Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae (original) (raw)
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Isolation of DNA Sequences Preferentially Expressed During Sporulation in Saccharomyces cerevisiae
Molecular and Cellular Biology, 1984
A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating aa cells and asporogenous aao cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the aa sporulation probe relative to the aa control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, aa, and aa cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified. Transcripts complementary to these genes are present only in aa cells after transfer to sporulation medium. Three of these clones contain two sporulation-specific genes. Three genes have been identified that are expressed in all cell types during vegetative growth and only in aa cells in sporulation medium. * Corresponding author. screened a yeast DNA library for genes expressed preferentially in MATa/MATot cells during sporulation. In this communication we report the identification of 14 sporulationspecific genes. MATERIALS AND METHODS Strains and culture conditions. Isogenic diploids of the S. cerevisiae strain AP-3 differing only at the MAT loci (MATa/ MA Ta, MA Ta/MA To, MA ToIMA Ta) were used throughout this study. The AP-3 genotype is adell+ ade2lade2 galll+ tyrl/+ lys2I+ his7/+ urall+ +Iura3 +/canl +Icyh2 +/leul CSP+ICSP+. These strains were derived by Hopper et al. (12, 13) and provided by J. Haber. Cells were grown
Molecular and Cellular Biology, 1986
A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was fo...
Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae
Molecular and cellular biology, 1982
An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.
Functional analysis of the sporulation-specific SPR6 gene of Saccharomyces cerevisiae
Current Genetics, 1990
The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation. The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size. This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused to SPR6 in the expected reading frame. SPR6 is inessential for sporulation; mutants that lack SPR6 activity sporulate normally and produce viable ascospores. Nonetheless, the SPR6 gene encodes a function that is relevant to sporulating cells; the wild-type allele can enhance sporulation in strains that are defective for several SPR functions. SPR6 is located on chromosome V, 14.4 centimorgans centromere-distal to MET6.
Molecular and Cellular Biology, 1987
We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta...
Identification and characterization of mutations affecting sporulation in Saccharomyces cerevisiae
Genetics, 1988
Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Saccharomyces cerevisiae, SCMS7-1. 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, SL64 1 , failed to sporulate at any temperature. Both mutants were blocked before premeiotic DNA synthesis, and both complemented spol, spo3, and spo7. Genetic analysis of the mutation in SL484 indicated linkage to TRP5 and placed the gene 10 map units from TRP5 on chromosome VII. A plasmid containing an insert which complements the mutation in SL484 fails to complement SL641. We therefore conclude that these two mutations are in separate genes and we propose to call these genes SPO17 and SPO18. These two genes are (with SP07, SP08, and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators RME and IME.
Messenger ribonucleic acid and protein metabolism during sporulation of Saccharomyces cerevisiae
Journal of Bacteriology
To investigate differences between growing yeasts and those undergoing sporulation, we compared several parameters of messenger ribonucleic acid (RNA) transcription and translation. The general properties of messenger RNA metabolism were not significantly altered by the starvation conditions accompanying sporulation. The average messenger RNA half-life, calculated from the kinetics of incorporation of [3H]adenine into polyadenylic acid-containing RNA, was 20 min in both cell populations. Furthermore, 1.3 to 1.4% of the total RNA was adenylated in both growing and sporulating cells. However, the proportion of RNA that could be translated in a wheat germ system slowly decreased during sporulation. Within 8 h after the induction of sporulation, isolated RNA stimulated half as much protein synthesis as the equivalent amount of vegetative RNA. There were significant differences in protein synthesis. The percentage of ribosomes in polysomes decreased threefold as the cells entered sporulation. This decrease began within 5 min of the initiation of sporulation, and the steady-state pattem was attained within 120 min. However, the ribosomes were not irreversibly inactivated; they could be reincorporated into polysomes by returning the sporulating cells to growth medium. Though unable to sporulate, strains homozygous for mating type, MATa/MATa, showed a similar decrease in the number of polysomes when placed in sporulation medium. Furthermore, the same shift toward monosomes was observed during stationary phase of growth. We conclude that the redistribution of ribosomes represents a general metabolic response to starvation. Our data indicate that the loss of polysomes is most likely caused by a decrease in the initiation of translation rather than a severe limitation in the amount of messenger RNA. Furthermore, the loss of polysomes is not due to the decreased synthesis of a major class of abundant proteins. Of the 400 vegetative proteins resolved by two-dimensional gel electrophoresis, only 19 were not synthesized by sporulating cells. Approximately 10 to 20% of the cells in a sporulating culture failed to complete ascus fornation. We have shown that [3S]methionine is incorporated equivalently into cells committed to sporulation and cells that fail to form asci. Furthermore, the proteins synthesized by these two populations were indistinguishable on one-dimensional gels. We compared proteins labeled by various protocols, including long-term and pulse-labeling during sporulation and prelabeling during vegetative growth before transfer to sporulation medium. The resulting two-dimensional gel patterns differed significantly. Many spots labeled by the long-term techniques may have arisen by protein processing. We suggest that pulse-labeling produces the most accurate reflection of instantaneous synthesis of proteins.
Yeast, 2002
A new high throughput mutant screening procedure for the detection of sporulation mutants was developed and used to analyse a set of 624 non-lethal homozygous deletion mutants created in the European joint research program EUROFAN. The screening procedure involved determination of LL- and DL-dityrosine, sporulation-specific compounds, which were shown to be robust markers of the extent and arrest stage of sporulation mutants. Secondary screens consisted of light microscopy to detect mature and immature spores and DAPI staining to monitor the progress of meiotic nuclear divisions. We discovered new phenotypic classes of mutants defective in spore wall synthesis that were not discovered by previous screens for sporulation mutants. The genes corresponding to the sporulation mutants fell in several functional classes, some of which were previously unknown to be involved in spore formation. Peroxisomes seem to play a role in spore wall synthesis. Mitochondria play a role in sporulation that is not simply restricted to supply of ATP from respiratory metabolism. The deletion mutants included in the set were functionally unknown at the start of EUROFAN; however, within the last few years the importance to sporulation of some of them was also reported by other authors. Taken together, about 8% of all single gene deletion mutants of non-essential genes of Saccharomyces cerevisiae seem to display a clear and reproducible sporulation phenotype.
Journal of Bacteriology
The nature and properties of the 20S ribonucleic acid which accumulates only during the sporulation of Saccharomyces cerevisiae were examined. The 20S ribonucleic acid (RNA) has a base composition considerably different from ribosomal RNA species and is virutally unmethylated. The 20S RNA did, however, exhibit approximately 70% homology with 18S RNA by RNA-deoxyribonucleic acid filter hybridization competitions. The 20S RNA showed a hybridization saturation plateau level 30 to 40% higher than 18S, consistent with measurements of the size difference in polyacrylamide gels. Pulse-chase experiments in the presence and absence of cycloheximide indicate that the 20S RNA has a presumptive relationship to the 20S ribosomal RNA precursor normally observed only in short pulse-labeling in vegetative cells.