Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified from S. pombe. This stimulation was species-specific, because S. pombe Mediator could not stimulate TFIIH purified from S. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.     

CaSRB9基因的克隆及其在酿酒酵母形态发生中的作用

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿酒酵母SRB9同源的新基因 ,命名为CaSRB9。该基因全长 4998bp ,编码一种16 6 5个氨基酸的蛋白质。在双倍体酿酒酵母中CaSRB9可以部分互补MAPK途径基因缺失株以及 flo8缺失株的菌丝生长缺陷 ;在单倍体酿酒酵母中表达能够互补 flo8缺失株的侵入生长缺陷 ,但在MAPK途径基因缺失株中不能形成侵入生长     

Identification of Candida albicans genes that induce Saccharomyces cerevisiae cell adhesion and morphogenesis

Morphogenesis and adhesion to host tissues and medical devices contribute to the virulence of Candida albicans, the most common fungal pathogen isolated from humans. However, identification of molecular mechanisms of C. albicans adhesion and morphogenesis has been impaired by the lack of effective molecular and genetic tools available for this organism. Saccharomyces cerevisiae provides an attractive model system for studying C. albicans adhesion and morphogenesis because of its well-characterized genetics and gene expression systems. To gain insight into the genetic mechanisms of C. albicans adhesion and morphogenesis, we used a parallel plate flow chamber to screen and quantitatively characterize attachment to polystyrene of an adhesion-deficient nonfilamentous flo8Delta S. cerevisiae strain expressing a C. albicans genomic library. We identified six C. albicans genes that are capable of promoting cell adhesion and pseudohyphal development in S. cerevisiae. We also analyzed the ability of these adhesion-promoting genes to regulate the expression of FLO11, which encodes an endogenous S. cerevisiae adhesin. One C. albicans gene, EAP1, appears to directly mediate adhesion and morphogenesis while the remaining five (EAP2, SWI1, MSB1, AAF1, and TEC1) upregulate expression of endogenous S. cerevisiae adhesins. These results suggest that S. cerevisiae is a useful system for molecular characterization of factors that regulate C. albicans adhesion and morphogenesis and that parallel plate flow chamber-based adhesion assays can be used in conjunction with genetic screens to identify molecular mechanisms regulating fungal cell adhesion.     

The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit

The amplification of the TLO (for telomere-associated) genes in Candida albicans, compared to its less pathogenic, close relative Candida dubliniensis, suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis.