AIM Capl p, encoded by CAP1 in Candida albicans, is highly homologous to Saccharomyces cerevisiae transcription factor Yapl p. It has been associated with tolerance to oxidative stress and resistance to a variety of t...AIM Capl p, encoded by CAP1 in Candida albicans, is highly homologous to Saccharomyces cerevisiae transcription factor Yapl p. It has been associated with tolerance to oxidative stress and resistance to a variety of toxicants previously. We used homemade microarray to reveal Capl p related genes in a broad spectrum as well as to lucubrate the functions of Capl p. METHODS Microarray analysis was used to identify differentially expressed genes between CAP1 deletion strain CJD21 and its parental strain CAI4. CAP1 over-expression strain was constructed to confirm the relationship between CAP1 and some differentially expressedgenes. Bioinformatics was applied to reveal promoters with Capl p binding site as well as the clusters of differentially expressed genes. RT-PCR and drug efflux analysis were used to lucubrate the functions of Caplp in Candida albicans.展开更多
HspA (also designated as SP21) of S.aurantiaca is synthesized during the developmental cell cycle or under stress conditions such as heat shock, oxygen limitation and indole treatment. Sequence alignment showed th...HspA (also designated as SP21) of S.aurantiaca is synthesized during the developmental cell cycle or under stress conditions such as heat shock, oxygen limitation and indole treatment. Sequence alignment showed that it belongs to α crystallin family of low molecular weight heat shock protein Immunoelectron microscopy revealed that HspA distributes mainly at the cell periphery in heat shocked cells and in fruiting body derived spores, either at the cell periphery or within the cytoplasm in indole induced cells. A unique transcription initiation site of the monocistronic hspA has been characterized by primer extension under heat shock and indole induced conditions. However, Northern analysis examined two transcripts under heat shock condition, but only one transcript under indole induction. Therefore, we suggest a posttranscriptional processing of hspA . messenger RNA. Promoter mapping results showed that the promoter region of hspA extends to about 200 bp upstream referring to the translation start site. No similarity has been found between hspA promoter and other known heat shock promoters of prokaryotes. The putative regulation regions upstream of hspA have been screened by gel shift assay. The result indicated that an enhancer may be involved in the expression of hspA . HspA has been produced in E.coli as a fusion protein. Size exclusion chromatography HPLC showed that HspA fusion protein forms an oligomeric complex of 26 subunits with a Mr of ca.560?kDa. When using citrate syntase (CS) as a model substrate to perform chaperone assay in vitro, HspA could interact with chemically denatured CS to form a stable complex and suppress the spontaneous aggregation of the denatured CS. In converse, when using insulin as a substrate, HspA could not prevent the spontaneous aggregation of β chain of chemically denatured insulin. We thus suppose that, physiologically, HspA prefers to bind with unfolded protein to prevent their precipitation. Taken together, the results demonstrated that HspA possesses the properties of a molecular chaperone. To investigate the function of HspA in vivo, null mutants of hspA have been constructed in which hspA has been replaced by neogene. However, no significant change in the phenotype has been observed with the mutants. This may due to a functional compensation of HspA existing in the S.aurantiaca cells.展开更多
文摘AIM Capl p, encoded by CAP1 in Candida albicans, is highly homologous to Saccharomyces cerevisiae transcription factor Yapl p. It has been associated with tolerance to oxidative stress and resistance to a variety of toxicants previously. We used homemade microarray to reveal Capl p related genes in a broad spectrum as well as to lucubrate the functions of Capl p. METHODS Microarray analysis was used to identify differentially expressed genes between CAP1 deletion strain CJD21 and its parental strain CAI4. CAP1 over-expression strain was constructed to confirm the relationship between CAP1 and some differentially expressedgenes. Bioinformatics was applied to reveal promoters with Capl p binding site as well as the clusters of differentially expressed genes. RT-PCR and drug efflux analysis were used to lucubrate the functions of Caplp in Candida albicans.
文摘HspA (also designated as SP21) of S.aurantiaca is synthesized during the developmental cell cycle or under stress conditions such as heat shock, oxygen limitation and indole treatment. Sequence alignment showed that it belongs to α crystallin family of low molecular weight heat shock protein Immunoelectron microscopy revealed that HspA distributes mainly at the cell periphery in heat shocked cells and in fruiting body derived spores, either at the cell periphery or within the cytoplasm in indole induced cells. A unique transcription initiation site of the monocistronic hspA has been characterized by primer extension under heat shock and indole induced conditions. However, Northern analysis examined two transcripts under heat shock condition, but only one transcript under indole induction. Therefore, we suggest a posttranscriptional processing of hspA . messenger RNA. Promoter mapping results showed that the promoter region of hspA extends to about 200 bp upstream referring to the translation start site. No similarity has been found between hspA promoter and other known heat shock promoters of prokaryotes. The putative regulation regions upstream of hspA have been screened by gel shift assay. The result indicated that an enhancer may be involved in the expression of hspA . HspA has been produced in E.coli as a fusion protein. Size exclusion chromatography HPLC showed that HspA fusion protein forms an oligomeric complex of 26 subunits with a Mr of ca.560?kDa. When using citrate syntase (CS) as a model substrate to perform chaperone assay in vitro, HspA could interact with chemically denatured CS to form a stable complex and suppress the spontaneous aggregation of the denatured CS. In converse, when using insulin as a substrate, HspA could not prevent the spontaneous aggregation of β chain of chemically denatured insulin. We thus suppose that, physiologically, HspA prefers to bind with unfolded protein to prevent their precipitation. Taken together, the results demonstrated that HspA possesses the properties of a molecular chaperone. To investigate the function of HspA in vivo, null mutants of hspA have been constructed in which hspA has been replaced by neogene. However, no significant change in the phenotype has been observed with the mutants. This may due to a functional compensation of HspA existing in the S.aurantiaca cells.
