Genetic and biochemical analysis of Cdc42p function in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
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Cell Cycle Proteins↗
Biomedical subjects
D I Johnson
Publications and source records attributed to D I Johnson.
Using external data and databases: issues and sources.
Third party payers, employers, and accrediting organizations such as the Joint Commission for Accreditation of Healthcare Organization have played an important role in moving health care organizations toward the use of outcomes to measure quality. In addition, other external forces, such as shrinking health care resources and an increasingly competitive health care environment, have pressured organizations to implement continuous quality improvement (CQI) programs. These programs are an important component in effective and efficient organizational operations. Health care organizations must provide the best quality at the lowest cost to survive today. CQI programs incorporate internal and external comparisons to demonstrate improvement. Internal and external comparisons are important to validate the organization's level of quality. The article concentrates on the use of external comparisons, sources of external data or databases, and issues involved in external comparisons or benchmarking.
Centers for Medicare and Medicaid Services, U.S.↗
Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42.
Cdc24p and Cdc42p are involved in the control of cell polarity during the Saccharomyces cerevisiae cell cycle. Cdc42p is a member of the Ras superfamily of GTPases and Cdc24p displays limited amino-acid sequence similarity with the Dbl proto-oncoprotein, which acts to stimulate guanine-nucleotide exchange on human Cdc42p. We have performed several genetic experiments to test whether Cdc24p and Cdc42p interact within the cell. First, overexpression of Cdc24p suppressed the dominant-negative cdc42D118A allele. Second, overexpression of wild-type CDC24 and CDC42 genes together was a lethal event resulting in a morphological phenotype of large, round, unbudded cells, indicating a loss of cell polarity. Third, a cdc24ts cdc42ts double mutant exhibited a synthetic-lethal phenotype at the semi-permissive temperature of 30 degrees C. These data suggest that Cdc24p and Cdc42p interact within the cell and that Cdc24p may be involved in the regulation of Cdc42p activity.
Alleles↗
Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product.
Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.
Alkyl and Aryl Transferases↗
Isolation and sequence analysis of CDC43, a gene involved in the control of cell polarity in Saccharomyces cerevisiae.
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Alkyl and Aryl Transferases↗
The tyrosine repressor negatively regulates aroH expression in Escherichia coli.
The levels of the tryptophan-sensitive isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli, encoded by the aroH gene, were elevated in tyrR and/or trpR mutants. The effect of tyrR and trpR lesions on aroH expression was confirmed by using a lacZ reporter system. The mutational elimination of either repressor led to a threefold increase in beta-galactosidase.
3-Deoxy-7-Phosphoheptulonate Synthase↗
Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.
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.
Amino Acid Sequence↗
Isolation and sequence analysis of CDC43, a gene involved in the control of cell polarity in Saccharomyces cerevisiae.
The Saccharomyces cerevisiae CDC43 gene product is involved in establishing cell polarity during the cell-division cycle. When grown at restrictive temperatures, temperature-sensitive cdc43 mutants are unable to form buds and display delocalized cell-surface deposition [Adams et al., J. Cell Biol. (1990) in press]. We have isolated a cdc43-complementing plasmid from a yeast genomic-DNA library and localized the CDC43 gene, by subcloning and transposon-mutagenesis experiments, to a 1.2-kb region of DNA that contained only one significant ATG-initiated open reading frame of 213 codons. The putative CDC43 gene product contains a possible nuclear-localization signal sequence, a cysteine-rich domain and a histidine-rich domain, and a region that is similar in structure to alpha-helix-turn-alpha-helix structural domains present in some prokaryotic and eukaryotic DNA-binding proteins.
Alkyl and Aryl Transferases↗
Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42.
We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.
Amino Acid Sequence↗
CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.
Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.
Cell Cycle↗
Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity.
The Saccharomyces cerevisiae CDC42 gene product is involved in the morphogenetic events of the cell division cycle; temperature-sensitive cdc42 mutants are unable to form buds and display delocalized cell-surface deposition at the restrictive temperature (Adams, A. E. M., D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R. Pringle. 1990. J. Cell Biol. 111:131-142). To begin a molecular analysis of CDC42 function, we have isolated the CDC42 gene from a yeast genomic DNA library. The use of the cloned DNA to create a deletion of CDC42 confirmed that the gene is essential. Overexpression of CDC42 under control of the GAL10 promoter was not grossly deleterious to cell growth but did perturb the normal pattern of selection of budding sites. Determination of the DNA and predicted amino acid sequences of CDC42 revealed a high degree of similarity in amino acid sequence to the ras and rho (Madaule, P., R. Axel, and A. M. Myers. 1987. Proc. Natl. Acad. Sci. 84:779-783) families of gene products. The similarities to ras proteins (approximately 40% identical or related amino acids overall) were most pronounced in the regions that have been implicated in GTP binding and hydrolysis and in the COOH-terminal modifications leading to membrane association, suggesting that CDC42 function also involves these biochemical properties. The similarities to the rho proteins (approximately 60% identical or related amino acids overall) were more widely distributed through the coding region, suggesting more extensive similarities in as yet undefined biochemical properties and functions.
Amino Acid Sequence↗
Mapping of the Saccharomyces cerevisiae CDC3, CDC25, and CDC42 genes to chromosome XII by chromosome blotting and tetrad analysis.
CDC3, CDC25 and CDC42 were localized to chromosome XII by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis. Meiotic tetrad analyses further localized these genes to the region distal to the RDN1 locus on the right arm of the chromosome. The STE11 gene, which had previously been mapped to chromosome XII (Chaleff and Tatchell, 1985), was found to be tightly linked to ILV5. The data suggest a map order of CEN12-RDN1-CDC42-(CDC25-CDC3)-(ILV5- STE11)-URA4. Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region of chromosome XII.
Blotting, Southern↗
Structural analysis of the ileR locus of Escherichia coli K12.
The Ile repressor protein negatively controls expression from the ilv and thr promoters of Escherichia coli K12. Its existence was inferred from an analysis of the phenotypes of the ileR mutant avr-16 (Johnson, D. I., and Somerville, R. L. (1983) J. Bacteriol. 155, 49-55). The nucleotide sequence of ileR, the structural gene for Ile repressor, has been determined. A DNA segment of 300 base pairs constitutes the ileR gene. The predicted gene product, a protein of 100 amino acids (molecular weight 11,823) has primary structural features reminiscent of other double-stranded DNA-binding regulatory proteins. S1 nuclease mapping of the 5' terminus of ileR mRNA revealed two discrete species whose startpoints differed by approximately 47 nucleotides. The ileR gene, like other repressors for amino acid biosynthetic systems, is autogenously regulated at the transcriptional level. Within the ileR promoter region lie two 18-base pair segments of DNA bearing significant homology to putative operator targets also found within the thr and ilv promoters. A second open reading frame capable of specifying a protein of 83 amino acids, designated orf83, is transcribed divergently from the ileR gene. There are 202 base pairs separating the first codons of the two genes. S1 nuclease mapping of the 5' terminus of orf83 mRNA revealed two discrete species whose startpoints differed by approximately 27 nucleotides. The upstream promoters for ileR and orf83 overlap in their -35 regions.
Amino Acid Sequence↗
New regulatory genes involved in the control of transcription initiation at the thr and ilv promoters of Escherichia coli K-12.
The gene ileR+, considered to encode a transacting protein involved in the regulation of the thr and ilv operons of Escherichia coli, has been cloned and localized to a 1.2 Kb BglII-SalI fragment of DNA. In strains harboring attenuation-defective fusions of lacZ to the promoter regions of the thr and ilv operons, ileR mutations lead to beta-galactosidase levels higher than those of the deattenuated parental strains. Reduced utilization of the thr and ilv promoters was observed in ileR cells harboring either ilvR+ plasmids or plasmids leading to the hyperproduction of Trp repressor. These results support the idea that ileR+ encodes a repressor protein that negatively affects the expression of the thr and ilv operons. Two additional trans-acting positive regulatory elements that act at the thr and ilv promoters have been identified by an analysis of deletion mutants. It thus appears that there exist positive as well as negative controlling elements that can act independently of attenuation to modulate the ilv and thr operons.
Cloning, Molecular↗
Evidence that repression mechanisms can exert control over the thr, leu, and ilv operons of Escherichia coli K-12.
Mutants of Escherichia coli K-12 resistant to either the threonine analog DL-alpha-amino-beta-hydroxyvaleric acid or the leucine analog 5',5',5'-trifluoro-DL-leucine were isolated. One DL-alpha-amino-beta-hydroxyvaleric acid-resistant mutant strain, designated SP572, constitutively expressed the thr and ilv operons. The mutant allele, avr-16, was localized between trpR and the thr operon at min 0. The wildtype allele of avr-16, designated ileR, is trans dominant. One 5',5',5'-trifluoro-DL-leucine-resistant mutant strain, designated FLR9, expressed the leu and ilv operons constitutively. The mutant allele, flr-9, is linked to entA at min 13. The constitutive expression of the thr, leu, and ilv operons in mutants avr-16 and flr-9 was partly reversed in cells harboring a plasmid, which leads to elevated levels of the trpR gene product, the Trp aporepressor protein. Operator-like sequences situated upstream from the transcription startpoints of the thr, leu, and ilv operons are plausible candidates for targets of systems of repressor-operator control functioning in parallel with attenuation.
Chromosome Mapping↗
Pre-issue assessment and counselling as a component of hearing-aid provision.
Historically hearing-aid uptake and use in the United Kingdom has been poor. One contributory factor is believed to have been lack of basic knowledge about hearing loss and the benefits and problems of amplification. The scheme operating in South Manchester is described, wherein potential hearing aid users receive counselling both before and after the provision of the aid. The pre-fitting interview is partly for assessment purposes--determining the communication problems of the individual and family in order that the most appropriate assistance can be provided--and partly for counselling purposes, including attitude modification (if appropriate) and development of realistic expectations. The post-fitting counselling utilizes selected and trained volunteers who visit hearing-aids users in their homes at carefully chosen intervals. Additionally the great majority (95%) of hearing-aid users are reviewed by the Hearing Therapist at the Clinic about 1 month after issue. Evidence from Studies carried out in Manchester suggest that such a service considerably increases both use of and satisfaction with hearing aids.
Attitude↗
Inhibition of carboxypeptidase A catalyzed peptide hydrolysis by 3-phenylpropanoate at activating and nonactivating substrate concentrations.
The carboxypeptidase A catalyzed hydrolyses of five structurally related dipeptide substrates in the presence of the inhibitor 3-phenylpropanoate have been studied. At nonactivating substrate concentrations, 3-phenylpropanoate is a mixed inhibitor of carbobenzoxyglycyl-L-phenylalanine hydrolysis and a noncompetitive inhibitor of the hydrolyses of benzoylglycyl-L-phenylalanine, cinnamoylglycyl-L-phenylalanine, hydrocinnamoylglycyl-L-phenylalanine, and acetylglycyl-L-phenylalanine. When carbobenzoxyglycyl-L-phenylalanine and benzoylglycyl-L-phenylalanine exhibit substrate activation, inhibition by 3-phenylpropanoate is mixed but appears to be mostly competitive. Proposed here is a site for the binding of 3-phenylpropanoate along with a kinetic mechanism consistent with these data.
Carboxypeptidases↗
Contraception and sterilization.
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Contraception↗