RESUMO
(1 --> 6)-beta-D-glucan is a key cell wall component of Saccharomyces cerevisiae and Candida albicans. Many genes are known to affect the levels or structure of this glucan, but their roles and a molecular description of the synthesis of (1 --> 6)-beta-D-glucan remain to be established and a method to measure (1 --> 6)-beta-D-glucan synthase activity in vitro would provide an enabling tool. Here, conditions for the detection of in vitro synthesis of this polymer are described. Crude membrane preparations from S. cerevisiae were isolated, and incubated in the presence of UDP-glucose and GTP. With anti-(1 --> 6)-beta-D-glucan-specific antibodies, a time-dependent increase in the amount of this glucan was demonstrated in a dot-blot assay, or through an inhibition enzyme immunoassay. Antibody specificity was validated by competition experiments using pustulan, a (1 --> 6)-beta-D-glucan, laminarin, a (1 --> 3)-beta-D-glucan, yeast mannan and glycogen. The identity of the reaction product was also demonstrated by its sensitivity to a recombinant (1 --> 6)-beta-D-glucanase. Extracts from mutants in 10 genes with a wide range of altered cell wall (1 --> 6)-beta-D-glucan levels were assayed for in vitro synthesis of the polymer. A strong correlation of in vitro synthase activity with in vivo glucan levels was found, providing genetic support for the specificity of the assay. The basis for the GTP-dependence of the synthase reaction was studied. Extracts from rho2, rho3, rho4 and rho5 null mutants had wild-type in vitro activity. In contrast, Rho1p overproduction led to increased in vitro synthesis, implicating Rho1p in the regulation of (1 --> 6)-beta-D-glucan synthesis.
Assuntos
Saccharomyces cerevisiae/metabolismo , beta-Glucanas/metabolismo , Western Blotting/métodos , Glucosiltransferases/metabolismo , Técnicas Imunoenzimáticas/métodos , beta-Glucanas/análiseRESUMO
In this study, we describe the effect of red and blue light on the timing of cell division, DNA synthesis, and activity and presence of cyclin-dependent kinases (CDKs), in synchronous cultures of the unicellular green alga Chlamydomonas reinhardtii. Cell division and DNA synthesis were found to occur later in cells grown in blue or white light, than in red light. CDK-like activity, measured using a histone H1 kinase assay, correspondingly occurred later in cultures that were grown in blue light compared to cultures grown in red light. The amount of CDK-like proteins, as detected using an antibody against the PSTAIRE motif, showed a maximum during the division phase. We conclude that the mechanism that causes the delay in the timing of cell division in blue light has its action before DNA replication takes place and also precedes the increase in CDK-like activity.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/efeitos da radiação , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/efeitos da radiação , Motivos de Aminoácidos , Animais , Western Blotting , Divisão Celular/fisiologia , Divisão Celular/efeitos da radiação , Células Cultivadas , Chlamydomonas reinhardtii/citologia , Quinases Ciclina-Dependentes/química , DNA/biossíntese , Luz , Proteínas Quinases/metabolismo , Fatores de TempoRESUMO
KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-beta-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-beta-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-beta-glucan-deficient strain fks1delta, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-beta-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-beta-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-beta-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-beta-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-beta-glucan levels in the wall.
Assuntos
Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Glucanos/análise , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , beta-Glucanas , Benzenossulfonatos , Parede Celular/química , Farmacorresistência Fúngica , Hidrolases/metabolismo , Hidrolases/farmacologia , Fatores Matadores de Levedura , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Micotoxinas/farmacologia , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoAssuntos
Leveduras/citologia , Divisão Celular , Parede Celular/metabolismo , Quitina/biossíntese , Quitina/metabolismo , Glucanos/biossíntese , Glucanos/metabolismo , Transdução de Sinais , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismoRESUMO
In this paper we study whether and how a number of arbitrarily chosen marker mutations interact in their effect on fitness, which is relevant for our understanding of the evolution of sex. If epistasis is synergistic, the main function of sex may be to facilitate selection against deleterious mutations. We use strains of the filamentous fungus Aspergillus niger with variable combinations of marker mutations that have been obtained by isolating segregants from a diploid between a wild-type strain and a related strain carrying a marker mutation on each of its eight chromosomes. The marker mutations include five auxotrophic and two resistance mutations. As a measure of fitness the mycelium growth rate on supplemented medium has been used. The results suggest that the marker mutations have independent effects on fitness, and hence they do not support the deterministic mutation hypothesis of the evolution of sex. The apparent linear relationship between mutation number and log fitness is the result of interactions of opposite type (i.e., synergistic and antagonistic) that cancel each other's effect. However, due to an isolation bias caused by the fact that not all possible strains with many mutations could be isolated, the results may be relatively biased towards an antagonistic relationship between mutation number and log fitness.