scholarly journals Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition.

Genetics ◽  
1992 ◽  
Vol 131 (1) ◽  
pp. 21-29 ◽  
Author(s):  
K Kitada ◽  
L H Johnston ◽  
T Sugino ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Genomic Library ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Allele Specific ◽  
Cell Cycle Transition ◽  
Mutant Cells

Abstract When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle. DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.

Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1590-1598
Author(s):  
M Patterson ◽  
R A Sclafani ◽  
W L Fangman ◽  
J Rosamond
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Genetic Recombination ◽  
Multiple Roles ◽  
Predicted Amino Acid Sequence ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Reading Frame ◽  
Genomic Insert

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

scholarly journals PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

1992 ◽  
Vol 12 (9) ◽  
pp. 3843-3856 ◽  
Author(s):  
J P O'Connor ◽  
C L Peebles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Genomic Library ◽  
Essential Gene ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Putative Open Reading Frame ◽  
Reading Frame ◽  
Trna Processing ◽  
Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

scholarly journals Temperature-Sensitive Lethal Pseudorevertants of ste Mutations in Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 115 (4) ◽  
pp. 627-636
Author(s):  
Margaret E Katz ◽  
Jill Ferguson ◽  
Steven I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Complementation Group ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Pheromone Response ◽  
Cycle Arrest ◽  
Homogeneous Cell ◽  
Complementation Groups ◽  
Mutant Cells

ABSTRACT A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes. Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles. These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles. Five mutations in a single complementation group were isolated as suppressors of ste2 alleles. None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations. In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells. Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.

scholarly journals Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
M Patterson ◽  
R A Sclafani ◽  
W L Fangman ◽  
J Rosamond
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Genetic Recombination ◽  
Multiple Roles ◽  
Predicted Amino Acid Sequence ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Reading Frame ◽  
Genomic Insert

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5

1993 ◽  
Vol 13 (7) ◽  
pp. 4445-4457
Author(s):  
K Kitada ◽  
A L Johnson ◽  
L H Johnston ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Suppressor Gene ◽  
Amino Acid Sequence Similarity ◽  
Nucleotide Sequence Analysis ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Chromosomal Dna ◽  
Multicopy Suppressor

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases. Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5. An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid. Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected. Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary. CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

scholarly journals The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function.

1990 ◽  
Vol 10 (5) ◽  
pp. 2214-2223 ◽  
Author(s):  
Y Wada ◽  
K Kitamoto ◽  
T Kanbe ◽  
K Tanaka ◽  
Y Anraku
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Library ◽  
Carboxypeptidase Y ◽  
Amino Acid Residues ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Lethal Event ◽  
Vacuolar Protein ◽  
And Function

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function

1990 ◽  
Vol 10 (5) ◽  
pp. 2214-2223
Author(s):  
Y Wada ◽  
K Kitamoto ◽  
T Kanbe ◽  
K Tanaka ◽  
Y Anraku
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Library ◽  
Carboxypeptidase Y ◽  
Amino Acid Residues ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Lethal Event ◽  
Vacuolar Protein ◽  
And Function

The SLP1 gene, which is involved in the expression of vacuolar functions in the yeast Saccharomyces cerevisiae (K. Kitamoto, K. Yoshizawa, Y. Ohsumi, and Y. Anraku, J. Bacteriol. 170:2687-2691, 1988), has been cloned from a yeast genomic library by complementation of the slp1-1 mutation. The isolated plasmid has a 7.8-kilobase BamHI-BamHI fragment that is sufficient to complement several characteristic phenotypes of the slp1-1 mutation. The fragment was integrated at the chromosomal SLP1 locus, indicating that it contains an authentic SLP1 gene. By DNA sequencing of the SLP1 gene, an open reading frame of 2,073 base pairs coding for a polypeptide of 691 amino acid residues (Mr, 79,270) was found. Gene disruption of the chromosomal SLP1 did not cause a lethal event. Vacuolar proteins in the delta slp1 mutant are not processed to vacuolar forms but remain in Golgi-modified forms. Carboxypeptidase Y in the delta slp1 mutant is localized mainly to the outsides of the cells. delta slp1 mutant cells have no prominent vacuolar structures but contain numerous vesicles in the cytoplasm, as seen by electron microscopy. Genetic and molecular biological analyses revealed that SLP1 is identical to VPS33, which is required for vacuolar protein sorting as reported by Robinson et al. (J. S. Robinson, D. J. Klionsky, L. M. Banta, and S. D. Emr, Mol. Cell. Biol. 8:4936-4948, 1988). These results indicate that the SLP1 (VPS33) gene is involved in the sorting of vacuolar proteins from the Golgi apparatus and their targeting to the vacuole and that it is required for the morphogenesis of vacuoles and subsequent expression of vacuolar functions.

PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing

1992 ◽  
Vol 12 (9) ◽  
pp. 3843-3856
Author(s):  
J P O'Connor ◽  
C L Peebles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Genomic Library ◽  
Essential Gene ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Putative Open Reading Frame ◽  
Reading Frame ◽  
Trna Processing ◽  
Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

scholarly journals A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5.

1993 ◽  
Vol 13 (7) ◽  
pp. 4445-4457 ◽  
Author(s):  
K Kitada ◽  
A L Johnson ◽  
L H Johnston ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Suppressor Gene ◽  
Amino Acid Sequence Similarity ◽  
Nucleotide Sequence Analysis ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Chromosomal Dna ◽  
Multicopy Suppressor

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases. Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5. An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid. Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected. Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary. CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

scholarly journals Translational maintenance of frame: mutants of Saccharomyces cerevisiae with altered -1 ribosomal frameshifting efficiencies.

Genetics ◽  
1994 ◽  
Vol 136 (1) ◽  
pp. 75-86 ◽  
Author(s):  
J D Dinman ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Open Reading Frame ◽  
Temperature Sensitive ◽  
Ribosomal Frameshift ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Daughter Cells ◽  
Mother And Daughter ◽  
Multiple Buds

Abstract A special site on the (+) strand of the L-A dsRNA virus induces about 2% of ribosomes translating the gag open reading frame to execute a -1 frameshift and thus produce the viral gag-pol fusion protein. Using constructs in which a -1 ribosomal frameshift at this site was necessary for expression of lacZ we isolated chromosomal mutants in which the efficiency of frameshifting was increased. These mutants comprise eight genes, named mof (maintenance of frame). The mof1-1, mof2-1, mof4-1, mof5-1 and mof6-1 strains cannot maintain M1 dsRNA at 30 degrees, but, paradoxically, do not lose L-A. The mof2-1, mof5-1 and mof6-1 strains are temperature sensitive for growth at 37 degrees, and all three show striking cell cycle phenotypes. The mof2-1 strains arrest with mother and daughter cells almost equal in size, mof5-1 arrests with multiple buds and mof6-1 arrests as single large unbudded cells. mof2-1 and mof5-1 strains are also Pet-. The mof mutations show differential effects on various frameshifting signals.

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