RESUMO
Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.
Assuntos
Proteínas Fúngicas/metabolismo , Paracoccidioides/citologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Paracoccidioides/genética , Paracoccidioides/fisiologia , Fagocitose , RNA Antissenso , Virulência , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Deleção Cromossômica , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Biblioteca Gênica , Dados de Sequência Molecular , Morfogênese , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
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.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Ciclo Celular , Núcleo Celular/ultraestrutura , Mapeamento Cromossômico , Cromossomos Fúngicos , Cruzamentos Genéticos , Teste de Complementação Genética , Genótipo , Morfogênese , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/ultraestruturaRESUMO
Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. These proteins act as molecular switches by responding to exogenous and/or endogenous signals and relaying those signals to activate downstream components of a biological pathway. The 11 current members of the Cdc42p family display between 75 and 100% amino acid identity and are functional as well as structural homologs. Cdc42p transduces signals to the actin cytoskeleton to initiate and maintain polarized gorwth and to mitogen-activated protein morphogenesis. In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an important role in multiple actin-dependent morphogenetic events such as bud emergence, mating-projection formation, and pseudohyphal growth. In mammalian cells, Cdc42p regulates a variety of actin-dependent events and induces the JNK/SAPK protein kinase cascade, which leads to the activation of transcription factors within the nucleus. Cdc42p mediates these processes through interactions with a myriad of downstream effectors, whose number and regulation we are just starting to understand. In addition, Cdc42p has been implicated in a number of human diseases through interactions with its regulators and downstream effectors. While much is known about Cdc42p structure and functional interactions, little is known about the mechanism(s) by which it transduces signals within the cell. Future research should focus on this question as well as on the detailed analysis of the interactions of Cdc42p with its regulators and downstream effectors.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclo Celular/fisiologia , Polaridade Celular , Células Eucarióticas/enzimologia , Proteínas de Ligação ao GTP/fisiologia , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Citoesqueleto/metabolismo , Imunofluorescência , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/química , Humanos , Dados de Sequência Molecular , Proteínas Quinases/metabolismo , Estrutura Secundária de Proteína , Especificidade da Espécie , Fatores de Transcrição , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton. The GTPase Cdc42 regulates actin rearrangements and signal transduction pathways in all eukaryotic cells [1], and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF). Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane [2] [3] at sites of polarized growth [4]. We show that Cdc24p labeled with green fluorescent protein (GFP-Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells. Unexpectedly, GFP-Cdc24p also localized to the nucleus and GFP-Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother-bud necks during cytokinesis. The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p. The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother-bud neck, and mating projection. Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the G(beta) subunit Ste4p. These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Sítios de Ligação , Ciclo Celular , Proteínas de Ciclo Celular/química , Núcleo Celular/metabolismo , Polaridade Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas de Fluorescência Verde , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.
Assuntos
Ciclo Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase , Proteínas de Fluorescência Verde , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , MAP Quinase Quinase Quinases , Dados de Sequência Molecular , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genéticaRESUMO
Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.
Assuntos
Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/crescimento & desenvolvimento , Alelos , Sequência de Aminoácidos , Sequência de Bases , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/genética , Genes Dominantes , Genes Fúngicos , Genes Letais , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Fenótipo , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , GTP Fosfo-Hidrolases/genética , Genes Dominantes , Genes Letais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Peptídeos/síntese química , Fenótipo , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/fisiologia , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2ts) grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in alpha-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.
Assuntos
Polaridade Celular , Proteínas Fúngicas/análise , Proteínas de Ligação ao GTP/análise , Saccharomyces cerevisiae/química , Frações Subcelulares/química , Sequência de Aminoácidos , Ciclo Celular , Sequência Consenso , Proteínas Fúngicas/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Reprodução Assexuada , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.
Assuntos
Proteínas Fúngicas/genética , GTP Fosfo-Hidrolases/metabolismo , Schizosaccharomyces/genética , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Proteínas Fúngicas/metabolismo , Chaperonas Moleculares , Dados de Sequência Molecular , Mutação , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe , Homologia de Sequência de Aminoácidos , Proteínas de Transporte VesicularRESUMO
Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4ls at 23 degrees. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet- vna mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4ls cells were elongated or had misshapen buds. A cdc24-4ls delta vma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4ls synthetic-lethality was not simply due to altered vacuole function. The cdc24-4ls mutant, like delta vma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca2+ as well as Na+ in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na+ tolerance.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Mutação/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Cloreto de Sódio/farmacologia , ATPases Vacuolares Próton-Translocadoras , Vacúolos/fisiologia , Cloreto de Cálcio/farmacologia , Carboxiliases/genética , Proteínas de Ciclo Celular/genética , Polaridade Celular/genética , Cruzamentos Genéticos , Genes Fúngicos/fisiologia , Genes Letais/fisiologia , Teste de Complementação Genética , Fenótipo , Proteínas Proto-Oncogênicas/genética , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
The Saccharomyces cerevisiae G protein beta gamma dimer, Ste4p/Ste18p, acts downstream of the alpha subunit, Gpa1p, to activate the pheromone response pathway and therefore must interact with a downstream effector. Synthetic sterile mutants that exacerbate the phenotype of ste4-ts mutations were isolated to identify proteins that functionally interact with Ste4p. The identification of a ste18 mutant indicated that this screen could identify proteins that interact directly with Ste4p. The other mutations were in STE5 and the STE20 kinase gene, which act near Ste4p in the pathway, and a new gene called STE21. ste20 null mutants showed residual mating, suggesting that another kinase may provide some function. Overexpression of Ste5p under galactose control activated the pheromone response pathway. This activation was dependent on Ste4p and Ste18p and partially dependent on Ste20p. These results cannot be explained by the linear pathway of Ste4p-->Ste20p-->Ste5p. Overexpression of Cdc42p resulted in a slight increase in pheromone induction of a reporter gene, and overexpression of activated forms of Cdc42p resulted in a further twofold increase. Mutations in pheromone response pathway components did not suppress the lethality associated with the activated CDC42 mutations, suggesting that this effect is independent of the pheromone response pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Genes Fúngicos , Feromônios/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Clonagem Molecular , Cruzamentos Genéticos , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/genética , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Mutagênese , Fenótipo , Plasmídeos , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Temperatura , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.
Assuntos
Divisão Celular , Proteínas Contráteis , Schizosaccharomyces/enzimologia , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Actinas/genética , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citoesqueleto/metabolismo , Proteínas de Fluorescência Verde , Immunoblotting , Proteínas Luminescentes/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Profilinas , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Tiazóis/farmacologia , Tiazolidinas , Fatores de Tempo , Tropomiosina/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alquil e Aril Transferases , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Mapeamento por RestriçãoAssuntos
Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alquil e Aril Transferases , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , DNA Fúngico , Dados de Sequência Molecular , Saccharomyces cerevisiae/citologiaAssuntos
Proteínas de Ciclo Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Genes Fúngicos , Glutationa Transferase/biossíntese , Histidina , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Sitios de Sequências Rotuladas , Frações Subcelulares/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
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.
Assuntos
Bases de Dados Bibliográficas , Atenção à Saúde/normas , Garantia da Qualidade dos Cuidados de Saúde , Centers for Medicare and Medicaid Services, U.S. , Redes de Comunicação de Computadores , Atenção à Saúde/tendências , Humanos , Joint Commission on Accreditation of Healthcare Organizations , Sistemas On-Line , Estados UnidosRESUMO
As financial constraints are placed on departments of nursing, cost-saving solutions must be found. Even as budgets shrink, state and federal agencies create standards that often are more easily tracked with flowsheets and forms. In the past, typesetters were contacted for form production. Today, in-house desk-top publishing can help to reduce costs. The tools may already be available in the word processor currently used. With a little time spent learning advanced features of word processors, attractive desk-top forms, newsletters, brochures, and patient information sheets can be produced. In this article, some of the features available to produce desk-top documents are discussed, as are other desk-top publishing programs.
Assuntos
Editoração , Processamento de Texto/instrumentação , Enfermagem , SoftwareRESUMO
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.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Genes Reguladores , Óperon , Transcrição Gênica , Colífagos/genética , Cruzamentos Genéticos , Elementos de DNA Transponíveis , Genótipo , Fenótipo , Plasmídeos , Transdução GenéticaRESUMO
Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1ts allele and identified multiple mutations in the coding region and 5' promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23 degrees C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42W97R mutant displayed a cold-sensitive, los-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42T58A and cdc42S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe: these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts.