scholarly journals Mutations in the homologous ZDS1 and ZDS2 genes affect cell cycle progression.

1996 ◽  
Vol 16 (10) ◽  
pp. 5254-5263 ◽  
Author(s):  
Y Yu ◽  
Y W Jiang ◽  
R J Wellinger ◽  
K Carlson ◽  
J M Roberts ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Null Mutation ◽  
Genetic Screens ◽  
Multicopy Suppressor ◽  
Plasmid Loss ◽  
Multicopy Suppressors ◽  
Rna Polymerase Ii Holoenzyme

The Saccharomyces cerevisiae ZDS1 and ZDS2 genes were identified as multicopy suppressors in distinct genetic screens but were found to encode highly similar proteins. We show that at semipermissive temperatures, a yeast strain with a cdc28-1N allele was uniquely deficient in plasmid maintenance in comparison with strains harboring other cdc28 thermolabile alleles. Quantitative analysis of plasmid loss rates in cdc28-1N strains carrying plasmids with multiple replication origins suggests that a defect in initiating DNA replication probably causes this plasmid loss phenotype. The ZDS1 gene was isolated as a multicopy suppressor of the cdc28-1N plasmid loss defect. A zds1 deletion exhibits genetic interactions with cdc28-1N but not with other cdc28 alleles. SIN4 encodes a protein which is part of the RNA polymerase II holoenzyme-mediator complex, and a sin4 null mutation has pleiotropic effects suggesting roles in transcriptional regulation and chromatin structure. The ZDS2 gene was isolated as a multicopy suppressor of the temperature-sensitive growth defect caused by the sin4 null mutation. Disruption of either ZDS1 or ZDS2 causes only modest phenotypes. However, a strain with both ZDS1 and ZDS2 disrupted is extremely slowly growing, has marked defects in bud morphology, and shows defects in completing S phase or entering mitosis.

scholarly journals Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4387-4395 ◽  
Author(s):  
D Mack ◽  
K Nishimura ◽  
B K Dennehey ◽  
T Arbogast ◽  
J Parkinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Lethality ◽  
Cell Polarization ◽  
Growth Defect ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Multicopy Suppressor ◽  
Bud Emergence ◽  
Multicopy Suppressors ◽  
Multiple Copies

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

SHM1: A multicopy suppressor of a temperature-sensitive null mutation in the HMG1-likeabf2 gene

Yeast ◽  
1996 ◽  
Vol 12 (12) ◽  
pp. 1239-1250 ◽  
Author(s):  
Ling-Rong Kao ◽  
Timothy L. Megraw ◽  
Chi-Bom Chae
Keyword(s):  
Temperature Sensitive ◽  
Null Mutation ◽  
Multicopy Suppressor

scholarly journals Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.

1995 ◽  
Vol 15 (1) ◽  
pp. 69-75 ◽  
Author(s):  
A E Adams ◽  
W Shen ◽  
C S Lin ◽  
J Leavitt ◽  
P Matsudaira
Keyword(s):  
Cell Types ◽  
Biochemical Properties ◽  
Actin Binding ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Null Mutation ◽  
Homologous Proteins ◽  
Functional Conservation ◽  
Cytoskeletal Component

The actin cytoskeleton is a fundamental component of eukaryotic cells, with both structural and motile roles. Actin and many of the actin-binding proteins found in different cell types are highly conserved, showing considerable similarity in both primary structure and biochemical properties. To make detailed comparisons between homologous proteins, it is necessary to know whether the various proteins are functionally, as well as structurally, conserved. Fimbrin is an example of a cytoskeletal component that, as shown by sequence determinations and biochemical characterizations, is conserved between organisms as diverse as Saccharomyces cerevisiae and humans. In this study, we examined whether the human homolog can substitute for the yeast protein in vivo. We report here that two isoforms of human fimbrin, also referred to as T- and L-plastin, can both substitute in vivo for yeast fimbrin, also known as Sac6p, whereas a third isoform, I-fimbrin (or I-plastin), cannot. We demonstrate that the human T- and L-fimbrins, in addition to complementing the temperature-sensitive growth defect of the sac6 null mutant, restore both normal cytoskeletal organization and cell shape to the mutant cells. In addition, we show that human T- and L-fimbrins can complement a sporulation defect caused by the sac6 null mutation. These findings indicate that there is a high degree of functional conservation in the cytoskeleton, even between organisms as diverse as S. cerevisiae and humans.

scholarly journals Interplay of Positive and Negative Regulators in Transcription Initiation by RNA Polymerase II Holoenzyme

1998 ◽  
Vol 18 (8) ◽  
pp. 4455-4462 ◽  
Author(s):  
Tong Ihn Lee ◽  
John J. Wyrick ◽  
Sang Seok Koh ◽  
Ezra G. Jennings ◽  
Ellen L. Gadbois ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Dynamic Balance ◽  
Regulation Of Gene Expression ◽  
Temperature Sensitive ◽  
Negative Regulators ◽  
Essential Components ◽  
Tata Box Binding Protein ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT Activation of protein-encoding genes involves recruitment of an RNA polymerase II holoenzyme to promoters. Since the Srb4 subunit of the holoenzyme is essential for expression of most class II genes and is a target of at least one transcriptional activator, we reasoned that suppressors of a temperature-sensitive mutation in Srb4 would identify other factors generally involved in regulation of gene expression. We report here that MED6 and SRB6, both of which encode essential components of the holoenzyme, are among the dominant suppressors and that the products of these genes interact physically with Srb4. The recessive suppressors include NCB1 (BUR6),NCB2, NOT1, NOT3, NOT5, and CAF1, which encode subunits of NC2 and the Not complex. NC2 and Not proteins are general negative regulators which interact with TATA box binding protein (TBP). Taken together, these results suggest that transcription initiation involves a dynamic balance between activation mediated by specific components of the holoenzyme and repression by multiple TBP-associated regulators.

scholarly journals Dhh1p, a Putative RNA Helicase, Associates with the General Transcription Factors Pop2p and Ccr4p from Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 571-579
Author(s):  
Hiroaki Hata ◽  
Hisayuki Mitsui ◽  
Hong Liu ◽  
Yongli Bai ◽  
Clyde L Denis ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Rna Helicase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Phenotypic Analysis ◽  
Multicopy Suppressor ◽  
General Transcription Factors ◽  
Close Relationship ◽  
Partial Suppression

Abstract The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression. Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells. In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes. Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex. Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway. The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5. These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.

scholarly journals Requirement for a Functional Interaction between Mediator Components Med6 and Srb4 in RNA Polymerase II Transcription

1998 ◽  
Vol 18 (9) ◽  
pp. 5364-5370 ◽  
Author(s):  
Young Chul Lee ◽  
Young-Joon Kim
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Functional Relationship ◽  
Specific Interaction ◽  
Class Ii ◽  
Temperature Sensitive ◽  
Functional Interaction ◽  
Activation Of Transcription ◽  
Allele Specific ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT Regulated transcription of class II genes of the yeastSaccharomyces cerevisiae requires the diverse functions of mediator complex. In particular, MED6 is essential for activated transcription from many class II promoters, suggesting that it functions as a key player in the relay of activator signals to the basal transcription machinery. To identify the functional relationship between MED6 and other transcriptional regulators, we conducted a genetic screen to isolate a suppressor of a temperature-sensitive (ts) med6 mutation. We identified an SRB4 allele as a dominant and allele-specific suppressor of med6-ts. A single missense mutation inSRB4 can specifically suppress transcriptional defects caused by the med6 ts mutation, indicating a functional interaction between these two mediator subunits in the activation of transcription. Biochemical analysis of mediator subassembly revealed that mediator can be dissociated into two tightly associated subcomplexes. The Med6 and Srb4 proteins are contained in the same subcomplex together with other dominant Srb proteins, consistent with their functional relationship revealed by the genetic study. Our results suggest not only the existence of a specific interaction between Med6 and Srb4 but also the requirement of this interaction in transcriptional regulation of RNA polymerase II holoenzyme.

scholarly journals Chd1 and yFACT Act in Opposition in Regulating Transcription

2007 ◽  
Vol 27 (18) ◽  
pp. 6279-6287 ◽  
Author(s):  
Debabrata Biswas ◽  
Rinku Dutta-Biswas ◽  
David J. Stillman
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Synthetic Lethality ◽  
Histone Methyltransferase ◽  
Tata Binding Protein ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Low Levels

ABSTRACT CHD1 encodes an ATP-dependent chromatin remodeler with two chromodomains. Deletion of CHD1 suppresses the temperature-sensitive growth defect caused by mutations in either SPT16 or POB3, which encode subunits of the yFACT chromatin-reorganizing complex. chd1 also suppresses synthetic defects caused by combining an spt16 mutation with other transcription factor mutations, including the synthetic lethality caused by combining an spt16 mutation with TATA binding protein (TBP) or TFIIA defects. Binding of TBP and RNA polymerase II to the GAL1 promoter is reduced in a pob3 mutant, resulting in low levels of GAL1 expression, and all three defects are suppressed by removing Chd1. These results suggest that Chd1 and yFACT have opposing roles in regulating TBP binding at promoters. Additionally, overexpression of Chd1 is tolerated in wild-type cells but is toxic in spt16 mutants. Further, both the ATPase and chromodomain are required for Chd1 activity in opposing yFACT function. Similar to the suppression by chd1, mutations in the SET2 histone methyltransferase also suppress defects caused by yFACT mutations. chd1 and set2 are additive in suppressing pob3, suggesting that Chd1 and Set2 act in distinct pathways. Although human Chd1 has been shown to bind to H3-K4-Me, we discuss evidence arguing that yeast Chd1 binds to neither H3-K4-Me nor H3-K36-Me.

scholarly journals Partners of Rpb8p, a Small Subunit Shared by Yeast RNA Polymerases I, II, and III

2001 ◽  
Vol 21 (17) ◽  
pp. 6056-6065 ◽  
Author(s):  
Jean-François Briand ◽  
Francisco Navarro ◽  
Peggy Rematier ◽  
Claire Boschiero ◽  
Sylvie Labarre ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Dosage ◽  
Small Subunit ◽  
Rna Polymerases ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Orphan Gene ◽  
Two Hybrid

ABSTRACT Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP..LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP..LW.GKQ provokes a temperature-sensitive defect. Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-Δ null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan geneYGR089w. A ygr089-Δ null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.

scholarly journals Activation of the Bur1-Bur2 Cyclin-Dependent Kinase Complex by Cak1

2002 ◽  
Vol 22 (19) ◽  
pp. 6750-6758 ◽  
Author(s):  
Sheng Yao ◽  
Gregory Prelich
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Single Gene ◽  
Eukaryotic Cell ◽  
Temperature Sensitive ◽  
Cycle Progression ◽  
Carboxy Terminal

ABSTRACT Cyclin-dependent kinases (Cdks) were originally identified as regulators of eukaryotic cell cycle progression, but several Cdks were subsequently shown to perform important roles as transcriptional regulators. While the mechanisms regulating the Cdks involved in cell cycle progression are well documented, much less is known regarding how the Cdks that are involved in transcription are regulated. In Saccharomyces cerevisiae, Bur1 and Bur2 comprise a Cdk complex that is involved in transcriptional regulation, presumably mediated by its phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. To investigate the regulation of Bur1 in vivo, we searched for high-copy-number suppressors of a bur1 temperature-sensitive mutation, identifying a single gene, CAK1. Cak1 is known to activate two other Cdks in yeast by phosphorylating a threonine within their conserved T-loop domains. Bur1 also has the conserved threonine within its T loop and is therefore a potential direct target of Cak1. Additional tests establish a direct functional interaction between Cak1 and the Bur1-Bur2 Cdk complex: Bur1 is phosphorylated in vivo, both the conserved Bur1 T-loop threonine and Cak1 are required for phosphorylation and Bur1 function in vivo, and recombinant Cak1 stimulates CTD kinase activity of the purified Bur1-Bur2 complex in vitro. Thus, both genetic and biochemical evidence demonstrate that Cak1 is a physiological regulator of the Bur1 kinase.

scholarly journals GPI7Involved in Glycosylphosphatidylinositol Biosynthesis Is Essential for Yeast Cell Separation

2004 ◽  
Vol 279 (50) ◽  
pp. 51869-51879 ◽  
Author(s):  
Morihisa Fujita ◽  
Takehiko Yoko-o ◽  
Michiyo Okamoto ◽  
Yoshifumi Jigami
Keyword(s):  
Cell Separation ◽  
Specific Growth ◽  
Growth Defect ◽  
Cell Cortex ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Loss Of Function ◽  
Septal Region ◽  
Multicopy Suppressors ◽  
Specific Proteins

GPI7is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) inSaccharomyces cerevisiae. We isolatedgpi7mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature.WSC1,RHO2,ROM2,GFA1, andCDC5genes were isolated as multicopy suppressors ofgpi7-2mutant. Multicopy suppressors could suppress the growth defect ofgpi7mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect ofgpi7mutants. Furthermore, deletion ofEGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of thegpi7mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating thatGPI7plays an important role in cell separation via the GPI-based modification of daughter-specific proteins inS. cerevisiae.

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