粟酒裂殖酵母his7+基因的克隆与操作作为分子遗传学研究的新选择标记
Cloning and manipulation of the Schizosaccharomyces pombe his7+ gene as a new selectable marker for molecular genetic studies.
作者信息
Apolinario E, Nocero M, Jin M, Hoffman C S
机构信息
Department of Biology, Boston College, Chestnut Hill, MA 02167.
出版信息
Curr Genet. 1993 Dec;24(6):491-5. doi: 10.1007/BF00351711.
Abstract
We have cloned the his7+ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366. The his7+ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7+ encodes a phosphoribosyl-AMP cyclohydrase. Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment. We describe the construction of plasmids to facilitate the use of his7+ as a selectable marker in S. pombe studies. Plasmid pEA2 carries his7+ cloned into the pUC18 polylinker. From either pEA2 or the original his7+ clone, pMN1, fragments carrying his7+ can be isolated using a variety of restriction enzymes for the construction of gene disruptions. Plasmid pEA500 is a cloning vector that carries his7+ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants.
摘要
我们通过对隐性突变等位基因his7-366的互补作用，克隆了裂殖酵母粟酒裂殖酵母的his7+基因。his7+基因能够互补大肠杆菌hisI基因的突变，这表明his7+编码一种磷酸核糖基-AMP环化水解酶。亚克隆实验将该基因定位到一个1.9kb的XbaI-BglII片段上。我们描述了质粒的构建，以促进his7+在粟酒裂殖酵母研究中作为选择标记的使用。质粒pEA2携带克隆到pUC18多克隆位点的his7+。从pEA2或原始的his7+克隆pMN1中，可以使用多种限制酶分离携带his7+的片段，用于构建基因破坏。质粒pEA500是一种克隆载体，它携带his7+和ars1，但仍保留使用蓝/白颜色筛选来鉴定重组体的能力。
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