DEVELOPMENT OF THE YEAST PICHIA-PASTORIS AS A MODEL ORGANISM FOR A GENETIC AND MOLECULAR ANALYSIS OF PEROXISOME ASSEMBLY
DEVELOPMENT OF THE YEAST PICHIA-PASTORIS AS A MODEL ORGANISM FOR A GENETIC AND MOLECULAR ANALYSIS OF PEROXISOME ASSEMBLY
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DOI：
10.1002/yea.320080805
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发表时间：
1992-08-01
期刊：
影响因子：
2.6
通讯作者：
SUBRAMANI, S
中科院分区：
文献类型：
--
作者：
GOULD, SJ;MCCOLLUM, D;SUBRAMANI, S
我们描述了过氧化物酶体组装（pas）缺陷的酵母毕赤酵母突变体的分离。巴斯德毕赤酵母的这些突变体只能通过它们不能在甲醇和油酸中生长来识别，其利用需要过氧化物酶体酶，并且通过电子显微镜和生化分级分离实验判断，通过不存在正常的过氧化物酶体来定义。这些突变体是单个位点遗传缺陷的结果，代表至少八个不同的互补组。通过简单筛选不能使用甲醇和油酸的突变体来分离巴斯德毕赤酵母的pas突变体代表了比其他生物体中可能的更有效的鉴定pas突变体的方法。为了充分利用这一优势，我们还开发了用于毕赤酵母遗传和分子操作的新试剂。其中包括一组具有基本上野生型遗传背景的营养缺陷型菌株、充当大肠杆菌-P 的质粒。毕赤酵母穿梭载体和基因组 DNA 文库，用于通过突变体的功能互补或核酸杂交分离毕赤酵母基因。大量 pas 突变体及其分子分析所需试剂的可用性应有助于分离和表征参与过氧化物酶体组装的基因。
We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotrophic strains with an essentially wild-type genetic background, plasmids that act as Escherichia coli-P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.