构建一种包含产朊假丝酵母自主复制序列的新型酵母克隆载体。

Construction of a new yeast cloning vector containing autonomous replication sequences from Candida utilis.

作者信息

Hsu W H, Magee P T, Magee B B, Reddy C A

出版信息

J Bacteriol. 1983 Jun;154(3):1033-9. doi: 10.1128/jb.154.3.1033-1039.1983.

DOI:10.1128/jb.154.3.1033-1039.1983

PMID:6304003

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC217572/

Abstract

DNA sequences from the Candida utilis genome which, when cloned into a yeast integration plasmid (YIp5), confer on YIp5 the ability to replicate autonomously in Saccharomyces cerevisiae are described. Several recombinant plasmids which transform S. cerevisiae YNN27 to Ura3+ with an efficiency of 2 X 10(3) transformants per microgram of DNA were obtained. One of the recombinant plasmids, pHMR22 (6.6 kilobases) contains ars (autonomous replication sequence), which is homologous with two different DNA fragments of the C. utilis genome but has no detectable homology to total DNA from Candida albicans, Pachysolen tannophilus, or S. cerevisiae. Restriction and subcloning analyses of pHMR22 showed that Sau3A destroys the functions of cloned ars whereas there are no BamHI, PstI, SalI, HindIII, EcoRI, or PvuII sites in the region of ars which is required for its functional integrity. Thus, pHMR22 appears to be a useful vector for cloning desired genes in S. cerevisiae.

摘要

描述了来自产朊假丝酵母基因组的DNA序列，当将其克隆到酵母整合质粒（YIp5）中时，赋予YIp5在酿酒酵母中自主复制的能力。获得了几种重组质粒，它们能将酿酒酵母YNN27转化为Ura3 +，转化效率为每微克DNA产生2×10³个转化子。其中一种重组质粒pHMR22（6.6千碱基）含有自主复制序列（ars），它与产朊假丝酵母基因组的两个不同DNA片段同源，但与白色念珠菌、嗜鞣管囊酵母或酿酒酵母的总DNA没有可检测到的同源性。对pHMR22的限制性酶切和亚克隆分析表明，Sau3A会破坏克隆的ars的功能，而在ars功能完整性所需区域没有BamHI、PstI、SalI、HindIII、EcoRI或PvuII位点。因此，pHMR22似乎是在酿酒酵母中克隆所需基因的有用载体。