在酵母中构建新的酵母载体并克隆肺炎克雷伯氏菌的 nif（氮固定）基因簇。

Construction of new yeast vectors and cloning of the nif (nitrogen fixation) gene cluster of Klebsiella pneumoniae in yeast.

机构信息

Laboratoire de Biologie Génétique et Moléculaire, Université de Paris Sud, Bâtiment 400, 91405, Orsay, France.

出版信息

Curr Genet. 1981 Jul;3(3):173-80. doi: 10.1007/BF00429819.

PMID:24190129

Abstract

Two vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif(+) phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.

摘要

构建了两个适合酵母转化的载体，称为 pG63.11（7.6 Kb）和 pHCG3（9.6 Kb）。pHCG3 载体具有 cosmid 特性。两个载体都包含一个 3.3 Kb 的 EcoRI-HindIII 片段，来自酵母，携带酵母 URA3 基因（1.1 Kb）和 2 µm 质粒的复制起点（2.2 Kb）。它们赋予氨苄青霉素抗性，并且含有 5 个独特的 EcoRI、HpaI、HindIII、BamHI 和 SalI 限制位点。用 cosmid pHCG3 克隆了分别由两个 17 和 26 Kb 的 HindIII 片段携带的肺炎克雷伯菌的固氮（nif）基因簇。所得 cosmid，称为 pGPC875（53 Kb），赋予大肠杆菌 Nif(+)表型，通过转化引入酵母。在转化体中未检测到乙炔还原活性。然而，已经表明，整个固氮信息可以在酵母中复制并保持完整，生长超过 50 代。