Senescence Mutants of Saccharomyces cerevisiae With a Defect in Telomere Replication Identify Three Additional EST Genes

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1399-1412 ◽  
Author(s):  
Thomas S Lendvay ◽  
Danna K Morris ◽  
Jeannie Sah ◽  
Bhuvana Balasubramanian ◽  
Victoria Lundblad
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomerase Activity ◽  
Gene Encoding ◽  
Epistasis Analysis ◽  
Mutant Strains ◽  
Primary Determinant ◽  
Complementation Groups ◽  
Telomere Replication ◽  
Senescence Phenotype ◽  
Novel Protein

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cermzsiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.

scholarly journals The Schizosaccharomyces pombe cho1+ Gene Encodes a Phospholipid Methyltransferase

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 553-562
Author(s):  
Margaret I Kanipes ◽  
John E Hill ◽  
Susan A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Analysis ◽  
Schizosaccharomyces Pombe ◽  
Gene Disruption ◽  
Structure And Function ◽  
Biosynthetic Enzyme ◽  
Gene Encoding ◽  
Complementation Groups ◽  
Eukaryotic Organisms ◽  
And Function

Abstract The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Δ) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.

scholarly journals Analysis of the interaction between piD261/Bud32, an evolutionarily conserved protein kinase of Saccharomyces cerevisiae, and the Grx4 glutaredoxin

2004 ◽  
Vol 377 (2) ◽  
pp. 395-405 ◽  
Author(s):  
Raffaele LOPREIATO ◽  
Sonia FACCHIN ◽  
Geppo SARTORI ◽  
Giorgio ARRIGONI ◽  
Stefano CASONATO ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Hybrid Approach ◽  
Inosine Monophosphate Dehydrogenase ◽  
Gene Encoding ◽  
Putative Protease ◽  
Structural Homologues ◽  
Novel Protein

The Saccharomyces cerevisiae piD261/Bud32 protein and its structural homologues, which are present along the Archaea–Eukarya lineage, constitute a novel protein kinase family (the piD261 family) distantly related in sequence to the eukaryotic protein kinase superfamily. It has been demonstrated that the yeast protein displays Ser/Thr phosphotransferase activity in vitro and contains all the invariant residues of the family. This novel protein kinase appears to play an important cellular role as deletion in yeast of the gene encoding piD261/Bud32 results in the alteration of fundamental processes such as cell growth and sporulation. In this work we show that the phosphotransferase activity of Bud32 is relevant to its functionality in vivo, but is not the unique role of the protein, since mutants which have lost catalytic activity but not native conformation can partially complement the disruption of the gene encoding piD261/Bud32. A two-hybrid approach has led to the identification of several proteins interacting with Bud32; in particular a glutaredoxin (Grx4), a putative glycoprotease (Ykr038/Kae1) and proteins of the Imd (inosine monophosphate dehydrogenase) family seem most plausible interactors. We further demonstrate that Grx4 directly interacts with Bud32 and that it is phosphorylated in vitro by Bud32 at Ser-134. The functional significance of the interaction between Bud32 and the putative protease Ykr038/Kae1 is supported by its evolutionary conservation.

scholarly journals A Novel Genetic Screen for snRNP Assembly Factors in Yeast Identifies a Conserved Protein, Sad1p, Also Required for Pre-mRNA Splicing

1999 ◽  
Vol 19 (3) ◽  
pp. 2008-2020 ◽  
Author(s):  
Zoi Lygerou ◽  
George Christophides ◽  
Bertrand Séraphin
Keyword(s):  
Restrictive Temperature ◽  
U6 Snrna ◽  
Assembly Pathway ◽  
Defective Mutant ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Putative Zinc Finger ◽  
Assembly Defect ◽  
Novel Protein

ABSTRACT The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 andprp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of β-galactosidase from reporter constructs is abolished, while extracts prepared fromsad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human,Caenorhabditis elegans, Arabidospis, andDrosophila.

scholarly journals Role for Vitamin B12 in Light Induction of Gene Expression in the Bacterium Myxococcus xanthus

2002 ◽  
Vol 184 (8) ◽  
pp. 2215-2224 ◽  
Author(s):  
María Cervantes ◽  
Francisco J. Murillo
Keyword(s):  
Myxococcus Xanthus ◽  
Carotenoid Biosynthesis ◽  
Inducible Promoter ◽  
Binding Domain ◽  
Gene Encoding ◽  
The Family ◽  
Mutant Strains ◽  
Carotenoid Genes ◽  
Carotenoid Synthesis ◽  
Novel Protein

ABSTRACT A light-inducible promoter (PB) drives the carB operon (carotenoid genes) of the bacterium Myxococcus xanthus. A gene encoding a regulator of carotenoid biosynthesis was identified by studying mutant strains carrying a transcriptional fusion to PB and deletions in three candidate genes. Our results prove that the identified gene, named carA, codes for a repressor of the PB promoter in the dark. They also show that the carA gene product does not participate in the light activation of two other promoters connected with carotenoid synthesis or its regulation in M. xanthus. CarA is a novel protein consisting of a DNA-binding domain of the family of MerR helix-turn-helix transcriptional regulators, directly joined to a cobalamin-binding domain. In support of this, we report here that the presence of vitamin B12 or some other cobalamin derivatives is absolutely required for activation of the PB promoter by light.

scholarly journals Differential Processing of Leading- and Lagging-Strand Ends at Saccharomyces cerevisiae Telomeres Revealed by the Absence of Rad27p Nuclease

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1583-1594
Author(s):  
Julie Parenteau ◽  
Raymund J Wellinger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Double Mutant ◽  
Telomerase Activity ◽  
Differential Processing ◽  
Okazaki Fragment Processing ◽  
Mutant Strains ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

Abstract Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres. Furthermore, the lengths of telomeric repeats become very heterogeneous. These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends. To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity. The results show that double-mutant strains died more rapidly than strains lacking only telomerase components. Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27Δ cells. The results from studies of the replication intermediates of a linear plasmid in rad27Δ cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis. These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.

scholarly journals Yeast Mutants Deficient in ER-Associated Degradation of the Z Variant of Alpha-1-Protease Inhibitor

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1355-1362 ◽  
Author(s):  
Ardythe A McCracken ◽  
Igor V Karpichev ◽  
James E Ernaga ◽  
Eric D Werner ◽  
Andrew G Dillin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Proteinase Inhibitor ◽  
Parent Strain ◽  
Mendelian Inheritance ◽  
Wild Type ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Yeast Mutants ◽  
Wild Type Yeast

Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, ∼30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.

The osmotic integrity of the yeast cell requires a functional PKC1 gene product

1992 ◽  
Vol 12 (11) ◽  
pp. 4896-4905
Author(s):  
G Paravicini ◽  
M Cooper ◽  
L Friedli ◽  
D J Smith ◽  
J L Carpentier ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Yeast Genome ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Electron Microscopic ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Mammalian Protein

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

scholarly journals The osmotic integrity of the yeast cell requires a functional PKC1 gene product.

1992 ◽  
Vol 12 (11) ◽  
pp. 4896-4905 ◽  
Author(s):  
G Paravicini ◽  
M Cooper ◽  
L Friedli ◽  
D J Smith ◽  
J L Carpentier ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Yeast Genome ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Electron Microscopic ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Mammalian Protein

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

scholarly journals Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation inSaccharomyces cerevisiae

2002 ◽  
Vol 13 (2) ◽  
pp. 503-514 ◽  
Author(s):  
Li-Lin Du ◽  
Peter Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Biological Processes ◽  
Cell Morphogenesis ◽  
Gene Encoding ◽  
And Function ◽  
Genetic Results ◽  
Novel Protein

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displayed defects similar to those ofcbk1 mutants. pag1 andcbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 andCBK1 act in the same pathway. Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes.

Substitutions in the Pheromone-Responsive Gβ Protein of Saccharomyces cerevisiae Confer a Defect in Recovery from Pheromone Treatment

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 947-961
Author(s):  
E Li ◽  
Eric Meldrum ◽  
Holly F Stratton ◽  
David E Stone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Negative Regulator ◽  
Mutant Form ◽  
Pheromone Response ◽  
Gene Encoding ◽  
Mating Response ◽  
Mutant Strains ◽  
Tight Linkage ◽  
Mating Signal ◽  
Mutant Forms

Abstract The pheromone-responsive Gα protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp−) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp− mutations showed tight linkage to the gene encoding the pheromone-responsive Gβ, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gβ dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gβγ subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gβγ, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gβγ.

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