Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe

1986 ◽  
Vol 6 (10) ◽  
pp. 3523-3530
Author(s):  
R Booher ◽  
D Beach
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Atp Binding Site ◽  
Leu2 Gene ◽  
Cdc2 Gene ◽  
A Site

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.

scholarly journals Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe.

1986 ◽  
Vol 6 (10) ◽  
pp. 3523-3530 ◽  
Author(s):  
R Booher ◽  
D Beach
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Atp Binding Site ◽  
Leu2 Gene ◽  
Cdc2 Gene ◽  
A Site

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

scholarly journals Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

1993 ◽  
Vol 13 (5) ◽  
pp. 2870-2881 ◽  
Author(s):  
L C Robinson ◽  
M M Menold ◽  
S Garrett ◽  
M R Culbertson
Keyword(s):  
Protein Kinase ◽  
Eukaryotic Cell ◽  
Casein Kinase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Casein Kinase I ◽  
Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe

1987 ◽  
Vol 7 (1) ◽  
pp. 504-511 ◽  
Author(s):  
J Hindley ◽  
G Phear ◽  
M Stein ◽  
D Beach
Keyword(s):  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Base Pairs ◽  
Direct Role ◽  
Protein Coding ◽  
Dna Fragment ◽  
Rna Transcript ◽  
Translational Product

Sucl+ was originally identified as a DNA sequence that, at high copy number, rescued Schizosaccharomyces pombe strains carrying certain temperature-sensitive alleles of the cdc2 cell cycle control gene. We determined the nucleotide sequence of a 1,083-base-pair Sucl+ DNA fragment and S1 mapped its 866-nucleotide RNA transcript. The protein-coding sequence of the gene is interrupted by two intervening sequences of 115 and 51 base pairs. The predicted translational product of the gene is a protein of 13 kilodaltons. A chromosomal gene disruption of Sucl+ was constructed in a diploid S. pombe strain. Germinating spores carrying a null allele of the gene were capable of very limited cell division, following which many cells became highly elongated. The Sucl+ gene was also strongly overexpressed under the control of a heterologous S. pombe promoter. Overexpression of Sucl+ is not lethal but causes a division delay such that cells are approximately twice the normal length at division. These data suggest that Sucl+ encodes a protein which plays a direct role in the cell division cycle of S. pombe.

scholarly journals Scw1p Antagonizes the Septation Initiation Network To Regulate Septum Formation and Cell Separation in the Fission Yeast Schizosaccharomyces pombe

2003 ◽  
Vol 2 (3) ◽  
pp. 510-520 ◽  
Author(s):  
Quan-Wen Jin ◽  
Dannel McCollum
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Separation ◽  
Deletion Mutant ◽  
Temperature Sensitive ◽  
Growth Defects ◽  
Septum Formation ◽  
Primary Septum ◽  
Septation Initiation Network ◽  
Ring Constriction

ABSTRACT Cytokinesis in the fission yeast Schizosaccharomyces pombe is regulated by a signaling pathway termed the septation initiation network (SIN). The SIN is essential for initiation of actomyosin ring constriction and septum formation. In a screen to search for mutations that can rescue the sid2-250 SIN mutant, we obtained scw1-18. Both the scw1-18 mutant and the scw1 deletion mutant (scw1Δ mutant), have defects in cell separation. Both the scw1-18 and scw1Δ mutations rescue the growth defects of not just the sid2-250 mutant but also the other temperature-sensitive SIN mutants. Other cytokinesis mutants, such as those defective for actomyosin ring formation, are not rescued by scw1Δ. scw1Δ does not seem to rescue the SIN by restoring SIN signaling defects. However, scw1Δ may function downstream of the SIN to promote septum formation, since scw1Δ can rescue the septum formation defects of the cps1-191β-1,3-glucan synthase mutant, which is required for synthesis of the primary septum.

scholarly journals Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src.

1986 ◽  
Vol 6 (3) ◽  
pp. 751-757 ◽  
Author(s):  
M P Kamps ◽  
B M Sefton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Atp Binding Site ◽  
Rous Sarcoma ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
Dependent Protein ◽  
Infected Cells

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src

1986 ◽  
Vol 6 (3) ◽  
pp. 751-757
Author(s):  
M P Kamps ◽  
B M Sefton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Atp Binding Site ◽  
Rous Sarcoma ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
Dependent Protein ◽  
Infected Cells

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis

1993 ◽  
Vol 13 (5) ◽  
pp. 2870-2881
Author(s):  
L C Robinson ◽  
M M Menold ◽  
S Garrett ◽  
M R Culbertson
Keyword(s):  
Protein Kinase ◽  
Eukaryotic Cell ◽  
Casein Kinase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Restrictive Temperature ◽  
Loss Of Function ◽  
Casein Kinase I ◽  
Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

Functional conservation of tRNase ZL among Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans

2009 ◽  
Vol 422 (3) ◽  
pp. 483-492 ◽  
Author(s):  
Zhen Zhao ◽  
Wenchen Su ◽  
Sheng Yuan ◽  
Ying Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Cancer Susceptibility ◽  
Short Form ◽  
Susceptibility Gene ◽  
Temperature Sensitive ◽  
Functional Conservation ◽  
Trnase Z

Although tRNase Z from various organisms was shown to process nuclear tRNA 3′ ends in vitro, only a very limited number of studies have reported its in vivo biological functions. tRNase Z is present in a short form, tRNase ZS, and a long form, tRNase ZL. Unlike Saccharomyces cerevisiae, which contains one tRNase ZL gene (scTRZ1) and humans, which contain one tRNase ZL encoded by the prostate-cancer susceptibility gene ELAC2 and one tRNase ZS, Schizosaccharomyces pombe contains two tRNase ZL genes, designated sptrz1+ and sptrz2+. We report that both sptrz1+ and sptrz2+ are essential for growth. Moreover, sptrz1+ is required for cell viability in the absence of Sla1p, which is thought to be required for endonuclease-mediated maturation of pre-tRNA 3′ ends in yeast. Both scTRZ1 and ELAC2 can complement a temperature-sensitive allele of sptrz1+, sptrz1–1, but not the sptrz1 null mutant, indicating that despite exhibiting species specificity, tRNase ZLs are functionally conserved among S. cerevisiae, S. pombe and humans. Overexpression of sptrz1+, scTRZ1 and ELAC2 can increase suppression of the UGA nonsense mutation ade6–704 through facilitating 3′ end processing of the defective suppressor tRNA that mediates suppression. Our findings reveal that 3′ end processing is a limiting step for defective tRNA maturation and demonstrate that overexpression of sptrz1+, scTRZ1 and ELAC2 can promote defective tRNA 3′ processing in vivo. Our results also support the notion that yeast tRNase ZL is absolutely required for 3′ end processing of at least a few pre-tRNAs even in the absence of Sla1p.

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