when an existing cassette is removed and replaced by a copy of the gene for the opposite mating type. This occurs in the original mother cell after the second division, and continues in generations thereafter. The transposable g or a information resides in silent loci which have been designated as HML (the usual locus of the gene for a) and HMR (usual a locus); these regions are normally repressed by the action of the SIR (silent information regulator) genes, of which there are four 20 complementation groups. Yet another large group that also exerts control over the process of mating, the STE genes, is thought to include the g and a pheromone regulatory genes and the genes involved in their processing and secretion (Pringle and Hartwell 1981; Thorner 1981; Herskowitz and Oshima 1981). Rare strains which are HMLg and HMRa occur and can also diploidize. The MAT locus, in addition to controlling mating and sporulation, is involved in the regulation of several related activities, and cells that are genetically identical in all aspects except at the MAT locus exhibit markedly different properties. Cell cycle arrest by the g and a pheromones (previously noted), g-a cell to cell agglutination, a-cell inactivation of a-factor, and budding patterns (a/a and a/a cells bud equatorially and g/a cells have polar budding), are some of these regulated functions. a/a cells, which do not produce or respond to pheromones, will undergo premeiotic DNA synthesis and sporulation while the homozygous diploids a/g and a/a will not; some processes are influenced neither by 9 nor a alone, but by an interaction between them. Thus, the function of the MAT locus has come to be viewed primarily as regulatory (MacKay and Manning 1974). The discovery of the RMEI locus (Hopper et al. 1975; Kassir and Simchen 1976), in which mutations permitted sporulation of a/a and a/a diploids, showed that MAT exerts some of its effects via this negative regulator. Homozygous rmel diploids sporulate regardless of their mating type, and haploids bearing rmel are able to undergo premeiotic DNA replication under sporulation conditions. Thus, RMEl was proposed (Rine et a1. 1981) to be an inhibitor of sporulation: the RMEI gene product is not functional in g/a diploids, but a/g and a/a diploids 21 express RMEl and cannot sporulate (Mitchell and Herskowitz 1986). MAT products 91 and a2 are both required for sporulation in wild type strains and act by repressing RMEI transcription (Mitchell and Herskowitz 1986). a haploids fail to repress because the MAToZ product is absent, and a haploids lack the ”Aral product; thus neither possesses sporulation capability. rmel haploids or haploids expressing al-a2 activity can enter the sporulation pathway and undergo DNA synthesis, recombination and some later meiotic events (Kassir and Simchen 1976; Hopper and Hall 1975; Roth and Fogel 1971; Wegstaff et a1 1982), but cannot complete normal meiosis because they lack homologous chromosomes (Roth and Fogel 1971; Wegstaff et a1. 1982). RMEI activity thereby prevents haploid entry into what would be an aberrant meiosis. Transcriptional repression of RMEI in a/a diploids is not complete; only a 10-20 fold reduction is seen. In contrast, other genes under the control of MAT exhibit a lOO-fold or greater decrease in transcription (Klar et a1 1981; Nasmyth et a1 1981; Jensen et a1 1983). This incomplete repression is not understood, but the residual transcription may have no function, since the null mutation (Mitchell and Herskowitz 1986) displays no unpredicted phenotype. RMEl responds to only one of the two known signals, starvation and diploidy, for successful sporulation. That is, cells with an inactivated RMEI are relieved of MAT control, but remain under nutritional regulation. Reports of a single gene PATI (RANl) in the fission yeast Schizosaccharyomyces pombe (Iino and Yamamoto 1985a; Nurse 1985; Iino and Yamamoto 1985b) which, when lost, relieved cells of both mating type and nutritional control of sporulation suggested that such a gene might also exist in S. cerevisiae. The PATl product Nutritional "’ 9 CDC25 9 Signal Product Inacuve Protein Klnase I I Arrest in G1 I hMflofls Adenyl Cyclase HAS; g Protein (gyfl) + Protein Klnase I I Blocks Meiosis I IMHOMs Figure 3. CAMP-dependent regulation of meiosis. is a negatively regulating protein kinase that lies between two positive regulators MEIZ and ”£13 in that sporulation pathway. In S. cerevisiae, disruptions of the CAMP-dependent protein kinase system are known to affect sporulation. CYRI, the adenyl cyclase gene (Matsumoto et a1. 1983) and its two known effectors CD025 (Broek et a1. 1987; Robinson et a1. 1987) and RASZ (Toda et a1. 1985) maintain cellular levels of cAMP (Figure 3). When CAMP levels are lowered by loss of any of these components, starvation control of meiosis is lost and sporulation is no longer suppressed by nitrogen. The regulatory unit of the CAMP-dependent protein kinase is encoded by the gene BCYl (Matsumoto et a1. 1983); homozygous bcyl cells do not undergo meiosis 23 and sporulation, and suppress mutations occurring earlier. Therefore, meiosis is negatively regulated by a protein kinase activity which is itself sensitive to environmental nutritional conditions. The IMEl gene in S. cerevisiae, a positive regulator of sporulation, is under the control of both MAT and starvation (Kassir et a1. 1988), and was isolated from a yeast genomic library because it permitted sporulation when transformed on a high copy-number plasmid into a MAT-defective strain. The creation of an rmel-imel double homozygote led to the simple model shown in Figure 4. Because this strain was unable to sporulate, the investigators concluded that RMEl normally represses IMEl. Further characterization of IMEI revealed that it is also environmentally regulated: i) no transcripts are found in cells growing in glucose, ii) acetate growth is accompanied by IMEI Nitrogen and Glucose —-I "“51 Signals '— Meiosis Figure 4. Regulation of meiosis by IMEI (Kassir et al. 1988). 24 IMMofic Recombination Starvation—\ \ \ a DNA ”—5—; Synthesis I MAT g " + j/,—————e>nmuosnn M a "M51 > Meiosis ll \———) Spore Wall Figure 5. Position of IMEl on the sporulation pathway. transcription, iii) under nitrogen starvation, IMEI transcription increases sharply in sporulation-competent g/a diploids whereas none is seen in g/g or alpha/alpha strains, thus demonstrating an IMEI response to MAT, iv) in vegetatively growing cells, neither MAT nor RMEl seems to affect IMEI transcription, and v) in rich medium, IMEl multicopy plasmids promote sporulation. Figure 5 incorporates the models shown in Figures 1 and 4, reflecting the regulation upon IMEI of both nutrition and MAT. The regulation by starvation of at least two early genes, IMEl and SP013, poses some interesting questions. Are IMEl and SP013 (and possibly others) regulated directly by the same product? Does this regulation result from DNA-specific binding, and if so, are there 25 analogous sequences located at or near each of the target genes? If the regulation is exercised by different mechanisms, are these then subject to a common regulator? The demonstration of the key role of IMEl has opened new avenues for the investigation of meiosis. First, the regulation by starvation may now be addressed more specifically by monitoring transcriptional responses of IMEl to various nutritional conditions and mutations. Second, newly isolated early sporulation genes may be positioned on the pathway by constructing double mutants with either RMEI and IMEl or both. We have tried such an approach with the early sporulation gene SP017 which was isolated by screening plasmids from a YCpSO yeast genomic library for the ability to complement the original spol7 mutant (Smith et al. 1988). After isolation and characterization of the gene, the double mutant spol7/spol7 rmeI/rmel was constructed and evaluated for the ability to sporulate. The rudimentary model of sporulation that has been proposed (Figure 1) reflects a lack of understanding of early regulation. This problem, which exists because of the lack of isolated early genes, can be approached by generating early sporulation mutants; such mutants, which are likely to affect regulation, would be useful in the isolation and characterization of early genes. An understanding of the interaction between negative regulatory genes (mutations in these permit sporulation under non-sporulation conditions) and positive regulatory genes (mutations in these prevent spore formation) depends upon the characterization of early gene activity. CHAPTER II (ARTICLE) ISOLATION OF SP017, A YEAST EARLY SPORULATION GENE By A.L. Kennedy and P.T. Magee Submitted to Molecular and Cellular Biology 26 ISOLATION OF SP017, A YEAST EARLY SPORULATION GENE By Allan L. Kennedy College of Osteopathic Medicine Michigan State University East Lansing, Michigan 48824 and P. T. Magee* College of Biological Sciences University of Minnesota St. Paul, Minnesota 55108 * Author to whom inquiries should be directed 27 ABSTRACT Diploids of the yeast Saccharomyces cerevisiae that bear the spol7 mutation are blocked in sporulation before premeiotic DNA synthesis occurs. We have isolated a plasmid bearing the SP017 gene sequence by its complementation of a spol7 mutant. An ADEZ/adeZ spolZ/spol7 recipient strain was transformed with a YCpSO yeast genomic library and sporulation-competent transformants were identified by their production of red papillae from white colonies on rich media after exposure to sporulation conditions. The complementing plasmid, p1912, was found to bear SP017 on a 5.8 kb yeast insert. Northern analysis demonstrated the presence of at least two bands, 2.7 and 2.1 kb, in mRNAs from wild- type cells under both sporulation and vegetative conditions, but at significantly higher levels in sporulating cells. Because SP017 is an early sporulation gene, a spol7/spol7 rmel/rmel strain was constructed to see if its effects are exerted by regulation of the early gene RMEI, a sporulation repressor. The double mutant is unable to sporulate under any conditions. Since the rmel mutation does not relieve the spol7 sporulation block, we conclude that SP017 does not exercise its effect by the inhibition of RMEI. 28 INTRODUCTION Sporulation in Saccharyomyces cerevisiae is a well-established system for the study of meiosis, and is easily induced in diploid cells by starvation. The diploidy requirement is met by two alleles at the mating-type locus, MATa and MATo. In haploids the pheromone products produced by these genes arrest cells of the opposite mating type in the G1 phase of the cell cycle so that mating may take place. Diploid cells bearing only MATa or MATo are unable to sporulate because of active repression by the RMEI gene product, a negative regulator of meiosis (17). The RMEl product is thought to act by inhibiting the effects of the inducer gene IMEl, which is sensitive also to nutritional status (14). The development of a more complete model of the early regulation of sporulation has been impeded by the dearth of well-characterized early genes. The pathway to sporulation proceeds through meiotic DNA synthesis, an event that can be blocked independently of mitotic DNA synthesis, although both pathways may employ the same synthetic machinery (20). Several mutant series have been identified in which premeiotic DNA synthesis is blocked. These include the sporulation mutants spo7, spo8 and spo9 (5), the cell division cycle mutants cdc4, cdc8 (26), cchl 29 30 (8), cch8 (26), and the meiosis mutants meil, meiZ, and mei3 (23). Smith et a1. (27) have generated two such mutant strains and have given these mutations the designations spol7 and sp018. Although many sporulation-specific genes have been isolated by the technique of differential hybridization (4, 9, 19), they are usually not absolutely required for successful sporulation, and thus are unlikely to be involved in the regulation of meiosis. We have now isolated the SP017 gene by transforming a spol7/spol7 ADEZ/adeZ strain with a genomic yeast library, and screening the transformants for sporulation-competence. Such strains subjected to sporulation conditions, then replica plated to rich medium, can be evaluated for meiosis because successful sporulation generates progeny that have undergone meiotic chromosome segregation, are adeZ/adeZ, and appear as red papillae on white colonies. This method was used by Fonzi and Sypherd (7) to clone the ornithine decarboxylase gene of Saccharyomyces cerevisiae. We have also constructed a spol7/spol7 rmel/rmel double mutant to evaluate the interaction between these two genes. The spol7 phenotype is epistatic to rmel. We thus conclude that the spol7 function lies either on a parallel pathway of sporulation events, or in the same pathway but downstream of the RMEI function. MATERIALS AND METHODS Yeast strains. See Table l. Plasmids. See Figure 1 and Table 2. Media and growth conditions, For culture maintenance and vegetative growth, cells were grown in YEPD (1% yeast extract, 1% Bact-peptone, and 3% glucose) liquid medium, or on YEPD plates (1.5% agar added). To increase the efficiency of sporulation, cells were first grown in presporulation acetate medium PSP2 (0.67% yeast nitrogen base without amino acids, 0.1% yeast extract, and 100 ml of 50mM potassium pthalate at pH 5.5 per liter; reference 22). After growth to 2-5 X 107 cells/ml, the cells were harvested, washed twice in sterile water, and transferred to sporulation medium SP2 (0.3% potassium acetate, 0.02% raffinose) and incubated in a rotary shaker. Cells were grown at either 30° (optimum) or 34°C, depending upon the desired conditions. Quantitative measurements of sporulation were carried out microscopically. At least 300 cells were counted for each point. Isolation of plasmid DNA from E. coli. Plasmids were maintained in E. coli strains DHl and DHS. Large scale isolations were prepared by the protocol of Maniatis et a1. (16), but with some revisions (15). Plasmids were prepared by inoculating 600 ml of Luria broth 31 32 (1% Tryptone, 0.5% yeast extract, 1% NaCl, ampicillin 20ug/m1) with a 1.0 m1 culture of the appropriate strain. After growth to stationary phase, cells were harvested and suspended in 8.0 ml (25% sucrose, 50mM Tris, pH 8.0). 0.6 ml of 10mg/ml lysozyme (freshly prepared in cold 0.25 M Tris pH8.0) was added. Cells were incubated on ice 10 min. After addition of 1.5 m1 of 0.25M NaEDTA pH8.0 and 4.8 ml Brij-DOC (50mMTris, 60mM NaEDTA, 1% Brij-35, 0.4% Na deoxycholate pH 8.0) the prep was centrifuged at 45K for 30 min, the supernatant extracted with phenol/chloroform, and precipitated by adding 0.25M NaAcetate (to a final concentration of 0.025M) followed by adding 2 volumes of 95% ethanol. The precipitate was pelleted (12K, 15min) and resuspended in 5 ml low TE (lOmM Trizma base, 0.1mM NaEDTA, pH8.0). RNA was digested with RNAse (50u1 of 10 mg/ml) at 37° for 30 min. After precipitation, pelleting, and resuspension in 5 ml low TE, a solution of 5.0 M NaCl was added to a final concentration of 1.5 M. 30% PEG (polyethylene glycol 8000 in 1.5 M NaCl) was added to a final volume of 25% of the total, and the solution was incubated at 0° for 30 min. The precipitate was pelleted at 10K, 4 min at 4 C, washed gently with 70% ethanol and resuspended in 2.0 ml of low TE. Transformation of yeast strains, Lithium acetate transformations were carried out by the method of Ito et a1 (1983) with some revisions. Yeast cells were grown in 100 m1 YEPD to 2 X 107 cells/m1, pelleted and washed once in TE (lOmM Tris-HCl pH 7.5, 1.0 mM NaEDTA). After resuspension in 5 m1 LiAc/TE (0.1 M lithium acetate, in TE), the cells were incubated at 30°C for 60 min, vortexed gently and 0.1 m1 aliquots were placed in 1.5 m1 microfuge tubes. 5 ul (50ug) carrier (salmon 33 sperm) DNA and 1-15 ul (1-10 ug) of transforming DNA were added, and the preparation was incubated 30 min at 30°C. 0.7 ml PEG/LiAc/TE (40% w/v polyethylene glycol 4000 in LiAc/TE) was added to each tube, they were gently vortexed, then incubated 60 min at 30°C. After 5 min at 42°C, the preparations were washed twice with 0.5 m1 TE, resuspended in 0.1 m1 TE, then plated on the appropriate media and incubated 3-4 days at 30° C. When higher transformation yields were desired, the protocol of Sherman et a1 (24) was followed; DNA fragments bearing genes disrupted with selectable markers were transformed into appropriate yeast strains by the spheroplasting method. 7 Preparation of RNA, Cells were grown to a concentration of 3 X 10 to 5 X 107 cells/ml in PSP2 (1.0 liter), then washed and transferred to SP2 (5 X 107 cells/ml). After 4 hrs, the cells were harvested by centrifugation and suspended in 6 mls of cold extraction buffer (100 mM Tris-HCl, 100 mM LiCl, 20 mM DTT, pH 7.5). This suspension was then added to a Braun bottle containing 5.0 mls of phenol (pH 7.5), 5.0 mls of chloroform, and 1.0 m1 of 10% sodium dodecyl sulfate (SDS). Glass beads were added to 2/3 volume and the mixture was subjected to 3 30 sec bursts in a Braun homogenizer. Calcium chloride was added to a final concentration of 0.01 M to precipitate the SDS, the lysate was transferred without the beads to a 30 ml Corex tube, and centrifuged for 30 min at 4°C, 11,500 RPM to remove other debris. The supernatant was ethanol precipitated, pelleted, and resuspended in 4.0 ml of DEPC treated water (made 0.1% diethylpyrocarbonate, incubated at 37°C overnight, then autoclaved). An equal volume of a 10% sarkosyl solution and 0.35 g CsCl were added, then this solution was layered 34 over 2.75 ml of 5.7 M CsCl, and centrifuged 16 hours at 15°C in an SW41 rotor at 25K RPM. The RNA pellet was resuspended in DEPC treated water, spectrophotometrically quantified, added to 2.5 volumes of 95% ethanol, and stored at -70°C. Southern analysis, Genomic yeast DNA was prepared by the method of Hereford et a1. (11), digested with the appropriate restriction enzyme, and subjected to agarose gel electrophoresis (16). Nitrocellulose paper was used for blotting (28). Preparation of DNA probes and hybridizations were carried out by the method of Maniatis et al (16). Desired fragments were isolated from agarose before being used for probe preparation. Northern analysis, RNAs isolated from appropriate strains were separated on a formaldehyde agarose gel, transferred to nitrocellulose paper and hybridized to DNA probes as described by Thomas (29). Poly(A)+ RNA was prepared as described (10). Enzymes and radioisotopes, Restriction enzymes, T4 DNA ligase and DNA polymerase I were purchased from either Bethesda Research Laboratories or New England Biolab. The suppliers' instructions were followed in their use. a-[32P] dATP was from New England Nuclear. OFAGE analysis, Orthogonal-field-alternation gel electrophoresis was performed by the method of Carle and Olson (3). Expressiop of RMEI in 52012 mutants, rme1::LacZ strains used were 35 grown on YEPD plates. 100 ul of X-gal (2%) was spread on each plate prior to inoculation. Plates were incubated for 2 days at 30°C, then at ambient temperature for 5-7 days. The rme1::LacZ colonies turned blue when RMEl was expressed. RESULTS STRATEGY FOR CLONING THE SP017 GENE The temperature sensitive sporulation mutant SL484 was first isolated in our laboratory by Smith et a1. (27). This mutation, which maps to chromosome VII, is of particular interest because cells which carry it are blocked in pre-meiotic, but not mitotic, DNA synthesis. We wanted to isolate the spol7 gene by complementation. Because most available yeast genomic libraries are on either LEUZ or URA3 vectors, we constructed a spol7/spol7 strain which was leu2/leu2 ura3/ura3 and also ADEZ/adeZ. After transformation with a genomic library, such a strain could be assessed for sporulation by first plating on sporulation medium, incubating, then replica plating to a rich medium. The appearance of red papillae at high frequency should take place only in colonies where sporulation competence was conferred by the transforming plasmid. To construct the strain, we sporulated SL484 at 30°C, the permissive temperature, dissected the tetrads, and mated the spores with W66-8A, an ade2 strain that bears other useful markers. Products of this mating were then sporulated and their tetrads dissected. Because both parents are homothallic (H0) any strains selected after these manipulations would be diploid, and homozygous at all loci (except MAT). Construction and characte ization of the ec ien train R9 W Of several hundred red (ade2/ade2) colonies from the mating between SL484 36 37 and W66-8A, twenty were found to be TRPS LEUl 1eu2. Since SP017 is linked to TRPS (27), we expected that the TRP5 progeny would be spol7. One of these, R9, was also found to be ura3, and its spol7 phenotype was verified. To establish heterozygosity at the ADE2 locus, R9 was plated on minimal medium with all requirements added except adenine. At the end of one week, four white colonies had arisen from the initial plating; of these, one (R92W) was selected and shown to produce red papillae after sporulation on plates at the permissive temperature, followed by replica-plating to YEPD. To determine if the ADE+ phenotype of R92W were due to a reversion or suppression event, we subjected the strain to further tests. Spores gave a 2:2 segregation of reds and whites; one of the white colonies (R92WH) was then sporulated and the spores were mated with an ADE2 haploid strain BB4. We reasoned that if suppression of ade2 were responsible for the ADE+ phenotype of R92W, some progeny of the mating between R92WH and BB4 should be red, because R92WH would carry the ade2 mutation. After mating and sporulation, seventeen tetrads from the cross were dissected and all spores gave ADE+ white colonies. We thus concluded that the ADE+ phenotype of R92W was due to a reversion in the ade2 gene. (It could, of course, also be due to a very tightly linked suppressor.) Isolation of SPOIZ. R92W was transformed with a YCpSO (CEN4 ARSl URA3) yeast genomic library obtained from Mark Rose (21). The average insert size of this library is 15-20 kb; thus we calculated a screen of 4,500 colonies was needed to cover 99% of the yeast genome, assuming a yeast genome size of 17 million base pairs. Transformants were subcultured to duplicate sporulation plates and incubated at permissive temperature 38 (30°C) and non-permissive temperature (34°C). After five days, these plates were replica plated to YEPD plates, incubated for three days at 30°C, then scored for production of red papillae. Initally, 14 positive transformants were isolated in a screen of only 2700 colonies. A second screen reduced the number of positives to eight. The plasmid from each strain was isolated and used to transform (13) the E.coli strain DHl. Three plasmids, p98, p1912 and p1940, were again transformed into the recipient strain R92W. These transformants were also able to sporulate at both permissive and nonpermissive temperatures. Restriction map comparisons of the plasmids gave a common pattern (data not shown), and we inferred that all contained identical or overlapping DNA inserts. To prove that sporulation competence was due to the plasmid, clones containing p98 and p1912 were plated on media containing S-fluoro- orotic acid (2). Cells that produce uracil in the presence of FDA are unable to survive, so any cells that survive no longer contain the URA3 plasmid. Three FOA survivors (R98FOA1, R98FOA2, R12FOA1) were tested and all three failed to generate red papillae at the nonpermissive temperature. This result, as well as the data in Table 3, clearly demonstrate the loss of sporulation ability. Plasmid p1912 was selected for further study because the size of its insert is only about 6 kb. Deletion of portions of this plasmid followed by transformation into R92W identified a region which contains SP017 (Figure 1 and Table 4). Plasmid p126S, which has a deletion between the two SalI sites, complements R92W. The failure of plasmids p126H (containing a HindIII - HindIII deletion) and p1268H (which has both deletions) to complement, however, suggests that sequences 39 essential to the function of SP017 span the HindIII site. Moreover, the plasmids pEMBB and YCpBB, which have the 2.4 kb BamHI - BglII fragment subcloned into the multicopy plasmid pEMBLY30 (Baldari and Cesareni 1985) and into the single copy plasmid YCp50, respectively, fail to complement. These data further suggest that SP017 and/or its regulatory region spans the BglII site. MAPPING OF SP017 The cloned SPOIZ sequence is on chrpmosome VII, Figure 2B is a Southern blot of yeast chromosomes which have been separated by orthogonal-field-alternation gel electrophoresis. After the stained gel pictured in Figure 2A was blotted, the blot was divided into three strips. The strip pictured in lane a in Figure 2B was probed with the sporulation-regulated gene SPRZ, which we have previously shown to be on chromosome XV (not published). Probes made from both the insert of p1912 (lane b) and TRP5 (lane c) give signals that demonstrate hybridization to the band identified as chromosome VII. We conclude that the sequence in p1912 is located on chromosome VII. The glppgg £2912 segugppg is linkgg tp T325, spol7 was previously shown to be linked to TRPS on on chromosome VII and the map distance was calculated to be 8.8 m.u. (27). To confirm that the sequence isolated was identical with SP017, plasmid pYilZ (Figure l) was constructed by inserting a 4.8 kb DNA fragment containing SP017 (BamHl- EcoRI) into the yeast integrating vector YIpS, which was then cleaved with BglII and used to transform (25) the ura3 haploid M1417-88. Of 93 40 transformants, three were rescreened, and one, M8B-Tl, was mated to spores of the ura3 trp5 strain W66-8A. Two of the four spores successfully mated with the transformed haploid and produced the diploids TleA and TleC, which were capable both of growth on nonsupplemented medium and of sporulation. We then asked whether URA3 from the integrated plasmid was linked to TRP5, as would be expected if the sequence directing integration were SP017. 31 tetrads from TleA and TleC were analyzed for cosegregation of URA3 and TRP5. 0f 27 tetrads showing a 2+:2- segregation for each of the two genes, 23 gave the parental ditype and 4 gave the tetra-type. The formula of Mortimer and Schild (18): cM _ __9Q (T+6NP2) 2 PD+NPD+T gives a linkage distance of 7.4 cM. This is in close agreement with the previously reported data of Smith et al. (27). THE FUNCTION OF SP017 SPQIZ is not 3426. We selected two ura3 spol7 progeny of the cross to test for complementation by the RAD6 gene, which is also located on chromosome VII (6), and is involved in sporulation. Data in Table 5 show that the URA3 RAD6 plasmid pR67, which we obtained from L. Prakash, did not complement cells bearing the spol7 mutation (strains PT4a67, PT4c67). Moreover, spol7 strains did not exhibit an increased sensitivity to UV irradiation (data not shown). 41 In vitro uta esis o SPOI con ers i he t u on transformed straips, A LEU2 disruptant (plasmid pLB174) was constructed by inserting the 3.0 kb LED? marker from YEp13 into the BglII site of pYil2 (Figure 1). After complete digestion with BamHI and partial digestion with EcoRI, the 7.8 kb fragment was isolated and transformed into the wild type diploid SCMS7-1 and the haploid BB4. Southern analysis was carried out by probing BamHI-EcoRI digested genomic DNA from the transformants with the radiolabeled 4.8 kb BamHI- EcoRI fragment from the p1912 insert which contains SP017. Results are shown in Figure 3. The transformants SLBO and BLBl gave two bands because of the EcoRI site in LEUZ. The sum of the sizes of the two bands, 4.4 kb and 3.6 kb, is approximately equal to the sum of the size of the wild-type band plus the size of the LEUZ insert, 4.8 kb and 3.0 kb). Transformants SLBl, SLB2, and SLB3 did not give the predicted bands, and were not analyzed further. Strain SLBO gave bands identical to those of the haploid transformant BLBl. In SLBO, however, the disrupted genes were probably integrated in both homologs; it is unable to sporulate at either 30°C or 34°C. Northgrm analysis or rhg disrmpraptg, Northern analysis suggests that the DNA included in the p1912 insert produces at least two messages, of approximately 2.7 and 2.1 kb (Figure 4). These messages are also expressed in vegetatively growing cells, though to a lesser extent (lane a), and expression seems to occur normally in an rmel/rmel diploid background (lane d). SLBO has lost both messages but gives one or possibly two smaller signals about 1.2 kb in length (lane c). The blot was also probed with DNA containing the URA3 gene (1.1 kb band); the intensity of this signal indicates that approximately the same 42 amount of RNA was loaded in each lane with the possible exception of SLBO, where the signal is somewhat fainter. InrgragriQnrhgryggn_££QLZ_pnd_flflfll. Kassir et a1. (14) demonstrated the interaction between RMEI and IMEI by constructing diploids homozygous for the two mutations imel and rmel. We obtained an a rmel- haploid (strain 2209) and an a rmel' haploid (strain 2210) from Mitchell (17), and made each spol7 by transformation with DNA bearing the disrupted gene (the BamHI-EcoRI fragment from plasmid pLBl74). Because the rmel disruptions were made by inserting LacZ (in-frame) into the coding region, cells attempting to express RMEI will form colonies that turn blue on plates containing X-gal. RMEI, a repressor of meiosis, is expressed in haploids; both strains 2209 and 2210 were blue in the presence of X-gal. Strains 2209-a and 2210-3, the spol7- disruptants, also turned blue. When the two spol7 haploids were mated, the resulting diploid (strain 221a3) was unable to sporulate and remained white on X-gal plates (Table 6). To prove that the strain we had derived was in fact the diploid product of a mating, we transformed it with the plasmid p1912 (our original spo17+ plasmid). Three transformants, 312-1, 312-2 and 312-3 were examined and found to be sporulation competent. DISCUSSION Using a yeast genomic library, we have isolated a plasmid that complements the sporulation deficient strain SL484. We know that the gene involved, which we have called SP017, is absolutely required for sporulation because wild-type diploids that have been rendered mutant at both SP017 loci are no longer able to sporulate. As we have previously reported, SP017 is among the earliest genes identified in the sporulation pathway, and presents a restriction map that is different from other characterized sporulation genes. Strains which have mutations in the RAD6 gene, which has been mapped to chromosome VII, also are unable to sporulate. The RAD6 plasmid pR67, however, does not complement spol7 strains, nor do spol7 strains exhibit increased sensitivity to UV irradiation as do rad6 mutants. We conclude that SP017 is not RAD6. As we reported previously (27) spol7 defines a complementation group different from those of spol, spo3 or spo7. Thus, SP017 appears to differ from all other characterized early sporulation genes. A variety of mutant genes give asporogenous phenotypes when their strains are sporulated. Thus, a ts Spo- phenotype can be due to a leaky allele of some gene which is in fact required for mitotic growth. The fact that both haploid and homozygous diploid disruptants of SP017 grow normally on several media (min, PSP2, and YEPD) at 24°, 30° and 34° suggests that if SP017 does have a mitotic phenotype it is a 43 44 relatively subtle one. The simplest explanation is that SP017 is a true sporulation gene. The low level of vegetatively produced message, on the other hand, might suggest a vegetative function for the gene. SP017 appears to be an early acting gene, since the mutant is blocked before premeiotic DNA synthesis. One possible role is that of a regulator of RMEI. If SP017 were involved in turning off RMEI in diploids, the loss of its function would block sporulation by failing to relieve RMEI inhibition of IMEI. If this model were correct, a spolZ/spol7 rmel/rmel strain should sporulate; however, the spol7 phenotype is epistatic to the rmel phenotype. Furthermore, the rme1::LacZ fusion is not expressed in spol7 diploids, demonstrating that the normal repression is operating. We conclude that spol7 functions either after RMEI or on a parallel pathway to regulate sporulation. The assay system we have used offers a useful tool for isolating cells which are sporulation-competent after transformation with a genomic library. Development of a recipient strain that is heterozygous at the ADEZ locus was accomplished by crossing the mutant SL484 with the ade2 trp5 strain W66-8A. This was possible because SL484 is a temperature sensitive spo mutant, and we could easily obtain haploid spores at the permissive temperature. Furthermore, selection of progeny from this cross was facilitated by the tight linkage of spol7 to TRPS, which permitted a rapid screen. We generated heterozygosity at the ADEZ locus by simply plating red ade2/ade2 spol7/spol7 products on medium lacking adenine. An ADE? heterozygote that could be transformed and screened by the method of Fonzi and Sypherd (7) was among the revertants. 45 The in vitro mutagenesis performed by inserting LEUZ into the coding region of SP017 resulted in the loss of two major mRNA bands (Figure 4, lane c), and the appearance of smaller molecules. The wild- type bands could represent different transcripts from SP017, or they could be from two different genes; both DNA strands might bear genes in the SP017 region. A simpler explanation would be that transcripts from a single gene in the region begin at two or more start sites, or if they have introns, that there are alternate splice sites. The nutritional sensitivity of IMEI expression shown by Kassir et a1 (14) has identified at least one mechanism by which nutrition regulates entry into meiosis. The preliminary experiments we have done do not suggest such tight nutritional regulation of SP017 expression. However, our initial data indicate that transcripts from the SP017 region are expressed at a significantly higher level in sporulating cells than in vegetatively growing cells, and we conclude that SP017 expression must be influenced by some other factors. If the SP017 gene product is a structural protein, it is required only for meiosis and not at all for mitosis. Such a protein could be involved in special aspects of premeiotic DNA synthesis or in the earlier sporulation-specific nuclear changes. Alternatively, its early expression could suggest a role in regulation. It is interesting to note that SP017 might not have passed a screen based on the differential hybridization of sporulation versus vegetative mRNAs, because its message is expressed at significant levels in vegetative as well as in sporulating cells. 46 ACKNOWLEDGEMENTS We thank Dr. Linda Smith for sharing some unpublished data, Dr. Mark Rose for providing the YCp50 genomic library, Dr. Louise Prakash for the RAD6 plasmid, and Dr. Aaron Mitchell for the rme::Lacz strains. We also thank Dr. Don Primerano, Dr. Erik Rikkerink and especially Dr. Anita Hopper for helpful discussions. This work was supported by Grant No. to PTM. ALK was supported by the Michigan State University College of Osteopathic Medicine. REFERENCES l. Baldari, C. and G. Cesareni. 1985. Plasmids pEMBlY: new single- stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene 35:27—32. 2. Boecke, J.D., F. LaCroute, and G.R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro—orotic acid resistance. Mol. Gen. Genet. 197: 345-346. 3. Carle, G., and M. Olson. 1985. An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA 82:3756-3760. 4. Clancy, M.J., B. Buten-Magee, D.J. Straight, A.L. Kennedy, R.M. Partridge, and P.T. Magee. 1983. Isolation of genes expressed preferentially during sporulation in the yeast Saccharyomyces cerevisiae. Proc. Natl. Acad. Sci. USA 80:3000-3004. S. Esposito, R.E., N. Frink, P. Bernstein, and M.S. Esposito. 1972. The genetic control of sporulation in Saccharyomyces. II. Dominance and complementation of mutants of meiosis and spore formation. Mol. Gen. Genet. 114:241-248. 6. Esposito, R.E., and S. Klapholz. 1981. Meiosis and ascospore 47 48 development. p. 211-287. In J.N. Strathern, E.W. Jones, and J.R. Broach (ed.), The molecular biology of the yeast Saccharyomyces: life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 7. Fonzi, W. and P. Sypherd. 1985. Expression of the gene for ornithine decarboxylase of Saccharyomyces cerevisiae in Escherichia coli. Mol. Cell. Biol. 5:161-166. 8. Game, J. 1976. Yeast cell-cycle mutant cdc21 is a temperature sensitive thymidylate auxotroph. Mol. Gen. Genet. 146:313-315. 9. Gottlin-Ninfa, E. and D.B. Kaback. 1986. Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharyomyces cerevisiae. Mol. Cell. Biol. 6:2185-2197. 10. Harper, J.F., M.J. Clancy, and P.T. Magee. 1980. Properties of polyadenylate-associated ribonucleic acid from Saccharyomyces cerevisiae ascospores. J. Bacteriol. 143:958-965. 11. Hereford, L.M., K. Fahrner, J. Woolford, Jr., M. Rosbash, and D. Kaback. 1979. Isolation of yeast histone genes H2A and H2B. Cell 18:1261-1271. 12. Hoffman, C.S., and F. Winston. 1987. A ten minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267-272. 49 13. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. 14. Kassir, Y., D. Granot, and G. Simchen. 1988. IMEI, a positive regulator gene of meiosis in S. cerevisiae. Cell 52:853-862. 15. Krieg, P. and D. Melton. 1985. Plasmid preparation. Promega Biotec 85/86 catalogue and applications guide, p. DB3. 16. Maniatis, T., E.F. Fritsch and J. Sambrook. 1982. Molecular cloning: a laboratory manual. (Cold Spring Harbor Press, New York.) 17. Mitchell, A. and I. Herskowitz. 1986. Activation of meiosis and sporulation by repression of the RMEI product in yeast. Nature (London) 319:738-742. 18. Mortimer, R.K., and D. Schild. 1981. Genetic mapping in Saccharomyces cerevisiae, pp. 11-26. In J.N. Strathern, E.W. Jones, and J.R. Broach (ed.), The molecular biology of the yeast Saccharomyces: life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 19. Percival-Smith, A. and J. Segall. 1986. Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharyomyces cerevisiae. Mol. 50 Cell. Biol. 6:2443-2451. 20. Pringle, J.R. and L.H. Hartwell. 1981. The Saccharyomyces cerevisiae cell cycle. p.97-142. In J.N. Strathern, E.W. Jones J.R. Broach (ed.), The molecular biology of the yeast Saccharyomyces cerevisiae: life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 21. Rose, M. and G.R. Fink. 1987. KARI, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell 48:1047-1060. 22. Roth, R. and H.O. Halvorson. 1969. Sporulation of yeast harvested during logarithmic growth. J. Bacteriol. 98:831-832. 23. Roth, R. 1973. Chromosome replication during meiosis: identification of gene function required for premeiotic DNA synthesis. Proc. Natl. Acad. Sci. USA 70:3087-3091. 24. Sherman, F., G.R. Fink, and J.B. Hicks. 1981. Methods in yeast genetics. Cold Spring Harbor Press, New York. 25. Shortle, D., J.B. Haber, and D. Botstein. 1982. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science 217:371-373. 26. Simchen, G. 1974. Are mitotic functions required in meiosis? 51 Genetics 76:745-753. 27. Smith, L., L. Robbins, A. Kennedy and P.T. Magee. 1988. Identification and characterization of mutations affecting sporulation in Saccharyomyces cerevisiae. Genetics 120:899-907. 28. Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 29. Thomas, P. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77(9):5201-5205. Table l. .msin Yeast Strains 52 _Smrss_ SCMS7 SL484 U66-8A R9 R92W R92WH BB4 M1417-88 MBB-Tl TleA TleC SLBO BLBl PT4a PT4b PT4c PT4d 2209 2210 2211 2209-a 2210-3 221a3 312-1 312-2 312-3 Siam mammals: HATE MO ade his‘ IeuZ mmmmmzmiz HATh no ads hlsé leu2 spoil MBIIQsdlesul HATa HO ade2 1e MIsiIQsdlesuZuLdmLZ HATo HO ade2 leu2 uraJ spol7 mmLm 12211 HATh MO ade2 1eu2 uraJ spol7 MIsHQIcHZmlmQIZ flATa HO 1eu2 ura3 sp017 HATa ho leu2 arg NAT! ho uraJ meta HATa ho uraJ meta SPOI7::URAJ mm++++ _ur.sJ___ HATo ho ade2 leul Iysl nerd trpfi uraJ spol7: :URAJ saae as TleA 84111110113111!!!“ Mmfimfiuralml ul lysZ meté trp5 uraJ canl M HATa no ads hisé leuZ spol7: :LEUZ HATa ho leuZ hisé erg spol7::LEU2 magnum HAIo HO uraJ spol7 ashram} HATh NO uraJ Same as PT4a Same as PT4b HATa rme1::LacZ hlsé-519 uraJ Ieu2 trpl HAIh rmeI::LacZ h1s4-519 uraJ 1eu2 trpl 2209 x 2210 HAT! rme1::LacZ hlsé-Sl9 uraJ 1eu2 trpl HATh rIe1::LacZ hls4-519 uraJ IeuZ trpl 2209-a x 2210-3 HAT-maniac: Same as 312-1 Same as 312-1 wmmmmw HATo rme1::Lac2 hlsé- 519 ura3 leuZ trpl canl spoil: :LEUZ canl spol7: :LEUZ canl spol7: :LEUZ Smith. 1985 Smith. 1985 Rothstein This work This work This work This laboratory This laboratory This work This work This work This work This work This work This work This work This work A. Mitchell A. Mitchell A. Mitchell This work This work This work This work This work This work 53 Table 2. Plasmids used and their sources. Plasmid Markers Sourcem, YCp50 library CEN4 ARSI URA3 BLA M. Rose p98 CEN4 ARSl URA3 BLA SP017 YCp50 library p1912 CEN4 ARSl URA3 BLA SP017 YCp50 library p1940 CEN4 ARSl URA3 BLA SP017 YCp50 library p126H CEN4 ARSI URA3 BLA spol7 This work pl26S CEN4 ARSI URA3 BLA SP017 This work p126SH CEN4 ARSI URA3 BLA spol7 This work pYi12 URA3 BLA SP017 This work pLBl74 URA3 BLA spol7::LEU2 This work pR67 CEN4 ARSl URA3 BLA RAD6 L. Prakash YCpBB CEN4 ARSI URA3 spol7 This work pEMBB LEU2 BLA spol7 This work 54 Table 3. Complementation of spol7 mutants by plasmids. Transforming Spprmlprlpm Strain DNA Markers 30° 34° W66-8A 85.0 49.3 SL484 18.3 1.0 R92W 20.7 0.0 R92W p98 SP017 URA3 52.0 13.7 R92W p1912 SP017 URA3 61.0 9.0 R92W p1940 SP017 URA3 79.0 6.0 R98FOA1 + - R98FOA2 + - R12FOA1 + - PT4c YRp12 TRPI URA3 + - 55 Table 4. Determination of the extent of the SP017 gene. Transforming g Spormlation Strain DNA 30°C 34°C R92W p126H 8.3 0.0 R92W p1268 23.3 7.3 R92W p126SH 11.7 0.0 56 Table 5. RAD6 does not complement spol7 mutations. Transforming g Spormlatiom Str_in DNA Markers 30°C 34°C PT4a 0.0 0.0 PT4b 31.0 30.0 PT4c 0.0 0.0 PT4d 25.0 21.0 PT4a67 pR67 RAD6 URA3 0.0 0.0 PT4c67 pR67 RAD6 URA3 4.0 0.0 PT4c YRp12 TRPI URA3 0.0 0.0 57 Table 6. Sporulation of the rme1::LacZ spol7::LEU2 double mutants. Color on Strain Genotype X-gal Sporulation 2209 a rmel::LacZ Blue - 2210 a rme1::LacZ Blue - 2211 2209 x 2210 White + 2209-a a rme1::LacZ spol7::LEU2 Blue - 2210-3 a rme1::LacZ spol7::LEU2 Blue - 221a3 2209-a x 2210-3 White - 312-1 221a3 + p1912 White + 312-2 221a3 + p1912 White + 312-3 221a3 + p1912 White + Figure 1. 58 Plasmid constructs. BamHI 83111 6181 EcoRI HindllI Hpall Sell 59 Figure l. Plasmid constructs. The heavy line indicates the YCp50 sequences; the light line is the insert carrying the SP017 gene. Plasmids pl26S and p126H were derived from p1912 by deleting the SalI and HindIII fragments, respectively. YCpBB was made by inserting the BglII-BamHI fragment into YCp50. The same fragment was inserted into the EMBLY vector pYE30 to give pEMBB. pYi12 was constructed by inserting the EcoRI-BamHI fragment into YIPS. SP017 was disrupted by inserting a copy of LEUZ into the BglII site; the resulting plasmid pLBl74, is not pictured. 60 -VII -XV Figure 2. OFAGE analysis. A. Genomic DNA separated by OFAGE and stained with ethidium bromide. B. Southern blot analysis of the DNA shown in A probed with nick translated DNA from one of the following genes: Lanes a, SPRZ; b, SP017; c, TRP5. Figure 3. 61 kbp 4.4’ 3.6 — Southern analysis. DNA from each strain was digested with EcoRI and 20 ug of digested DNA were loaded into each lane of an agarose gel and separated by electrophoresis. A Southern blot was prepared and probed with the radiolabeled HindIII-BglII fragment from the SP017 sequence. Lanes 8, BB4; b, BLBl; m 83% m man a fl3&.fl $31 Figure 4. 62 (unasl Northern analysis. 20 ug of poly (A+) RNA was loaded in in each lane of a 1.2% agarose denaturing gel and electrophoresed. A Northern blot from the gel was probed with the radiolabeled HindIII-BglII fragment (1.2 kb) from p1912 and autoradiographed. The blot was later probed with the 1.1 kb URA3 gene. Sources of RNA were as follows: Lanes a, SCMS7-l (SP017/SP017) vegetative cells; b, SCMS7-l cells after 4 hrs in sporulation medium; c, SLBO (spol7::LEU2/spol7::LEUZ) cells after 4 hrs in sporulation medium; d, 2211 (spol7/spol7 rme1::LacZ/rme1::LacZ) cells after 4 hours in sporulation medium; e, tRNA (control). CHAPTER III DISCUSSION 63 DISCUSSION The isolation of SP017 was accomplished with relative ease because the donditional nature of the original spol7 mutant, SL484, allowed the mutation to be introduced into a new genetic background from which the gene could be identified. After its isolation, SP017 was disrupted by in vitro mutagenesis and transformed into several wild type strains. Diploidized mutants generated by this method are unable to sporulate at any temperature, and it was concluded that the SP017 product is absolutely required for sporulation. The fact that mRNAs of nonsporulating disruptants differ markedly from wild type SP017 messages also supports this conclusion. Does SP017 have a mitotic functiomz A possible role for SP017 in the mitotic cell cycle is suggested by the presence of SP017 mRNAs in vegetative cells, even though all spol7 mutants seem to grow normally. An alternative explanation for the vegetative expression is that SP017 is not tightly regulated by the cell. This is in contrast to the early gene IMEI, which is so tightly regulated that its messages do not appear in glucose-grown cells (Kassir et a1. 1988). Of course, IMEI is regulated by at least two mechanisms, starvation and repression by RMEl; thus, tight control of its expression might be expected. Expression of the repressor gene RMEI does not exhibit such tight 64 65 regulation. Although RMEI is freely expressed in haploids to prevent entry into sporulation, there remains a residual level of expression in a/a cells (a 10 to 20-fold reduction is seen). RMEI is negatively regulated by the al-a2 product, which has been shown to repress two other genes, MATal and H0; interestingly, the reduction of these transcript levels is greater than lOO-fold. Mitchell and Herskowitz (1986) were unable to find any functional significance for the residual RMEl transcription in a/a cells. It seems curious that the MAT locus would tightly regulate the expression of some genes, yet permit residual expression of others. Although SP017 is probably not regulated by the same mechanism as RMEI, its residual expression in vegetative cells is of interest. The extent of its induction during sporulation has yet to be determined. There is another hint of a possible mitotic role for SP017. Figures 1 and 2 (Appendix D) are photographs of plates which contain colonies that have been exposed to permissive and nonpermissive temperatures under sporulation conditions. Several points of interest can be noted. During the initial screen of transformants, a difference in plate growth was noted. This can be seen in the colonies at positions 15, 16 and 17. These are strains that have lost the SP017 plasmid after growth on S-FOA. As expected, no red papillae are produced on either plate. However, growth of the colonies that have been subjected to sporulation conditions at 34°C is much poorer than those subjected to 30°C. During the screen this difference was so striking that growth alone was sufficient to identify clones maintaining a SP017 plasmid. This difference was not seen, however, in SL484 (the original spol7 66 mutant). It may be that this feature is a characteristic of the new genetic background of R92W; a modifier gene may have been introduced or a mutation may have occurred during or after the cross with W66-8A. It should be noted that colonies from strain R92W do not exhibit the growth differential on these plates although usually the difference was quite marked. To further examine these growth characteristics, strain SLBl, which is a heterozygous (SP017/spol7) transformant, was sporulated and growth of the progeny was evaluated in rich liquid medium and in liquid acetate medium. No significant differences in the rate of growth between the spol7/spol7 or the SP017/SP017 strains could be seen, nor was there any apparent loss of viability in rich, acetate, or sporulation media (data not shown). spol7/spol7 cells did appear to be slightly smaller, but a quantitative analysis was not carried out. Strain SLBl was later found not to have exactly the predicted bands after its disruption (Chapter 11, Figure 3, lane d), but the spol7 disruption is evident, and its progeny segregated 2:2 for sporulation- competence. The growth phenotype of strain R92W remains a mystery and awaits further investigation. Consistency of the generation of red papillae, Production of red papillae in most of the transformants (colonies at positions 7 through 14 and 18 through 29) can be noted on both plates. Some transformants occasionally fail to produce red papillae (eg., colony No. 13) after exposure to either temperature. This could be due to the variation in sporulation efficiency that is sometimes seen in cultures, even when sporulated in liquid medium. Another 67 explanation is that the SP017 plasmids borne by these strains are different. This is known to be true because most of the restriction enzymes used in mapping gave different restriction patterns in different plasmids. Plasmid p1912 (strain 1912, position 13) was selected for further study because of the small size of its insert. In fact, most plasmids which passed the screen had inserts which were much larger. The variation in the plasmids could account for the variability seen in the production of the red papillae. The five strains at positions 25 through 29 were transformed with the multicopy plasmid pRB367, which contains most of the insert (BamHI- EcoRI fragment) from plasmid p1912. These strains consistently gave good growth and sporulation. Whether this vigor is due to the small size of the insert or because of the multicopy effect of the plasmid is not known. Is SPOIZ also subjecr to multiple regulprionz Transcription of both SP013 and IMEI begins very soon after introduction into sporulation medium, and both genes are known to be regulated by two mechanisms, nutrition and the MAT locus. It seems possible that the SP017 product is involved in the cell's response to the available nutrition. Could its expression also be regulated by nutritional status? The masking effects of MAT regulation on SP017, if it exists, could be removed by analyzing SP017 expression in a/a and a/a diploids after their introduction into starvation medium. Wha he elat twee E It seemed reasonable to ask how the effects of SP017 might be 68 related to the established model of sporulation, and this question was addressed by creating a spol7/spol7 rmel/rmel strain and evaluating its ability to sporulate. The results did not suggest a role for SP017 in the regulation of RMEI. It would be very interesting to determine the level of SP017 expression in imel mutants after sporulation induction. Conversely, the level of IMEI expression in spol7 mutants could be determined. If no interaction between IMEI and SP017 exists, SP017 may prove to be very interesting, indeed. APPENDIX A 69 Isolation of genes expressed preferentially during sporulation m the yeast Saccharomyces cerevisiae (microbial development/cDNA probe/finesse“ phone hybridization/meiosis and W) MARY]. CLANCY‘, BEATRICE BL'TEN-MACEE, DAVID]. Snuucrn', ALLAN L. KENNEDY, RocsnM. Pun-runes, AND P. T. Mncss Deputmeatofhticrobs‘ologyaadl’ublicflealth. NMState um.umwupmuol ComnioatedbyAdrien M. Sereaasrle. 1983 ABSTRACT A library of Seccharoqces cereaist'ae DNA is thevectorAGIaroaflwasprobedforsequencescomplcmeuar-y to cDNA made from poly(A)° RNA isolated from the well-sper- ulating yeast strain API a/a. The RNA was isolated from cells that had been incubated 7, 9.1!. and II! II '- sporulatr'on media. DNA complementary to poly(A)° RNA from «In (nonsporulat'ng) Al’lwasusedasacantrolnndddbacteriaphagethatgavcsstrmr response with a/a cDNA than with all! cDNA were obtained ‘m a screeningof three yeast genomes worth of DNA. Two of the bacteriopliageappearedtooontairia/a-specifrcgenes,iithatthey hybridized to cDNA from vegetative a/a RNA. The rest appeared tocorrespoadtoa/a genes expressed preferentially during sper- ulation. Restrictionendorarcleaseanalysisoffourofthecloaed sequencesrevealedasinglemajorrep’uoftranscriptionineach; thmengimrangedinshefmm25b4.0kilobues. RNAblat analysisshowedthat, inthreeofthefowcascs,transcriptsoftwo differentsiaeswerel'iomologmatothechmedsequence. Inallfeu’ cases,the transcriptsappearedatabout7hrandwere decreasingin smountby 13hr. Thesereaalts provide evidcneefor transcriptional control of genes expressed during sporulation and foratlesstonegroupofgenesthatist'aedonataboutthetime ofmciosislinsporulatioa. The ascomycete Saccharomyoes cerevisiac has a relatively com- plexlifecycletlmtincludesamorpinlop‘ullydelinedcellcycle. mating and zygote formation. and meiosis and ascosporogcne- sis. The combined techniques of genetics and molecular biol- ogy have been used to elucidate several aspects of this life cycle and have led to a number of advances in our understanding of the cell cycle (1) and the control of mating type (2-6). The study of meiosis and sporulation (7). however. has heretofore lagged. partly because of the dilficulty of isolating and characterizing mutations in genes expressed only in diploids (8). Meiosis and ascosporogenesis is a complex process involving macromolecular synthesis (9. 10). recombination (l l). and turn- over of preexisting cellular components (12). It occurs in cells expressing both a and a mating type alleles on a shift to me- dium lacking ammonia and glucose but containing a respirablc carbon source. Cells expressing only one MAT allele (haploid a or a. diploid a/a or a/a cells) do not undergo the process; such cells fail to undergo premeiotic DNA synthesis (13. 14) or any of the subsequent events that have been monitored. Meiosis and sporulation are therefore under the control of the mating type locus. although nutritional signals must also play a role in the initiation of the process. The product of the developmental program. an ascus con- taining (usually) four haploid spores. is morphologically distinct from vegetative cells. In a typical (well-sporulating) diploid, fil- lbcpublicstioncostsofthisarticlewercdefrayedin by charge payment'lbisuticlemust thereforebe benbymedmwrtise- neat" inaooordmcewithIBUmSC “Maolelytoindicatethisfict. 3000 one of the cells achieve this differentiated state in a relatively synchronous process tlnt lasts 16—24hr underthecoaditions we use (15). Physiological studies have shown that premeiotic S phase occupies the period from 8 to 7 hr (in the culture as a whole. theSper-iodofindividualcellsappearstolastaboutl hr). recombination occurs over roughly the same period. be- ginning and ending about half an hour later than S phase. and meiosis l takes about 120 min, from 8 to 10 hr (9. l2). Meiosis ll follows immediately and is completed by 12 hr. Immature asciaredetectablc at thrinsomecells. and ~80‘lvofthefinal levclofasci isfound in thecultureby 16hr. withthcremaiader forming over the next 8 hr. Several attempts to identify specific gene products involved in this process have failed. despite the existence of numerous mutations affecting gene products needed in sporulation but not for vegetative growth (8). One- and two-dimensional gel analyses of proteins synthesised during a pulse label ofspor ulating cells have identified few (16) or no (l2. 17. 18) proteins specific to sporulation. This may be because the abundance of these gene products is simply too low to be detected on gels. Recently. workers in this laboratory have identified an en- zyme. sporulation amyloglucosidase (SAC), that occurs only' In all: cells in sporulation medium and appearance is de- pendent on the progression of the cells well into the pachytene stage of meiotic prophasc (19,20). It seemed worthwhile to ask Whether a group of genes was expressed during the time that this enzyme activity appears. how large this hypothetical group might be. and whether these genes share common (c.g., duration of transcription) and structural properties. We have therefore screened a Saccharomyces oereoisr’ae—A Charon 28 (21) library for bacteriophage containing'sequences specifically expressed from 7 to 13 hr in sporulation medium. Although we have not yet identified the SAC gene. we have found 46 hybrid phage that contain sequences more highly ex- pressed in sporulating MATa/MATa cells than in otherwise isogcneic MATa/MATacells. Ibeclnractcrintionofsixofthesc sequencesisreportedherc MATERIALS AND METHODS and Enzymes. ["PldCl' P (>4!) Ci/mmol; 1 Ci - 37 CBq) was from New England Nuclear. Restriction en- zymes and oligo(dT) primer were from Bethesda Research lab- oratories and were used as recommended by the supplier. DNA polymerase and avian myeloblastosis virus reverse transcriptasc were from Boehringcr Mannheim and Life Sciences. respec- tivcly. Nitrocellulose sheets aid filter dist: were from Schlcicber Abbreviations: SAC. sporulation amyloglucosidase; kb. kilobase pairs. °Presentaddress: Dept ofMicrobiology. UniversityofNotreDame NotreDame. IN46556. Genetics: Chantal. & Schuell. Formamide for hybridintion (Aldrich) was deion- ized with Amberlite (Aldrich) before use. Drosaphila cytoplas- mic RNA was a gift from Jerry Dodgson. Growth Conditions mad Preparation of RNA. The Esche- richincolistrainusedbrgowthofthebacteriophagewasKHm a derivative of K-12. S. cerevisiae strains used were APl a/a and its isogeneic asporogenous derivative APl a/a (12). The cells were ordinarily gown and sporulated as described (15) in acetate pregrowth medium. Vegetative or sporulating yeast cells were harvested by centrifugation. washed in ice-cold sterile distilled water. and broken by mixing in a Vortex with gins beads (18) or in a Bronwill homogenizer (22). Poly(A)’ RNA was pre- pared as described (22). Library Construction. A S. cerevisiae library was con- structed in A Charon %. DNA from S. cerevisiae strain A1122 was extracted by the method of Hereford et al. (23) and digested with Mbo 1 to yield fragments of ~ 15 kilobase pairs (kb). Fif- teen- to 20-ltb fragments were isolated by sucrose gradient cen- trifugation. and these were ligated to Charon 28 arms prepared by digestion with BamHI (24). The ligated DNA was packaged in vitm (25). plated. and amplified. Minipreparations of A DNA were used for all experiments. essentially as in Maniatis et at (24). Differential Plaque Hybridization. The method used wu essentially that of St. John and Davis (26). Aliquots of the li- brary were plated onto E. coli KH802 on NZCY M (24) and grown to yield ~3oo plaques per plate. Isolated plaques were picked using sterile toothpicks into an ordered array on freshly poured lawns of E. coli KH802 (~50 pl of an overnight culture) and allowed to grow for 8-12 hr. This amplification was necessary to allow detection of the hybridization signals from a majority of the plaques; only lO—M of the plaques yielded signals when filters were made directly from the original plaque plates whereas >90!» of the clones gave detectable signals with both probes when patch plates were used (26). Duplicate nitrocellulose fil- ter replicas were prepared from the plates essentially by the method of Benton and Davis (27). Hybridization was carried out as in Engel and Dodgson (28). Preparation of Hybridization Probes. ”P-labeled cDNA was prepared from poly(A)’ RNA by the method of StJohn and Davis (26). except that 40 mM sodium pyrophosphate was in- cluded in the reaction mixture. The reaction was started by the addition of reverse transcriptase. allowed to proceed at 40°C for (SO-W min. and terminated by the addition of base. Hydrolysis of RNA and separation of the cDNA from the unincorporated nucleotides was done as in St. John and Davis (26). Probes were medataconcentrationdlto2ng/ml. Nick-mlationofcloned 70 Proc. NsdAadSctUSAwUSfl-‘D Ml DNAwasaccomplishedbythemetbodofRigbyctal(29). Agarose Gel Electrophoresis and Nitrocelhdose Filter Hy- llidiaation. Agarose (Bethesda Research Laboratories) gels were run submerged in 40 mM Tris-HCl/20 mM NaOAc/ 1 mM EDTA. pH 7.5. at 60-lm mA and stained with ethidium bro- mide (l rig/ml). The gels were 0.4%. 0.7%. or 1.2% agarose. depending on the experiment. DNA from agarose gels was transferred to nitrocellulose by the method of Southern (30) and hybridized to cDNA prepared as described by St. john and Davis (fi). For RNA blots. ~5 ug of poly(A)‘ RNA per lane was denatured with glyoxal (3i) and loaded onto 1.5-ml agarose gels (30) and electrophoresed. Then. the RNA was transferred to nitrocellulose by the method of Thomas (32) and hybridized to nick-translated DNA as de- scribed above. Dot blots were prepared by spotting S-ul ali- quota of the A phage stock onto nitrocellulose filters. The DNA was denatured and neutralized. The filters were halted as de- scribed for differential plaque hybridization and then hybrid- izedtocDNAprobespreparedasdescribedabove. RESULTS DifferentialPlaquellybridiaationasaScreenforSporu- lation-Specil‘rc Genes. Differential plaque hybridization. a modification of the plaque filter hybridization method of Ben- ton and Davis (27). allows the isolation of genes whose RNAs are present at increased concentration in one population of cells compared with another. This method involves the preparation of two [nPchNA probes. one against each of the RNA prep- arations to be compared. and hybridization of these probes to duplicate nitrocellulose filter replicas of a collection of A clones on a Petri plate. Clones that hybridize strongly to one probe but not to the other contain genes that are expressed at different levels in the two populations. This method has been used suc- cessfully in yeast to isolate galactose-inducible genes (26) and genes expressed at high levels in low-phosphate medium (33). We have used differential plaque hybridization to screen a A Charon Q-S. cerevisine library for sequences expressed pref- erentially during sporulation. A [”PchNA probe was pre against RNA isolated from MATa/MATa cells after 6—13 hr of incubation in sporulation medium (Fig. l) and. as a compari- son. a similar probe was made from an otherwise virtually iso- geneic MATa/MATa strain that is incapable of sporulation. The RNA preparation from the sporulating MATa/MATa cells should contain sequences transcribed specifically during spor- ulation that the MATa/MATa cells would laclr. These might include those coding for products required for premeiotic DNA deaoocece PRIEDTlC-D IEIGNC MVBIOI89 ASCOSPORI DNA SYN!” NRIATION O 2 Q 8 I ID l2 l4 "WIS L L ‘1“ 1‘" fir‘ ’F A ’F'h aria . SAME.” o z 4 8 I l0 l2 l4 NOUI! a L 1 A F—f—TL s T 1 a" SAME! Fla. 1. Preparation of hybridization One-liter cultures of the sporulation-proficient strain APl a/a (Upper) and the asporogenous APl n/n strain (lower) were grown to 2 x 10 cells/ml and shifted tosporulation medium. The cells were allowed to progre- into spas-ulation and 500- ml samples were taken at the indicatedtimesforthepnpantionofRNAThesamplesfromeachculturswerepooledt‘orthspreparationofpoMA)‘ RNA. and [a-‘PleDNA probes were prepared against these RNAs and used for differential plaque hybridisation. 3002 Geneties:Clancyetol synthesis. recombination. the two meiotic divisions. and spore formation. aswellasRNAsthatarestored in thesporein prep- aration for dormancy and germination. The RNA sequences common to both probes would include those homologous to housekeeping genes or to genes induced by starvation. That these latter are the predominant sequences is suggested by the fact that two-dimensional gel electrophoresis of the proteins made in sporulation medium by sporulating-proficient and sporulation-deficient cells gives similar or identical patterns (16- 18). Isolation of Sporulation-Specific Genes. Aliquots of the li- brary were plated in top agar onto E. coli 101802. individual clones were picked and amplified by patching into an ordered array onto freshly poured lawns of KH802 and duplicate nio trocellulose filters were prepared and hybridized. Clones that gave stronger signals with the MATa/MATa probe were picked and retested. The second test was necessary to purify the phage and also served to eliminate false positives. which represented >50!» of the clones chosen on the first test. whether the same cDNA preparation was used for both the first and the second test or the probes were different. A sample autoradiograph from such an experiment is shown in Fig. 2. Plaques I and 2 gave stronger signals with the a/a probe than with the mrrespond- ing a/a probe on the original plate. On retesting, however. only clone 2 isolates hybridized preferentially with the a/a probe. Such clones were retained and phage stocks were prepared. We screened ~3.7(l) plaques and retained a total of 46 clones that appeared to contain differentially expressed genes. These clones varied at least lO-fold in the intensities of the hybrid- intion signals obtained as well as in the intensity differences between the a/a and a/rx probes. The dot blots (Fig. 3A and 8) show that some clones (e.g.. nos. 1. ll. 16. 34. and 42) gave extremely intense signals with the a/a cDNA but weak signals with the corresponding a/a probe while others (eg., nos. 2. A Get as. . “it..." 2;: ha. 2. Diflerential plaque filter hybridisation asa screen for spor- ulationqrecif'ic genes. Aliquots of the A library were patched onto 5. coii K8802. and du licate nitrocellulose filter replicas were prepared and hybridized tol )cDNA probes prepared against RNA from spor- ulating MATa/MAT‘a cells and nonsporulating MATo/MATa cells. The hybridized blots were washed and exposed to x-ray film and the signals obtained with the two probes were compared. Clones 1' and 2‘ (these numbers referto theorderoftestingandarenotrelated toclone numbers referred to in the text) appeared to contain sporulation-in- ducedgeneson thefirstwstu)butonlyclone2appearodpositiveon the second td (B). 71 Proc. Natl. Acad. Sci. USA NUS”) 4. unmmmrmhmmwm ought to represent some 15 genes. assuming that each is rep- resented only once per haploid genome and that no highly ex- pessedgenesthatarenotsporulation specificareloatedncarby. It is possible that the differentially expressed genes isolated in this screen were actually MATa/MATa specific. rather than sporulation specific. because the only criterion for selection was that the clones hybridize more strongly to the probe made from MATa/MATa cells in sporulation medium than to the corre- sponding MATa/MATa probe. That this is not the me is shown in Fig. 3 c and D. (”Pk-DNA probes were prepared to poly(A)’ RNA from vegetatively growing MATa/MATa and MATrx/ MATo cells and the signals obtained when the 46 clones were hybridized to the two probes were compared. Most clones hy- bridized equally well to the two vegetative probes and more weakly to both than to the cDNA from sporulating cells. Clones 7 and 69 were exceptions to this since they hybridized strongly as both MATa/MATa probes and weakly to both MATa/MATa probes. These two clones. therefore. contain a/rx rather than sporulation-specific genes. Clone 16 was surprising. since it hy- bridized more strongly to the MATa/MATa probe prepared from vegetativecells than toacomparableprobefrorn a/acells. Subsequent analysis showed that the apparent a/o-specifrc se- quence was located ~5 kb from the sporulation-specific se- quence within the same clone (see below). Restriction Maps and Time of Appearance of the Sporu- lation-Specific Transcripts. We wanted to determine whether each clone contained a single sporulation-specific gene or sev- eral and that time during sporulation at which the correspond. ing transcripts appeared. Six clones. nos. 1. ll. 16. 32. 34. and 42 were chosen for this analysis. because they gave particularly clear differential signals. Restriction maps were constructed and the locations of the differentially transcribed girtion in each wu determined by Southern blotting ((1)) using[ PIcDNA from sporulating MATa/MATa cells as the probe. As shown in Fig. A B 1‘- 0 -""'° 2 a 0.. a 3 O .‘e‘. 30 4. 0 "° 5,coeoe D 6 .000 ‘ 7.00.0’ “ ‘ 8.0.. ." a abcdef C D ' g; 006.}. ... ' .. ' .'..0' .090. .... .... C ' g . 0.0‘ ~ 0 o aOooQ. 0.0a. i 000* a Us. a Pro. 3. Dotblotanalyaisofthe46elonea. Dotblotawerepreparsd from insisted phage that appeared to mntain "ulation-inducible gen. and hybridized to P'PchNA probes prepared against poly(A)‘ RNA from MATa/MA‘I‘o cells undergoing sporulation (A). MATa/MATa alla incubated in sporulation medium (B). MATa/MA‘I‘n cells growing ventstivoly (C). and MATa/MATa growing vegetatively (D). The clan. referred to in the text are 1. position Ia: 2.1b; 4. 1c; 7. Id; II. 1e; 18. lf;32. 2c;34.2f;42.3f;and69.4f.£qualamountsdm13 DNAwero included as mntrob (position 8f). 1b hop t H nun an in FL e ---“.W‘ ‘- Xsp re 9! an 1‘ ex 1 I ......... “A AA “ H.-.-.--o.-- F—s s— Xep 11 9! L! ’5! 4 5 L _ -———4 ken 42 E! x r {F L o——-—s ~ ""“"“" hep 32 5J5" Eff K' — ksoa4 9 :9 5! L?!“ as! 9 —— Pic. 4. Restriction enzyme analysis of selected dense. Single and doublerestriction enaymedigsstaofthspurifiedt DNAswereana- lyssd on 1.2% and 0.4% horizontal gels. Sire standards were rsdrictionmme-digestedtandYEPla DNAa Enzymesuasdware EEcoRl; B.Baml-II;H.HindIIl;X.Xbo1; R.Kpn LThedottedlinss representtheACharonZBarmsandtheheavylinesreprssentyeast DNA. The thick lines shown below the maps reprunt the approximate locations of the sporulation-specific sequences within each phage. as determined by Southern blotting of the gels followed by hybridisation to the aP-labeletl NATO/MATH: and MATn/MATn probes used to iso- late the dense originally. Clones 11 and42 contained HindIII sitesbut these are not shown on the maps. 4. clones 11 and 42 ntly contain the same sporulation- specific gene. as do 1 and 16. Clones 32 and 34 differ from each other as well as from the other clones. The maps of thetwopairsofclones(nos. land 16;nos. 11and42)aresimilar to each other. although not identical. In each case. the spor- ulation-specific gene could be located on a single restriction ‘ fragment <3 kb long. as shown by the bars in Fig. 4. The small size suggests that each clone contains only one. or possibly two. closely linked sporulation-specific genes. The restriction fragments flanking those containing the spor- ulation-specific gene also hybridized to the s/a probe but at a greatly diminished intensity. It was not possible to determine whether this was the result of low levels of transcription from a flanking gene or whether the transcribed portion of the spor- ulation-specific gene extended past the fragments shown. In all cases except clone 34. the indicated fragment also hybridized to the a/a probe to some extent. This may reflect a low level of transcription d the 'sporulation gene" by MATa/MATa cells or the presence of an adjacent transcription unit that is ex- pressedus in common between MATa/MATa and MATa/MATa cc . We have used RNA blot analysis (34. 32) to determine the time of appearance of the transcripts corresponding to these genes and their sizes. API a/a cells growing vegetatively were shifted to sporulation medium and aliquots were removed from the culture at intervals during sporulation for the preparation of poly(A)’ RNA. This RNA was denatured with glyoxal. elec- trophoresed on 1.5% agarose gels (31). and blotted to nitro- 72 Proc. Natl. Acad. Sci. USA scam) arm A B C 135191113 136791113 135191113 ' s 1703a,}; . . 1. I.“ 9"? y b ' . v o.” - - . ‘r l 4 — ' H r f‘ ‘4 . “l, L? . ' . '. a . ,. ~ 3", , - ~ “r“: '4 f ' «.‘3 $34.: ' their? the 6. RNAbhtandyshofdmlth).34(Bl.and42(CLNy(Al‘ RNA was prepared from MATa/MATo cells incubated in mutilation msdiumfor1.3.5.7.9. 11.and 13hr. five-microgramaamplesofthess RNAs were denatured with glyoxal. on 1.5% gels. blotted to nitrocellulose. and hybridized to cloned DNAs labeled in vitro with ln-"PldC'I'P by nick-translation. Autoradiograpby was dsneat -70'Cfor3—8dayswithone intensifierscresn. Numberson thsleftot'thegehindicatssiass(kbldt.bedsvelopmsntally regulated tremipta. cellulose. The blots were hybridized to A clone DNA that had been labeled in oitro with [J'PldCl'I’ by nickotmslation (28). As shown in Fig. 5. clones 16 and 34 each hybridized to two developmentally regulated transcripts rather than one. and clone 32 also hybridized to two distinct transcripts (data not shown). Clone42hybridiaedtoonemajortranscript. butotherhintbmds were also present. We do not know whether the multiple bands represent transcription of the same gene from different pro- moters. processing of the larger transcript. or hybridization of the cloned DNA to messages from two closely linked devel- opmentally regulated genes. It is also possible that the second transcripts are from different but related genes elsewhere in the genome. None of these transcripts was detected in blots made to RNA from a/a cells. although such transaipts may have escaped detection because they were present at low lev- els. DISCUSSION Although a considerable amount is known about the physiology ofthedevelopmentalpathway lcadingtorecombinatimi. meiosis. and ascosporogenesis in S. cerecisiae and extensive genetic analysis has been done (8). molecular approaches have been somewhat unproductive. The genetics has provided part of the explanation for this lack of progress in that it has been calcu- lated that a relatively small number of genes are sporulation s Taste. required for the process but otherwise dispens- e (8). 1f the proteins that are the products of these genes are present in low abundance. the failure to see specific proteins on two-dimensional gels is easily explained. We sought to iden- tify the genes involved in sporulation directly. by differential plaque hybridization. with the hope that this method would be sensitive enough to detect sporulation-specific sequences. Al- though there is no doubt that our screen would fail to detect certain classes of genes. it has found a number of sequences $atarepreferentially expressed in a/acells insporulation me- urn. Therearetwornajorgroupsofgenesthatwillhaveescaped ourscreen. Oneoftheseistheclassthatcomprisesgeneslo- cated close to a gene actively transcribed in «In cells in spor- ulation medium. On the average. 15% of our clones gave a sig- nal with a/a cDNA so strong that we would not have been able to detect an a/a difference. Thus. we may have missed 5—10 clones. The more important class contains genes whose tran- scripts are present in very low abundance or unstable to iso- m4 Cenetiazqancyetd lationanchNA syntheticprncedure. Weestimatethstwecan detectgeneswhosetransaiptsarepresentinS-IOcopiespsr cell; since >905 of all clones pve detectable signals in the irri- tialscreen.wemustbedetectingmostofthe middleabun- dance sequences and possibly lower abundance sequences a well. Many of the less highly expressed transcripts may have escapedourscreen. however. lnaddition. ofcourxe. thosem whose transcripts are not polyadenylylated will not be repre- sented among the sequences in our probe. The four sequences we have studied have several charac- teristir: in common. Most interestingly. transcripts of all of them appear between 5—7 hr after the shift to sporulation medium anddecrcaseinabundanceby 11-13hr. Thefactthatourprobe was prepared from mRNA isolated from cells at various stages of sporulation. the earliest of which was at 7 hr. may account for this coincidence. On the other hand. it may be that a sig- nificant number of sporulation-specific genes actually begin to be transcribed at this time. and our group is a representative sample. The fact that the SAC gene product appears at about this time. as shown by methods independent of the hybridin- tion screen used here. is interesting in this context. A second significant characteristic of three of these se- quences is the presence of two coordinately controlled tran- scripts. A number of possible explanations exist for these tran- scripts. One is that we are seeing either processing events or a failure to terminate transcription part of the time. Another possibility is that there are two promoters for each gene. both of which are developmentally regulated. Finally. it is possible that each clone contains two clustered genes. both of which are sporulation specific. The last possibility might be examined by R-looping experiments. Whatever the explanation. it seems likely that the phenomenon may have some developmental signifi- cance; while dual transcripts from a single gene are not un- known in yeast (35. 36). they are rare and it seems improbable that three separate genes would exhibit this behavior by chance. The role of these differentially expressed genes in the spor- ulation process is not known. It is possible that they represent genes whose products are required for the meiotic divisions or spore formation; they may also represent transcripts that are stored in the spore in preparation for germination. It is known that the spore contains a large number of transcripts (22) and that these include histone RNAs as well as those for glycolytic enzyme (D. Kaback. personal communication). Some of the clones we have identified may correspond to these RNAs. The success of this screening method in identifying devel- opmentally controlled genes whose transcripts are present in relatively low abundance is somewhat surprising. Although analogous methods have been used to identify conidiation-spe- cilic genes in Aspergillus (37). in that system the probe was en- riched by “cascade hybridization" for specific sequences (38). By amplifying the "receptor" DNA on the filter by simple patching. we have been able to bypass the enrichment step. We umdmanmdbnmuMnmdMmmwmdwwd of genes described here may give important insights into spor- ulation in particular and eukaryotic development in general. We gratefully acknowledge the aid of Dr. E. 1". Fritsch in the con- struction of the library. We thank 1erry Dodpon and Ronald Patterson for advice and many helpful discussions. Larry Snyder for critically 73 ProcNatlAcarlSci. USA ”(1983) readingthsmarmacr'qrt. lidthedepartmentdcleriulstafl'irrhelpin preparingit. ThiswuhenssupportedbyNatioaalScienceFouadation Crust PCM 781258104. A.L.K. and 0.1.5. were supported byagr-rt from the National Foundation-March of Dimes to the Michigm State University Collep J Human Medicine. This is journal article no. 10.443 from the Michigan Agicultural Experiment Station. Hartwell. l... H. (1974) Bacterial Rec. as. 164-1”. Haber. 1. E. s George. 1. r. (1979) Genetics as. 13.35. Hicks. 1.. Str‘hern. 1. M. s Klar. A. 1. s. (1979) Nature (lander!) 83. 478—483. Nasmyth. K. A. I Tatchcll. S. K. (1”) Cell 19. 753-754. Oshima. T. I Takano. 1. (1m) Genetics 94. 859-870. Rine.1.. Sm. C. F..1r.. I Herskowitz. l. (1%” Mol Cell Biol 1. QSB-W. Croes. A. F. (1%7) Plants 79. 209-226. Esposito. M. S. I Esposito. R. E. (1974) Genetics 73. 215-2fi. Esposito. M. 5.. Es ito. R. E.. Arnaud. M. I Halvorson. H. o. (1969) 1. ' loo. 180-186. Magee. r. r. a Hopper. A. K. (1974) 1. Bacterial m. 952-960. Game. 1. C.. Zamb. T. 1.. Braun. R. 1.. Resnick. M. I Roth. R. M. 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L M- Fahrner, K.. Woolford.1..1r.. Rosbch. M. I Kahck. D. (19791Cell13. 1%1-1271. 7A. Maniatis. T.. Fritsch. E. I". I Sarnbrook. 1. (lml)A Manual lrl Genetic Engineering: Molecular Cloning (Cold Spring Harbor Laboratory. Cold Spring Harbor. NY). Hahn. B. I Murray. K. (19771PrxNatlAadeUSA7fi3fi4— 3263 51. john. T. 1’. I Davis. R. W. (1979) Cell 1‘. 443—452. Benton. W. D. I Davis. R. W. (1977) Science 1“. lw-lu Engel. 1. o. s Dodgson. 1. s. (1981) Me. Natl Acad. Sci. USA 73. 2856-2“. Rigby. r. w. 1.. Dieckrnann. M.. Rhodes. c. a Berg. 9. (1977); Mol Biol 11. 237—51. Southern. E. M. (1975) 1. 1401 set as. Sin—517. McMaster. c. K. a Carmidiael. c. A (tmrrucNarlAcstd USA 74, 4835—4838. Thomas. P. S. UM) Proc. Natl Acad. Sci. USA 77. 5201-5205. Kramer. R. A. I Anderson. N. (1980) Proc. NetlAcsd. Sa' USA 77. 6541—6545. Alwine. 1. C.. Kemp. D. 1. I Stark. C. R (1977) Proc. Natl Acad. Sci. USA 75. 53511-5354. Carlson. M. I Botstein. D. (1%21 Cell 33. 145-154. Perleman. D. I Halvorson. H. O. (1%1) Cell 25. 525-515. Zimmerman. C. R.. Orr. W.. Leclerc. R.. Barnard. E. I Tim- berlake. W. (m0) Cell 21. 713-715. .T‘rmberlske. W. E. (180) Dec. Biol 78. 497-510. 99s 999 ewr ~— 2"? 5 ~— 9s» .38 .8 .3338 i3 8 $43.88 1‘ .81?» APPENDIX B APPENDIX B DISRUPTION OF THE SPORULATION-SPECIFIC GENE SPRZ A lambda clone bearing a DNA fragment which contains the sporulation-specific gene SPRZ was among those isolated by the differential hybridization method (Appendix A). Because of its strong signal during the screen it was selected for further study and subcloned into an E.coli plasmid. A large (5.7 kb) fragment was deleted from the original DNA insert and replaced with a selectable marker (URA3) to see if disruption would affect sporulation. The disrupted fragment was transformed into a homothallic wild type strain that was then sporulated and the asci were harvested. Upon tetrad dissection, progeny were found to segregate 2:2 for sporulation- competence. Northern analysis revealed the presence of two transcripts from the region, and the genes were designated SPRZ (SPorulation- Regulated) and HKPZ (HouseKeePing). HKPZ is expressed significantly during vegetative growth as well as during sporulation. A restriction map of the isolated DNA fragment is pictured at the top of Figure 1. Beneath it are wavy lines that represent the expressed messages and their transcriptional orientation. Shown at the bottom of Figure l is the 5.7 kb HindIII-HindIII deletion that was thought, initially, to be responsible for loss of sporulation capability. Four LEUZ disruptions are also pictured. Surprisingly, none of the LEUZ disruptions interfered with 74 75 diploidized. This result led to a further search of the region for other genes, and the sterile gene STE4 was discovered. The location of STEA is shown in Figure 2. The asporogenous phenotype of the strains that are homozygous for the 5.7 kb deletion is due to the disruption of 8184. Without a functional STEé, these strains are sterile (cannot mate) and remain in the haploid state, unable to sporulate. The functions of SPRZ and HKPZ remain unknown. m Bil b“ P‘ HI. b" N -m "fill PM band! N v-Mfl M Dal Dill M P“ ”M Hits. ~Eeel "I! Loans a a a a 4 l <-v~e um: Dora p.‘ W on: ma. “—7 . t" tea as: r_ r L, um: J .‘ can mung. t uxfifl‘ .asux over-ram «- . - - /I__-... -- -‘—\ as [, Lguz :] ' 68mm» ___;‘°“ W W. 5“. _____ 7 .... L,‘ on: ] one -- l - - - -- rm | LEU: 1 Ian“ out to. and Ian“ Figure 1. Gene disruption at the SPRZ locus. EU 76 dill; ill i .3 WWW- Figure 2. 8TE4 SPRZ Location of the STE4 transcript. APPENDIX C PLEASE NOTE: Copyrighted materials in this document have not been filmed at the request of the author. They are available for consultation. however. in the author's university library. These consust of pages: 7 7 - 8 5 UM-I weimhtmmmoum 77 Identification and Characterization of Mutations Affecting Sporulation in Saccharornyces cerevisiae Linda M. Smith,‘ L. G. Robbins,’ A. Kennedy‘ and P. T. Magee'| ‘ Department ofMierobiolag and 'Departaseru onaolog. Michigan State University. East Lansing, Michigan 48824 Manuscript received May 4. 1988 Revised copy accepted August 18. 1988 ABSTRACT Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Sachem-tyres cerevisiae. SCMS'l-l. Two were found. both of which were deficient in sporulation at 34’. One. SL484. sporulated to 50% normal levels at 30' but less than 5% at 34' or 22 '. The other. SL641. failed to sporulate at any temperature. Both mutants were blocked before premeiotic DNA synthesis. and both complemented spol, spas. and spa7. Genetic analysis of the mutation in 81.484 indicated linkage to rm and placed the gene 10 map units from TRPS on chromosome VII. A plasmid containing an insert which complements the mutation in 51.484 fails to complement SL641. We therefore conclude that these two mutations are in separate genes and we propose to call these genes SP017 and SPOIB. These two genes are (with SPO‘I. SPO8. and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators ME and (ME. ACCHAROMYCES cerevisiae normally can grow mi- totically in either the haploid or diploid state. Mating type is conferred by two alleles. the a allele and the a allele. at the mating-type locus. MAT. MATa and MATar haploid strains produce mating phero- mones. a-factor and a-factor respectively. and can mate to form diploid cells of the genotype MATa/ MATo which do not produce pheromones or mate. When incubated in a medium that lacks nitrogen and contains a carbon source that can be respired. such as acetate. MATa/MATar diploids will divide meiotically to give rise to four haploid ascospores. A number of biochemical processes have been observed in sporu- lating cells. including DNA synthesis. RNA and pro- tein synthesis and degradation (Esrosrro and KM- rrrou 1981; Rssmcrt or al. 1984; Werrt~Trrosrrsorv and DAWES 1984; WILLIAMSON et al. 1983). alterations in the synthesis or activity of particular proteins (DAwrs et al. 1983; Esrasrro and Rumors 1981; On 1982; RBNICK et al. 1984). modification of his- tones (Mums and Wm'rmaeacert 1982). synthesis of storage molecules such as lipids and carbohydrates (KAN: and [torn 1974; Honest at al. 1974) and the eventual degradation of glycogen as mature asco- spores appear (Hams! er al. 1974). Some of the biochemical events seen in sporulating cells are also observed in asporogenous cells incubated in sporula- tion medium and are thought to be a response to the starvation conditions that induce sporulation. Other events are specific to sporulation; these include DNA 'Presentaddress: ofGeneticsandCelliology.Univerfly dumumumssroewemwm beaddreued. Genetics txst 999-901 (December. lass) synthesis (Ram and LUSNAR 1970; Rasmcx er al. 1984). RNA degradation (Hone! et al. 1974). syn- thesis of neutral lipids after about 12 hr of incubation (Eseosrro and KLAPHOLZ 1981). synthesis of a spore- surface-specific antigen (DAWES et al. 1983). and gly- cogen degradation (Honest et al. 1974). There are also reports of meiosis-specific increases in the activity of DNA polymerases 1 and 11, two different deoxyri- bonucleases (Rumor et al. 1984). and LBJ-glucon- ases (Du. RAY et al. 1980; HIEN and Fun 1983). as well as a change in the properties of RNA polymerase 11 (Mace: 1974). and a loss of mitochondrial circular RNAs (Scuaoeoex. BREl‘l'ENBACH and Saiwevarv 1983). This complex series of events has recently been shown to depend on two central regulatory genes. RME and 1M5. M5 is a negative regulator of meiosis; mutations in this gene allow haploids or diploids homozygous at the mating type locus to sporulate (Mrrcrreu. and Hensrtowrrz 1986). [MB is a positive regulator which leads to sporulation when present in a high copy number plasmid even when R.ME is pre- sent and active (MAacotsrtee er al. 1986). Both these genes act before premeiotic DNA synthesis. Mutations in three SPO genes have been shown to block cells before premeiotic 5; these mutants are spo7; spo8 and 31:09 (Esposrro and Kumrou 1981). The molecular function of these genes is unknown. COLONNA and Mace: (1978) first described the enzyme responsible for the extensive degradation of glycogen specific to sporulating cells. The glycogen- degrading enzyme. sporulation amyloglucosidase (SAG). appears after 8-10 hr of incubation in spons- TAIL! 1 Genotyp- of S. sensisias strains and in thb pap. Strain Genotype Source SCMS‘M u. (HO la' -1 12 an ode) 11m laboratory SL484 Same as SCMS7-l except spo‘ This study SL641 Same as SCMS7-l except spo' This study 6-3A a/a (HO ara’ lys' ut‘ odex Th'n study upon) XMM-l2b nsstl Killer+srg9 ilv3 aral I. Hzasxowrrz RC757 n ism-I aul use can] on I. Husxowrrz WG6-8A a/n (HO odd-I (cal ups-2 It. Rm nra3-l last4ol lys2-l) 74-IA a/a (HO amt-l :pI-I) 3.1-2. Esrosrro 8940 I/a(HOsrg4-l acct spas-l) R.E.Farosrro C5243 l/a (HO srg4-1 sps7-I) R. L Bruno lation medium. its appearance coincides with the on- set of glycogen degradation and with the first appear- ance of mature spores. The analysis of sporulation mutants (R. E. Esrosrro. personal communication) suggests that the sporulation pathway branches after the initial event of premeiotic DNA synthesis. The event(s) critical for SAG appearance occurs in the pachytene stage of meiosis (CLANCY. SMITH and MA- CE: 1982). and is therefore very close to this decision point. The enzyme. however. is not required for cells to complete sporulation (YAMASIIrI‘A and [Maw 1985). In this study. we have capitalized on SAG's position in the sporulation pathway to identify mutations blocked early in sporulation. Two mutants were iso- lated: SL484. which is temperature-sensitive for SAG production and for sporulation. and SL641. which fails to produce SAC and sporulate under any condi- tions. Spheroplast fusion was used to analyze SL641. MATERIALS AND METHODS Yeast strains: The strains ofS. msvisiu used in these experiments and their genotypes are 'given in Table 1. Media, growth and sporulation 0 cells: Standard yeast media were essentially as described by SHERMAN. Fma and HICKS (1981). For some sporulation experiments. the pre- sporulation (P5P) medium of Rant and HALvoasou (1969) was used. In other experiments. YEPacetate [10 g yeast extract. 10 g peptone. 10 g potassium acetate and 100 ml 0.1 H phthalate buffer (pH 5.2) per liter] was used to adapt cells to growth on acetate. Cells were sporulated in liquid SPM (COLONNA and thou: l978) at the indicated temper- ature after pregrowth in either PSP or YEPacetate as de« scribed previously (CLANCY. SMITH and MAcu 1982). To sporulate cells on solid medium. patches of cells were grown overnight on YEPD. then replica-plated onto PSP and in- cubated 24-48 hr. The patches were then transferred to SPM using toothpicks. and incubated for three days. Preparation of cell extracts: Washed cells were sus- pended at a cell density of 1 x 10’ to 3 X 10’ cells per ml in 0.1 M sodium citrate buffer (pH 6.2). hereafter called citrate buffer. that contained 0.3 mg per ml of the inhibitor phenylmethylsulfonylfluoride (PMSF) and the pro- tease inhibitor aprotinin (Sigma Chemical Co.) at a concen- 78 L. M. Smith st al. tration of 200 KU (kallikreinin units) per ml. Cells were broken by blending in a vortex with glass beads (0.45 mm diameter; 8. Braun Melsugen AG. Germany). The cells were alternately vortexed for 15 sec and cooled on ice for 15 sec until they had been vortexed a total of 3-5 min. Breakage was always greater than 90%. The broken cell suspension was immediately centrifuged at 12.000 X g for 20 min and then at 45,000 rpm for 2 hr in a Beckman type 65 rotor. The supernatant was dialyzed for 16-24 hr at 4' against two changes of citrate buffer. The extract was generally assayed for SAG immediately after dialysis. SAC and protein assays: Qualitative and quantitative assays for SAG were done using a coupled assay system which has been described previously (CLANCY. Surnt and MA“: 1982; COLONNA and MAcu; 1978). For qualitative analyses. a whole cell assay was employed. In these assays. 0.2 ml of citrate buffer containing 0.33 mu p-chloromer- curibenzoate (PCMB) was added to cells permabilized by air drying on Whatman 3MM filter paper (Mowsstowrrz 1976). Duplicate 2.1 cm filters were made in each experi- ment. To one filter. 0.4 ml of a 1% solution of glycogen (Sigma. type II from oyster) in citrate buffer was added. To the second filter. 0.4 ml of citrate buffer was added. The final volume of the reaction mixture for each filter was 0.6 ml. The filters were incubated at 30' or 34' for 18-24 hr and then 1.2 ml of glucose oxidase reagent was added. After 30-60 min of incubation at 30'. 0.8 ml of concentrated HCl was added and the reaction mix vortexed immediately. The duplicate filters were compared. and SAG activity was indicated by formation of greater color by the cells incu- bated with glycogen. For quantitative determinations. aliquots of a cell extract (0.05—0J ml per 0.6 ml total reaction volume) were incu- bated at 22'. 30'. or 34‘ in an assay mixture containing citrate buffer. 0.66% glycogen and 0.33 mil PCMB. At various times. 0.6 ml samples were removed and boiled for 10 min. The samples were centrifuged and the amount of glucose present in the supernatant was determined using glucose oxidase reagent (COLONNA and MAcu 1978). D- Glucose was used as a standard. Specific activity was ex- pressed in milliunits r mg of protein. where one unit is defined as 1 mmol 0 glucose released per minute. Protein was determined by the method of Lowav st al. (1951). Isolation and mutagenesis of spores: A 300-m1 culture was sporulated and the asci harvested by centrifugation. The asci were resuspended in 10 ml of 24 mu phosphate buffer (pH 7.0) containing 20 ul of 2-mercaptoethanol and 15 mg Zymolyase 60.000 (Kirin 8rewery.japan). The digese tion mixture was incubated at 30‘ with gentle shaking for 1 hr. The spores were harvested by centrifugation and washed three times with sterile distilled water. Aggregates of spores were disrupted by sonicating the spore suspension for three 30-sec bursts. The spores were washed two times in a sterile 1% solution of Tween 80 in distilled water. then finally washed two times in sterile distilled water and plated onto YEPD at a concentration of 1000-2000 spores per plate. The spores were mutagenized by UV irradiation for 60 sec (10% survival). and incubated in the dark at 34'. The surviving ascospore colonies that were to be screened for SAG activity were inoculated onto solid YEPD. incu- bated overnight. then replicated twice to premoistened 3MM Whatman filter paper lying atop plates of P5P con- taining histidine. leucine and adenine. After two days of incubation. the ascospore colonies growing on the filters were shifted to SPM by simply transferring the filter to leucine-supplemented SPM plates. The colonies were incu- bated in SPM for 3 days. then the filters were removed and air dried for 4-5 hr at room temperature. The individual Sporulation Mutants in Yeast 901 colonies were separated and assayed using the whole cel assay for SAG. . Test for pheromone production: The ability to produce mating pheromones was determined using the procedure of erAcus and stsxowrrz (1981). Patches of cells to be tested were replica-plated onto 88MB plates spread with either XMB4-12b or RC757. Nuclear staining and measurement of DNA synthesis Progress through meiosis was monitored cytologically with the fluorescent dye 4.6-diamidino-2-phenylindole (DAPI) as described previously (CLANCY. SMITII and IMAGE: 1982). DNA synthesis was measured by collecting triplicate. one ml samples of cells incubated in SPM at the indicated times. The cells were frozen and stored until DNA content was assayed using the fluorescent compound 3.5-diaminoben- zoic acid (DABA) (Hunt. LINDNu and Rams I975). Calf thymus DNA was used as a standard. Spheroplast fusion: A modification of the procedure used by KAItAIt and MAcu'.(1982) was used. A lO-ml culture of cells was grown overnight in YEPD at 30'. then diluted with 15 ml fresh YEPD and incubated for 1.5-3 hr to allow the cells to reenter log phase growth. Approximately I x 10’ to 3 x 10’ cells were harvested. washed once in distilled water. once in sorbitol-phosphate [l M sorbitol. 0.1 It potase sium phosphate (pH 7.5)] and then resuspended in sorbitol- phosphate containing 5 mg Zymolyase 5.000 (Kirin Brew. ery. japan) and 12.5 ul 2-mercaptoethanol. The cells were incubated at room temperature in the spheroplasting mix until greater than 90% of the cells were converted to spher- oplasts. This was determined by checking the degree of lysis in either 5% sodium dodecyl sulfate or distilled water. The spheroplasts were washed five times with sorbitol phosphate by centrifugation for 10 min at approximately 700 x g. All subsequent ccntrifugations were done in this manner. Spheroplasts to be fused were mixed in a 1:1 ratio. Gener- ally. between 1 x 10' and 5 x 10' spheroplasts ofeach strain were used. The mixed spheroplasts were incubated for 20 min at room temperature. then centrifuged and resuse pended in five m1 of fusion mix [30% (wt/vol) polyethylene glycol 4.000. 10 Inst CaCls. and 10 Inst Tris (pH 7.5)]. The spheroplasts were incubated in the fusion mix for 1 hr at room temperature and then stored overnight at 4'. Prior to being plated. the spheroplasts were centrifuged. resus- pended and incubated for 20 min at room temperature in 5 m1 SOS. a recovery broth (SHHMAN. PINK and HICKS 1981). They were then centrifuged. resuspended in I ml of sorbitol-phosphate and either 0.01 or 0.1 ml of spheroplasts was mixed with 5 ml molten minimal agar medium contain- ing 1 M sorbitol (MIN-sorbitol) held at 50'. then poured immediately onto M lN-sorbitol plates. The plates were in- cubated at 30’ for 4-10 days. Prototrophic colonies were streaked onto MIN plates and in some cases transferred to YEPD plates where they were stored at 4' until needed. Segregation analysis of mutants: Strains heterozygous for the sporulation defect were constructed by either spore to-spore matings or spheroplast fusions. The heterozygotes were sporulated and the tetrads dissected by micromanipu- lation. Nutritional requirements were determined by rep- lica-plating from YEPD master plates to various nutrient drop-out plates. The SAC phenotype and the ability to sporulate were determined in most experiments by incubat- ing IO-ml cultures at either 30' or 34' in SPM after pregrowth in YEPacetate. Cultures were examined micro- scopically to ascertain the percent sporulation and SA was assayed using the whole cell assay by spotting 50 ul of cell suspension on 3MM Whatman filter paper circles (2.3 cm) and air drying for 2-4 hr. In some experiments. only the percentsporulationwasdeterminedaftercellsweresporu- lated on solid SPM. Complementation tests: Strains heterozygous for two different sporulation defects were constructed by either spore~to-spore matings or fusions. These strains were sporulated in lO-ml cultures at 25'. 30" or 34'. The ability to sporulate and the percent sporulation was deter- mined. SAG was assayed using the whole cell assay after spotting cells onto 3MM Whatman filter paper circles and air drying as described above. Transformation: Yeast transformation was carried out as described by Hmnan. Htcxs and Flux (1978). RESULTS Mutagenesis: Homothallic strains can be used to isolate recessive and dominant mutations in genes that function only (in diploids (Esposrro and 55mm 1969). Through the action of the homothallism allele. H0, any mutation generated by mutagenizing the haploid ascospores of the homothallic strain will be present in a homozygous condition in the diploid colony that arises from each spore. Ascospores from the homothallic strain SCMS7-l were mutagenized by UV irradiation. The 2528 sur- viving ascospore colonies were prescreened in two ways. First. the ability of the survivors to grow in MIN medium that contained histidine. leucine. and adenine was tested. to eliminate nutritional defects other than those present in the parent strain. Second. the colonies were tested for their ability to grow on YPG. a me- dium that is commonly used to determine respiratory competence (SHERMAN. FINIt and HICIts 1981). since cells must be able to respire in order to sporulate. A total of 2168 colonies were identified by these two criteria for screening for SAG activity. Screen for SAG activity: Duplicates of ach colony were sporulated on 3MM Whatman filter paper. then air dried and assayed for SAG. Included were MATa/ MA Ta (positive) and MA Tti/MATa (negative) controls. F or each ascospore colony tested. one of the duplicates was assayed in a reaction mixture containing glycogen. the substrate for SAC. and the other was assayed in a reaction mixture lacking glycogen. If a visual compar- ison showed that the duplicate assayed with glycogen produced more color than the duplicate assayed with- out glycogen. the colony was scored as positive for SAG. If the duplicates had an equal amount of color. the colony was scored as negative for SAG. Using this screening procedure. 296 putative SAG-minus colo- nies were identified. One hundred forty-one of these were retested for SAG activity in liquid culture at 34' using the whole cell assay. Twenty-one colonies were deficient for SAC and were characterized further. Pheromone production: The screening procedure used in this study asked only whether or not the cells produced SAG. Thus. in addition to the expected identification of defects in early sporulation functions and in the structural and regulatory sequences for 902 TAILB 2 SpedflcaetlvityofSAGinmutantstrainsandintheirpasent Specil'tcactivityfollowingtimeinqroruhtionmeditml (1"): Strain o 24 4s 72 SL484 0.170 34‘ 1.13 2.52 8.34 30' 3.22 19.9 35.1 22' 0.35 0.56 0.81 SL641 0.138 34' 1.06 1.10 1.99 30‘ 0.157‘ 1.04‘ 1.81' 22' 0.531‘ 0.267“ 0.312“ SCMS7-1 0.415 34’ 48.6 47.8 46.2 ‘ 30‘ 28.0 28.6 27.4 22' 54.8‘ 88.2‘ 111‘ Cells were incubated in SPM at the temperature indicated after pregrowth in PSP. ‘ Resqu of a single experiment. SAG. mutants defective in H0. MAT. and mating functions might also be isolated. Such nonsporulation- specific mutants can be differentiated from the spor- ulation mutants based on the production of mating pheromones and on ploidy. Of the 21 SAG-minus colonies identified after re- testing for SAG activity in liquid cultures. two. SL484 and $1.64 1 . did not produce either mating pheromone and were characterized further. The ploidy of these strains will be considered later. Specific activity of SAG in mutant strains: SL484. SL641 and their parent were incubated at 22'. 30° or 34‘ in SPM. At the indicated times. samples of cells were harvested and used to determine the specific activity of SAG (Table 2). Unless otherwise indicated. the results of two or more experiments are expressed as the mean specific activity. Most of the extracts were assayed at all three temperatures. No significant dif- ference was found between the different assay tem- peratures and only the results obtained when the extracts were assayed at 30" are presented. When incubated in SPM at 22° and 34'. SL484 had, at the most. one-fifth the specific activity of the parent strain, SCM7-I. However. at 30’ . and after 72 hr in SPM. SL484 exhibited wild type levels of SAG. The only difference between SL484 and SCMS7-l at 30' was that SCMS7-l attained full activity by 24 hr and changed little thereafter. SL484. on the other hand, did not reach maximal activity until after ap- proximately 48 hours of incubation. Thus. SL484 is temperature sensitive for SAG production. with 30' being the permissive temperature. SL641 exhibited approximately the same activity at all temperatures and at all incubation times. Its specific activity varied from 1/15 to 1/200 of wild-type activity. Thus. SL641 is not temperature sensitive for SAG production. Sporulation and meiotic behavior of the mutants: The ability of the mutant strains to sporulate is shown 80 L. M. Smith st al. TABLES Sporulationabllityofmutantsandtbeirparent Percent sporulation at fol- lowurg time in SPM (hr): Strain Temperature 24 48 72 SL484 34' 0.3 1.3 2.3 30' 3.9 21.5 38.7 22' 0' 0.6 1.3 SL641 34‘ 0 0 0 30' 0 0 0 22' 0 0 0 SCMS'l-l 34' 35.1 47.1 46.8 30' 38.2 67.9 69.4 22' 44.4‘ 72.0‘ 73.6‘ Cells were incubated in SPM at the indicated temperature after pregrowth in PSP. Unless otherwise noted. the values presented aretheaverageofatleasttwodeterminatioru. ‘ Resqu of a single experiment. TABLE 4 Progre- of the mutants through meiosis Strain Hours in Percent SPM celh SL484 SL641 SCMS'l-l 24 Motto 99.0 99.0 21.0 Bi 1.0 0.3 20.3 Tetra 0 0.7 25.2 Asci 0 0 33.4 48 Motto 99.3 97.3 33.9 Bi 0.7 2.0 4.6 Tetra 0 0.7 4.3 Asci 0 0 57.2 72 Mono 100 89.7 20.6 Bi 0 7.9 6.0 Tetra 0 2.3 12.3 Asci 0 0 61.1 Cells were incubated in SPM at 34' after pregrowth in PSP at 30'. in Table 3. The results presented are the average of at least two determinations. SL641 failed to sporulate at all three temperatures. SL484 sporulated very poorly at 22° and 34°. but. paralleling the SAG activity. it sporulated at nearly wild-type levels at 30'. The progression of the mutants along the meiotic pathway was also examined. The nuclei of the cells were stained with the fluorescent stain DAPI which binds DNA and renders the nuclei visible when the . cells are observed by fluorescence microscopy. The results of one experiment are shown in Table 4. For both mutants. most cells remained mononucleate. For SL641. the small percentage of cells (8%) that appears to progress through one or both nuclear divisions should be regarded with caution. Mitochondrial DNA is also stained by DAPI and we have observed that when cells are incubated in SPM but are unable to sporulate. large vacuoles are formed and the mito- Sporulation Mutants in Yeast 10* . O .2 I.s+ a \ C ... m I\ .1 O .J ta) 0 fi 0 x 4 Z O o 1 h-- I 0.. V V V V V V V r V I Tfi 1”. V V 2 4 6 8 IO 12 24 TIME (H) Home 1.—Premeiotic DNA synthesis in mutant strains and their parent. Cells were pregrown in PSP then incubated at 34' in SPM. At the indicated times. loml samples of cells were removed and frozen until assayed for DNA content. DNA content 'n ex- pressed as the ratio of DNA at each time point (Tx) to DNA at time zero (To). Premeiotic DNA synthesis in the parent SCMS'l-l (O—O). and the mutants SL484 (0—0) and SL641 H chondria are concentrated in a small area in the cell. Under such conditions, it is sometimes difficult to distinguish a group of mitochondria from the nucleus. Thus. these results are best interpreted as an indica- tion that neither of the mutants progressed through the first meiotic division. Premeiotic DNA synthesis: The occurrence of pre- meiotic synthesis in SL484 and SL641 was deter- mined. Cells incubated in SPM at 34' were sampled after various times of incubation. and the DNA con- tent of the cells measured using the fluorescent com- pound DABA. The results are shown in Figure l. The parent strain began synthesizing DNA almost immediately. and completed the synthesis within six hours of incubation. During that time. the DNA con- tent almost doubled. The failure to double completely the DNA content reflects the fraction of cells that failed to enter the meiotic pathway. In contrast. nei- ther SL484 nor SL641 synthesized DNA. The DNA content of the strains at zero time was 0.301. 0.446. and 0.286 g of DNA per 1 x 10' cells for SL484. SL641. and SCMS7-1. respectively. These values are within 72-112% of the values reported for diploid S. cerevisior strains. using the same and other methods‘ of measurement (LAUER. Rosters AND Kwrz 1977; than. Kiss and Fumczv 1978). Segregation of tbe SL484 defect: Strains heterozy- 81 903 TABLE 5 Linkage analy‘ of SL484 defect Linkage to: '17 NH? 11‘ HQ W H184 4 3 8 Unlinked URA3 8 7 10 Unlinked MET 4 2 7 15 Unlinked LYS2 4 4 l4 Unlinked TRP5 20 0 5 10 m.u. ' PD ,is parental ditype ascus. ' NPD is nonparental ditype acus. ‘ TT is tetratype ascus. ‘ Mapdistance is the recombination frequency times 100. gous for the SL484 defect were constructed by spore- to-spore matings of SL484 and the wild type strain W66-8A. The diploids produced by such matings are heterozygous at a number of loci. All ascospores pro- duced by SL484/W66-8a diploids contain the HO allele and diploidize. Therefore. each ascospore col~ any can be tested for its ability to sporulate and produce SAG. SL484/W66-Sa diploids were sporu- lated and the tetrads characterized for nutritional genetic markers and for their ability so sporulate after incubation on SPM plates for three days at 34'. SAG activity was not assayed. All phenotypes. including the ability to sporulate segregated 2+:2-. These results indicate that the sporulation deficiency in SL484 is the result of a defect in a single gene. Linkage of the SL484 defect to H184. URA3. MET4. LYS2 and TRP5 was tested. The requirements for leucine and adenine were not studied. since two genes were segregating for each phenotype and these genes are not directly distinguishable in the progeny. The numbers of parental ditype. nonparental ditype. and tetratype asci are shown in Table 5. Only tetrads showing 2+:2- segregation for each gene were con- sidered. For the ”[84. URA3. MET 4 and LYS2 genes. the ratio of parental ditypes to nonparental ditypes was approximately 1:1 (by x' analyses) indicating that these genes are not linked to the SL484 defect. How- ever. with the TRP5 gene. most of the tetrads were parental ditypes suggesting that the two genes are linked and 10 map units apart on chromosome VII. We call the gene affected by mutation in SL484 SP017. Ploidy of the mutants: The DNA contents of SL484 and SL641 suggest that both are diploids. The genetic analyses of the mutants also support this con- clusion. SL484 sporulated at 30' . and its spores mated to give rise to diploids that segregated various genetic markers in a normal diploid fashion. Since SL641 was unable to sporulate. genetic analysis was undertaken by fusing spheroplasts of SL641 and W66-8A (SMITH 1985). The normal tetraploid segregation observed for the SAG-deficient phenotype in the SL641/W66- 8a derived fusant is consistent with the conclusion that 904 L. M. Smith st of. SL641 is diploid. The viabilities of spores produced by the SL641 fusant also support this conclusion. If SL641 were haploid. fusion with W66-8A would have created a triploid strain. Sporulation of triploid yeast strains generally yields less than 15% viable spores (PARRY and Cox 1970) while tetraploids exhibit good spore viability. If SL641 were diploid. then the fusion product would be tetraploid. The SL641 derived- fusant exhibited grater than 50% spore viability. The segregation of sporulation ability from the SL641/ W66-8a fusant suggested that the SL641 defect was due to a single mutation. When compared in pairs with each of three markers from W66o8a. 1,32. ura3, and his4. the sporulation defect segregated as a single gene. in that the numbers of the type I. II. and III asci were not significantly different from the expected values (data not shown) (ROMAN, PHILLIPS and 8mm 1955). Furthermore. trp5 segregated aberrantly in the fusion examined. suggesting that the fusion was aneuploid for chromosome VII. Since the defect sege rcgated normally. while the chromosome in which spol 7 is located did not. it seems highly unlikely that the two mutations affect the same gene (see below). We are therefore calling the affected gene SP018. _ Complementation tests: Three strains. C52-4B. 74- 1A and 89-10. which are defective in premeiotic DNA synthesis (spo7). the first meiotic division (spol). and spore formation (spo3). respectively. were available in this laboratory for complementation tests. It was pre- viously determined that at 34'. only spa? mutants do not exhibit SAG activity (Custer. SMITH and MA“: 1982) (data not shown). Thus. though spal and spe3 mutants fail to sporulate. they progress far enough along the sporulation pathway to produce SAG. We asked then, whether the spa mutants would comple- ment the SAG and/or sporulation deficient phenotype of SL484 and SL641. trans-Heterozygotes of spo7 and SL484. and sp03 and SL484 were constructed by spore-to-spore matings. A tetraploid heterozygous for both the spol and the SL484 defects was constructed by fusing spheroplasts of the two strains. trans-Het- erozygotes of SL641 and the three sporulation mu. tants were also constructed by spheroplast fusion. Ten-milliliter cultures of the heterozygotes were in- cubated at 34° and at the permissive temperature. either 25' or 30'. in SPM and examined for SAG activity using the whole cell assay for SAG. The results are shown in Table 6. All six heterozygotes produced SAC and sporulated to the same magnitude at both the restrictive and permissive temperatures. Although the percent sporulation was low for the SL484/spol, SL641/spo7 and SL641/spol fusants. none of the parents sporulate to this degree at 34'. It is important to note that the spo7 strain. C52-4B. used in this study is unconditionally asporogenous in our heads. This may explain the low level of sporulation in the heter- TABLI 6 wmsuueusmr napal.sps3.and spa? meal- W Sporulation defect % Sporuhtion SAG Activity SL484 x ...r DNA synthes'u 35.1 4» sun It epr 16.3 4- 51.484 x epl Meiosis 1 17.0 +‘ sum x spl 4.2' +' SL484 x spe3‘ Spore formation 50.5 + 81.541 x spa: 55.1 + ‘CrossesofSL484 weredoneassporevspore matings. Croasesof SL641 were doneasspheroplast fusions. Unlessotherwise noted.a culture of cells growing in YEPacetate was shifted into SPM and half of the cuhure was incubated at 34° and the other half was incubated at the permissive temperature. either 25' or 30'. After 48 h of incuhtion. the cultures were examined for lation and SAG was assayed using the whole cell assay descri in autumn AND um All cultures sporulated and produced SAG at the permissive tenmerature. 'Cells were pregrownin PSPthenshifted intoSPMandincu- bated as above. ‘ Results after 24 hrofincubation. ozygotes. These results indicate that the SL484 and the SL641 mutations are probably not in the SPOI. SP03 and SPO7 genes. although we cannot rule out intragenic complementation. SL484 and SL641 contain different mutations: Given their similar phenotypes. it was important to show that SL484 and 81.641 contained different mu- tations. Tetraploid segregation data from SL641/ W66-8a fusants (not shown) suggested that the genes had different map positions. but explanations for these results. such as chromosome loss upon fusion. could not be ruled out. Since SL641 will not sporulate under any tested conditions. we used the technique of spher- oplast fusion to test for complementation between the two mutants. 1f the two mutations were in the same gene. the fusants would be sporulation-defective at temperatures nonpermissive for SL484. and sporula- tion 'at an SL484-permissive temperature should yield tetrads containing only SPO' spores. A derivative of the cross SL484 x W66-8A. strain 6-3A. which was urn3 lys2 Iart4 odeX spol7 HO. was fused with SL641 as described in Materials and Meth- ods. The yield was low; only three prototrophs were obtained. Each of these failed to sporulate at 34' suggesting absence of complementation. but the fu- sants were aberrant; DNA measurements showed that each population was highly heterogeneous in DNA content. with value ranging from triploid to hexap- loid. This variability was not diminished by continued recloning of the strains. The fusants did sporulate at permissive temperature. and 7 asci were dissected. Only one of these gave 4 viable spores. The results for all are shown in Table 7. Although neither the parents of the fusant nor the fusant were able to sporulate at 34'. many of the progeny were SPO‘ at Sporulation Mutants in Yeast 905 TAILI‘I Sporulusanol-eiotleproganyfra-afnsantofSWIXHA Sporulation M O E I. O a in" +i+++ +l+ +++ +1- + (all 2 spored) gig-Vactng-rnuvh‘c |++ll l+++l s. time» tr ++ ++ The fusant was sporulated at 50' and 7 tetrads were dissected. All spores were tested for sporulation (but not SAG production) at 30' and 34'. Only tetrad 7 gave 4 viable spores. TAIL! I Sporulationofrpdandrpallallatransfor-edwitbpla-ib containing a patadveSN" gene Time in pneu- latinn medium ...—.— No. ofea- Strain Temperature 24 hr 48 h periments SL ‘84 30’ GP 59 3 34' l l 9 SL MI 30' 0 0 4 34° 0 0 ‘ Figures are 5 sporulation. Strains were transformed and spor- ulated as described in sinuous mo stations. The fragment containing the putative spsl7 gene was carried as a 2st plasmid containing LED? as a selectable yeast marker. both 30' and 34'. We interpret this to mean that the mutations in SL641 and SL484 are not allelic. al- though we cannot explain the behavior of the original fusants. Auxotrophic marker segregation was normal. that is followed expectations for segregation for a tetraploid. in the spores. We have recently cloned SPOI? (A. KENNEDY and P. T. Masts. unpublished data) and have shown that a plasmid containing an insert which fully complements SL484 does not come plement SL641 (Table 8). We therefore conclude that SL641 contains a mutation in a gene different from SPOl7. DISCUSSION We have described the isolation and characteriza- tion of two SAG-deficient mutants of S. cerevisiae. SL484 and SL641. SL484 and SL641 had similar phenotypes. Neither synthesized DNA. completed meiosis l. or formed asci when incubated in sporula- tion medium at 34'. 51.484 differed from SL641 by being temperature-sensitive for both sporulation and SAG production. The 2+:2- segregation pattern of the sporulation- defective phenotype in cells heterozygous for the SL484 defect shows that the phenotype is caused by a mutation in a single gene. We call this gene SPOI 7. The mutation lies l0 map units from the TRP5 gene on chromosome VII. SL641 is a nonconditional mu- tant so genetic analyses have been done using tetra- ploid strains constructed by fusing spheroplasts of SL6“ with spheroplasts of other homothallic diploid strains. Preliminary results indicate that the SL64] phenotype is also the result of a defect in a single gene and that this gene is different from SP017; hence we are calling this gene SPOIB. We cannot rule out at this time the possibility that the mutation in SL641 is in SP07 and that the sporulation of the heterozygote was due to intragenic complementation. Spheroplast fusions between a spore from the SL484/W66-8a cross and SL641 gave ambiguous re- sults. A fusant heterozygous for both the spa” and spa18 defects failed to sporulate at 34'; however. interpretation of negative results in such a comple- mentation test is not straightforward. It has been our experience that tetraploids constructed by fusing two MATa/MATa diploids are often aneuploid (Sun's! I985). Thus. even if the two mutations were in differ- ent complementation groups. the fusant derived in this study could be trisomic for one of the spa-bearing chromosomes. for example spal7/spol7/+. and spal8/spal8/+/+ for the other mutation. Then the heterozygote might fail to sporulate at 34' beause of dosage effects at the SPOl7 locus. If such a heterozy- gote sporulated at 30° (as does the spa l 7 parent). then it would segregate progeny that were able to sporulate at the nonpermissive temperature. We observed that the SL484/SL64l-derived fusant was able to sporu- late at 30' and that it segregated progeny that spor- ulated at 34°. The fact that only l/7 asci yielded 4 viable spores is consistent with the aneuploid hypoth~ esis. Another explanation for the behavior of the SP017/SL641 fusant is that the two mutations are in fact different alleles of the same gene and that the sporulation-competent segregants were produced by intragenic recombination or complementation which restored the wild type gene. We feel that this is not the case. however. for two reasons. The first is that the frequency of sporulation-competent segregants 906 was too high. The second is that a study on the segregation of chromosomes in a fusant produced by SL641 and W66-8A showed that chromosome VII (the SP017-bearing chromosome) missegregated while the SL641 defect segregated normally. This suggests that the SL641 mutation is not located on chromosome VII (SMITH 1985). Finally. complemen- tation tests with the cloned SP017 gene indicate that the SL641 defect is not in SP017. The SAG gene has recently been cloned on the basis of its homology to the starch-degrading amylo- glucosidase of S. diastolicus. It has been shown to be transcriptionally controlled during sporulation. How- ever. it is dispensable for sporulation in that diploids homozygous for a disruption in the gene still sporulate normally (YAMASHITA and FURUI 1985). In this it resembles some other genes which are transcribed exclusively or mainly during sporulation but which are dispensable for the developmental process (D. Pamuumo. A. KENNEDY and P. T. Mscu. unpub- lished data; M. J. CLANCY personal communication; COTI'UN-NINFA and Ritual: 1986; PERCIVAL-SMITH and SEGALI. 1986). Since neither mutant discussed here sporulate: at 34° and since both are blocked early. it seems unlikely that their defects are in the structural gene for SAG. Furthermore. both grow normally at the temperatures tested: therefore. they are not defective in cell-cycle genes. It is probable then that both are defective in some early sporulation function. probably one which functions before DNA synthesis. This would lead. of course. to a failure in recombination-competence. and the ability to form various meiotic structures such as spindle pole bodies and synaptonemal complexes (DAVIDOW and BYERS I984; Brostro and Kuumou 1981). With the ex- ception of spo7. which was complemented by both SL484 and SL641. the relationship to other early functioning genes has not been examined. Strains carrying mutations in SP08 and SPO9 are not available at this time. so complementation tests could not be performed to establish the relationship between SL484 and SL641 and these genes. Since the mutation in SL484 maps on chromosome VII. while [ME was found to be located on chromosome X (Mut- couxu ct al. 1986). these 2 genes are not identical. We cannot rule out the identity of the gene altered in SL641 with (ME. Characterization of SP017 and SP018 is likely to help to sort out the early steps in the transition from mitotic to meiotic growth in a/a S. cerevisiae. How- ever. the functions of the gene products defined by the mutations in SL484 and SL641 are not clear to us at this time. Both mutations block cells before DNA synthesis; thus. along with spo7. spa8. and sp09. they are among the earliest spa mutations isolated. The early part of the pathway to meiosis and sporulation 84 L. M. Smith ct al. has not been well studied. but since these mutations (or at least these alleles of them) do not affect vege- tative growth. one might assume that they are after RME and [MB or that SP018 is allelic with the latter. If they are farther down the pathway. they may en- code protein factors that are either involved in pre- meiotic DNA synthesis or that regulate the entry of cells into that part of the developmental pathway. An alternative role for one or both of these genes is in the regulatory pathway that senses the state of starvation of the cells. No genes analogous to RMEI or [ME have been identified for this pathway. but the fact that rural mutations do not bypass nutritional control suggests that there is a separate regulatory network operating. Tests of epistasis with mutations known to overide this control [ras2-val (KATAORA. :1 al. 1984). and qu (Mnsuuoro. Una and ISI'IIKAWA 1983)] will give some insight into whether SP017 and SP018 are a part of that pathway. This work was supported by US. Public Halth Service Grant GMSSSOS. We are grateful to Professor Mary Clancy for many helpful discussions. This is jourml Article No. 12122 from the Michigan Agricultural Experiment Station. LITERATURE CITED Cumcv. M. 1.. L. M. Sums and P. T. Milan. 1982 Develo opmental regulation of a sporulation-specific enzyme activity in SaccAaroatyccs cerevisiae. Mol. Cell. Biol. 2: I7l-I78. GOIDNNA. W. 1.. and P. T. MAG". I978 Glycogenolytic enzymes in sporulating yeast. j. Bacteriol. 134: 844-853. Davroow. L. S.. and 8. 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Yastasurra. 1.. and S. Fuxur. 1985 Transcriptional controlof the sporulationapecilic glucoamybse gene in the yeast SeccAere- nyces cerevisiae. Mol. Cell. Biol. 5: 3069-3073. Communicating editor: D. B01311"! APPENDIX D APPENDIX D SP017, ADDITIONAL DATA I. Productia of red 3 i lae in s o la sformants. Figures 1 and 2 are photographs of plates that were inoculated with the various strains used in this work and subjected to identical sporulation conditions, but at different temperatures, as described in Chapter II. Several observations can be made about the plate experiments: a) Strain R92W consistently failed to produce red papillae at both the permissive and nonpermissive temperatures. It did, however, sporulate in liquid cultures at the permissive temperature. The parent strain SL484 is a rather poor sporulator at the permissive temperature. Thus, R92W, without an intact copy of SP017, may be incapable of sporulating on plates. b) In the initial screen, transformants producing red papillae consistently grew better after exposure to sporulation conditions at 34°C than those that failed to produce red papillae. This suggested that SP017 might be involved in nutritional regulation. The difference in growth is demonstrated quite clearly in 98FOA-1, 2, and 3, strains that were generated by plating transformant 98, which carried a SP017 plasmid, on media containing 5-f1uaro-arotic acid. The surviving 86 87 strains, having lost the URA3 plasmid, were sporulation-deficient, failed to produce red papillae, and exhibited poor growth on rich media after exposure to sporulation at 34°C (Figure 2, colonies 15, 16 and 17). c) Strain SL484 consistently gave good vegetative growth after exposure to sporulation conditions at both 30°and 34°C. d) R92W usually failed to grow well after exposure to sporulation conditions at 34°C, but it appears to have grown well in the particular experiment pictured in Figures 1 and 2. e) Some strains (eg., transformants 98 and 208) were not consistent in the production of red papillae in the plate experiments. This is probably due to a variation in sporulation efficiency among experiments, as is frequently observed even in carefully controlled liquid experiments. f) The colonies at positions 25-29 are transformants containing the multicopy plasmid pEMBB. These strains were exceptionally consistent in growth and in the production of red papillae. II. LacZ expression in gmel 32012 double mutants. RMEI expression was evaluated in sp017 mutants and the results are shown in Figure 3. The rmel strains (obtained from Aaron Mitchell) were disrupted by inserting the LacZ gene (in-frame) into the RMEI gene. The haploid strain 2210 (a rme1::LacZ) turns blue when grown on media containing X-gal (not 88 pictured). Haploid strain 2210-3 (a rme1::LacZ spol7::LEU2) is pictured on the left in Figure 3. Even though this strain is spol7, the colonies remain blue on X-gal medium. Thus, RMEI is expressed normally in spol7 haploids. Strain 221a3 is the product of a mating between two rme1::LacZ spol7::LEU2 haploids, and is pictured in Figure 3. These colonies remain white on X-gal medium, thus implying that normal repression of RMEI occurs in spol7 diploids. 89 H wusmflm 90 «Has _.¢H_ ecu” .ma. ”mm .«H anzunxsuma .m~.m~ hm ,HH ace ,cu oee .c~ anau, .nu as .m omou ,NN as .o mow AJaN m .A com .ou.‘. “seam .o ~a-o¢mH .mfi «aqua .m Ha-~H¢H ..mH . swam «a n-4cmma "NH as .m «-4oaoa.. ea ... n-5m20m .N H-.ou voudHn.oonmouwco u «no! mouauasu may :.o.om um whou.n you omuundoau.wco noodau «mmuov no.mxo«n£uoou. onuuoua no“? wouauaaoasu one} newcoaoo .0.0m um acouuuvaoo coaumazuonu. ou ouauonxo nouns «cannon Houcoawuoaxo «nodua> mo savouo .H ousmwm 91 92 mama .ea ases .MH. “mm .- nnzua\s~au .a~-m~ an .HH ace .qu as; .oH cmau .mN as Is snow; .mu «m .u ecu .HN o ..5 so“. .om Hess» .c «s-o¢aa. .ad. «aqua. .m ~a-~HaH. .as . swam .e n- mo nujbuu ..u ousmqm 93 Figure 3 94 .mmHNN An ecu m-oH- an no“: umxmouum was How-x am no H3 COH fiuws wouMHSOOGw whoa mouoaa 9mm» maowhuoaon «Noam m can vaoaaon Nwomm m aw cowmmouaxo ~m2N .ufioaaov .m ouswam REFERENCES REFERENCES Beggs, J.D., J. van den Berg, A. van Ooyen and C. 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