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首页> 外文OA文献 >Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.
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Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

机译：PEP3的分离和鉴定，PEP3是酿酒酵母中液泡生物发生所必需的基因。

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The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.
机译：通过互补pep3-12菌株中的羧肽酶Y缺乏症，从野生型基因组文库中克隆了酿酒酵母PEP3基因。亚克隆互补结果将PEP3基因定位在一个3.8-kb DNA片段上。确定了该片段的DNA序列。 2754 bp的开放阅读框预测PEP3基因产物是亲水的107 kDa蛋白，与任何已知蛋白均无显着相似性。 PEP3预测的蛋白在其C末端附近有一个锌指（CX2CX13CX2C），与腺病毒E1a锌指具有间距和轻微的序列相似性。放射性标记的PEP3 DNA探针与对数期和双生滞后期细胞提取物中的3.1 kb RNA转录物杂交。带有pep3缺失/破坏等位基因的细胞是可行的，蛋白酶A，蛋白酶B和羧肽酶Y抗原的水平降低，可抑制的碱性磷酸酶活性降低，通过荧光显微镜和电子显微镜观察到的正常液泡样细胞器非常少，但丰度为羧基荧光素双乙酸盐染色的极小囊泡在37°C时受到严​​重抑制，不能形成孢子（如纯合子）。表达双功能PEP3 :: SUC2融合蛋白的细胞的分级分离表明，PEP3基因产物在对数期和静止期细胞中均以低丰度存在，并且是液泡状外周膜蛋白。序列同一性证实PEP3和VPS18（J.S.Robinson，T.R.Graham，和S.D.Emr，Mol.Cell.Biol.11：5813-5824，1991）是相同的基因。

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