Colthurst D R, Schauder B S, Hayes M V, Tuite M F
Biological Laboratory, University of Kent, Canterbury, UK.
Mol Microbiol. 1992 Apr;6(8):1025-33. doi: 10.1111/j.1365-2958.1992.tb02168.x.
As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.
与许多其他真菌一样,包括出芽酵母酿酒酵母,二态性真菌白色念珠菌编码新型翻译因子延伸因子3(EF-3)。使用快速亲和层析方法,从白色念珠菌中纯化出均一的EF-3,其表观分子量为128 kDa。针对白色念珠菌EF-3产生的多克隆抗体也与酿酒酵母的EF-3发生交叉反应。同样,在Southern杂交分析中,酿酒酵母TEF3基因(编码EF-3)与白色念珠菌的基因组DNA发生交叉杂交,表明白色念珠菌基因组中存在一个与TEF3密切相关的单一基因。通过使用0.7 kb聚合酶链反应扩增的DNA片段筛选白色念珠菌基因文库克隆了该基因。对克隆片段的200 bp进行DNA序列分析,结果显示一个开放阅读框,在编码的65个氨基酸区域内,推测的白色念珠菌EF-3基因与其酿酒酵母对应基因之间的预测氨基酸同一性为51%。通过证明其能够拯救原本无法存活的酿酒酵母tef3:HIS3缺失突变体,证实了克隆的白色念珠菌序列确实编码EF-3。因此,白色念珠菌的EF-3与酿酒酵母的EF-3在结构和功能上均显示出相似性。