Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

scholarly journals Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

scholarly journals DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4675-4684 ◽  
Author(s):  
F R Cross
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Critical Size ◽  
Size Control ◽  
Mutant Gene ◽  
G1 Phase ◽  
Wild Type ◽  
Alpha Factor ◽  
Multiple Copies ◽  
Kinetics Of

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

scholarly journals Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p.

1995 ◽  
Vol 128 (4) ◽  
pp. 509-523 ◽  
Author(s):  
R Erdmann ◽  
G Blobel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Growth Defect ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
Daughter Cells ◽  
Sds Page ◽  
Multiple Buds ◽  
Media Form ◽  
Tubular Membranes

We have purified peroxisomal membranes from Saccharomyces cerevisiae after induction of peroxisomes in oleic acid-containing media. About 30 distinct proteins could be discerned among the HPLC- and SDS-PAGE-separated proteins of the high salt-extracted peroxisomal membranes. The most abundant of these, Pmp27p, was purified and the corresponding gene PMP27 was cloned and sequenced. Its primary structure is 32% identical to PMP31 and PMP32 of the yeast Candida biodinii (Moreno, M., R. Lark, K. L. Campbell, and M. J. Goodman. 1994. Yeast. 10:1447-1457). Immunoelectron microscopic localization of Pmp27p showed labeling of the peroxisomal membrane, but also of matrix-less and matrix containing tubular membranes nearby. Electronmicroscopical data suggest that some of these tubular extensions might interconnect peroxisomes to form a peroxisomal reticulum. Cells with a disrupted PMP27 gene (delta pmp27) still grew well on glucose or ethanol, but they failed to grow on oleate although peroxisomes were still induced by transfer to oleate-containing media. The induced peroxisomes of delta pmp27 cells were fewer but considerably larger than those of wild-type cells, suggesting that Pmp27p may be involved in parceling of peroxisomes into regular quanta. delta pmp27 cells cultured in oleate-containing media form multiple buds, of which virtually all are peroxisome deficient. The growth defect of delta pmp27 cells on oleic acid appears to result from the inability to segregate the giant peroxisomes to daughter cells.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766 ◽  
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

scholarly journals Mutant Tax Protein from Bovine Leukemia Virus with Enhanced Ability To Activate the Expression of c-fos

2002 ◽  
Vol 76 (5) ◽  
pp. 2557-2562 ◽  
Author(s):  
Shigeru Tajima ◽  
Yoko Aida
Keyword(s):  
Bovine Leukemia Virus ◽  
Mutational Analysis ◽  
Leukemia Virus ◽  
Upstream Sequence ◽  
Wild Type ◽  
Cell Leukemia Virus Type ◽  
Mutant Proteins ◽  
Tax Protein ◽  
Human T Cell ◽  
Carg Box

ABSTRACT Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. We previously identified several mutants of the BLV Tax protein with an ability to transactivate transcription via the BLV enhancer that is significantly greater than that of the wild-type Tax protein. Moreover, the mutant proteins also activated other viral enhancers, such as the enhancer of human T-cell leukemia virus type 1, which cannot be activated by wild-type BLV Tax. In this study, we demonstrated that the mutant proteins but not wild-type protein activate the upstream sequence of the human c-fos gene, which contains two major cis-acting elements, the CArG box and cyclic AMP-responsive element (CRE) motif. The mutant protein also strongly increased levels of endogenous c-fos mRNA in both human and bovine cell lines. On the other hand, the wild-type Tax protein has no activity to activate the expression of human c-fos, indicating that wild-type BLV Tax might discriminate between human and bovine c-fos promoter sequences. Deletion and point-mutational analysis of the cis-acting elements revealed that both the CArG box and the CRE motif were indispensable for the activation of c-fos by the mutant BLV Tax protein. Our results suggest that the mutant BLV Tax proteins might not only have the ability to enhance the production of virus particles but might also have increased ability to induce leukemia.

scholarly journals Complete Virion Assembly with Scaffolding Proteins Altered in the Ability To Perform a Critical Conformational Switch

2009 ◽  
Vol 83 (15) ◽  
pp. 7391-7396 ◽  
Author(s):  
James E. Cherwa ◽  
Bentley A. Fane
Keyword(s):  
Amino Acid ◽  
Scaffolding Proteins ◽  
Wild Type ◽  
Conformational Switch ◽  
Mutant Proteins ◽  
Virion Assembly ◽  
Glycine Residue ◽  
Atomic Structures ◽  
Dominant Lethal ◽  
Α Helix

ABSTRACT In the φX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30° kink to form in α-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses.

scholarly journals Aggregation of prion protein with insertion mutations is proportional to the number of inserts

2007 ◽  
Vol 403 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Shuiliang Yu ◽  
Shaoman Yin ◽  
Chaoyang Li ◽  
Poki Wong ◽  
Binggong Chang ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Biochemical Properties ◽  
Mutant Protein ◽  
Insertion Mutant ◽  
Wild Type ◽  
Mutant Proteins ◽  
Severity Of The Disease ◽  
Α Helix ◽  
Insertion Mutations

Mutation in the prion gene, PRNP, accounts for approx. 10–15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrPC (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP8OR, and the other with five more octapeptide repeats, rPrP10OR. We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and α-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrPC. Accordingly, rPrP10OR is also more efficient in promoting the aggregation of rPrPC than rPrP8OR. These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.

DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (11) ◽  
pp. 4675-4684
Author(s):  
F R Cross
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Critical Size ◽  
Size Control ◽  
Mutant Gene ◽  
G1 Phase ◽  
Wild Type ◽  
Alpha Factor ◽  
Multiple Copies ◽  
Kinetics Of

The mating pheromone alpha-factor arrests Saccharomyces cerevisiae MATa cells in the G1 phase of the cell cycle. Size control is also exerted in G1, since cells do not exit G1 until they have attained a critical size. A dominant mutation (DAF1-1) which causes both alpha-factor resistance and small cell size (volume about 0.6-fold that of the wild type) has been isolated and characterized genetically and by molecular cloning. Several alpha-factor-induced mRNAs were induced equivalently in daf1+ and DAF1-1 cells. The DAF1-1 mutation consisted of a termination codon two-thirds of the way through the daf1+ coding sequence. A chromosomal deletion of DAF1 produced by gene transplacement increased cell volume about 1.5-fold; thus, DAF1-1 may be a hyperactive or deregulated allele of a nonessential gene involved in G1 size control. Multiple copies of DAF1-1 also greatly reduced the duration of the G1 phase of the cell cycle.

scholarly journals Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2443-2451 ◽  
Author(s):  
A Percival-Smith ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intergenic Region ◽  
Mutational Analysis ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Regulatory Sequences ◽  
Wild Type ◽  
Base Pairs ◽  
Differential Hybridization ◽  
Dna Library

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

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