Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae

Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc 4, 7, and 28) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the "initiation" of DNA synthesis. Mutations in two genes (cdc 8 and 21) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 13 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature.     

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).     

The PRP31 gene encodes a novel protein required for pre-mRNA splicing in Saccharomyces cerevisiae.

The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.     

Six novel genes necessary for pre-mRNA splicing in Saccharomyces cerevisiae.

We have identified six new genes whose products are necessary for the splicing of nuclear pre-mRNA in the yeast Saccharomyces cerevisiae. A collection of 426 temperature-sensitive yeast strains was generated by EMS mutagenesis. These mutants were screened for pre-mRNA splicing defects by an RNA gel blot assay, using the intron- containing CRY1 and ACT1 genes as hybridization probes. We identified 20 temperature-sensitive mutants defective in pre-mRNA splicing. Twelve appear to be allelic to the previously identified prp2, prp3, prp6, prp16/prp23, prp18, prp19 or prp26 mutations that cause defects in spliceosome assembly or the first or second step of splicing. One is allelic to SNR14 encoding U4 snRNA. Six new complementation groups, prp29-prp34, were identified. Each of these mutants accumulates unspliced pre-mRNA at 37 degrees C and thus is blocked in spliceosome assembly or early steps of pre-mRNA splicing before the first cleavage and ligation reaction. The prp29 mutation is suppressed by multicopy PRP2 and displays incomplete patterns of complementation with prp2 alleles, suggesting that the PRP29 gene product may interact with that of PRP2. There are now at least 42 different gene products, including the five spliceosomal snRNAs and 37 different proteins that are necessary for pre-mRNA splicing in Saccharomyces cerevisiae. However, the number of yeast genes identifiable by this approach has not yet been exhausted.     

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.     

GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae.

The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24:263-315, 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function.     

Possible role of histidine in the L-proline transport system of Saccharomyces cerevisiae

The L-proline transport system of Saccharomyces cerevisiae is shown to be specifically inactivated upon incubation of intact yeast cells with the histidine modifier diethylpyrocarbonate. The extent of inactivation is half-maximum at 0.5 mM diethylpyrocarbonate for an incubation of 2 min at 30 degrees C and pH 6.0. Under the same conditions, the time dependence of inactivation is monophasic with the second-order rate constant of 5.5 M-1 X s-1 and the maximum rate Jmax of L-proline transport is lowered by about 50%, while the KT value remains unchanged. Moreover, L-proline afforded significant protection against diethylpyrocarbonate inactivation. The complete reactivation of a partially inactivated L-proline transport system by neutral hydroxylamine and the elimination of the possibility that the modification of other amino acid residues are responsible for the inactivation, suggested that the transport protein inactivation occurs solely by a modification of histidine residues.     

ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae

ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation. ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus. In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell. Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant. Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant. A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane. These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.     

Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae.

To further our studies of protein sorting and biogenesis of the lysosome-like vacuole in yeast, we have isolated spontaneous mutations in 11 new VPL complementation groups, as well as additional alleles of the eight previously described VPL genes. These mutants were identified by selecting for cells that mislocalize vacuolar proteins to the cell surface. Morphological examination of the vpl mutants indicated that most contain vacuoles of normal appearance; however, some of the mutants generally lack a large vacuole, and instead accumulate smaller organelles. Of the 19 VPL complementation groups, 12 were found to be identical to 12 of 33 VPT complementation groups identified in a separate study. Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups. Collectively, mutants in these four collections define 49 complementation groups required to deliver or retain soluble vacuolar enzymes, including carboxypeptidase Y (CPY) and proteinase A. We have also isolated 462 new mutants that lack normal levels of vacuolar CPY activity. Among these latter mutants, only pep4 mutants were found to be specifically defective in vacuolar zymogen activation. We conclude that there is a large number of gene products required for sorting or retention of vacuolar proteins in yeast, and only a single gene, PEP4, that is essential for activation of CPY and other vacuolar zymogens.     

Efficient construction of homozygous diploid strains identifies genes required for the hyper-filamentous phenotype in Saccharomyces cerevisiae

Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch) and a PSTE18-URA3 gene (counter selection marker for diploids). Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA) plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.