Ostrander D B, Gorman J A
Department of Microbial Molecular Biology, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ 08543-4000, USA.
Yeast. 1997 Jul;13(9):871-80. doi: 10.1002/(SICI)1097-0061(199707)13:9<871::AID-YEA127>3.0.CO;2-2.
We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells.
我们已经分离出白色念珠菌的肌动蛋白结合蛋白基因PFY1。基于高度同源区域设计的简并寡核苷酸引物被用于获得该基因的聚合酶链反应扩增拷贝。然后将其用作探针从白色念珠菌基因组文库中分离该基因。我们的研究表明，全长基因在大肠杆菌中不稳定。对几个克隆进行了测序，预测的氨基酸序列显示与其他生物的肌动蛋白结合蛋白具有同源性，最显著的是酿酒酵母。Northern分析表明该基因在白色念珠菌中表达。在用酿酒酵母启动子替换白色念珠菌启动子之前，在酿酒酵母细胞中表达该基因的尝试均未成功。该基因在需要酿酒酵母肌动蛋白结合蛋白的细胞中表现出功能互补性。当该基因在酿酒酵母细胞中表达时，针对分离出的白色念珠菌肌动蛋白结合蛋白产生的抗体识别出预测分子量的蛋白质。
