Tomenchok D M, Brandriss M C
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103.
J Bacteriol. 1987 Dec;169(12):5364-72. doi: 10.1128/jb.169.12.5364-5372.1987.
The PRO1, PRO2, and PRO3 genes were isolated by functional complementation of pro1, pro2, and pro3 (proline-requiring) strains of Saccharomyces cerevisiae. Independent clones with overlapping inserts were isolated from S. cerevisiae genomic libraries in YEp24 (2 microns) and YCp50 (CEN) plasmids. The identity of each gene was determined by gene disruption, and Southern hybridization and genetic analyses confirmed that the bona fide genes had been cloned. Plasmids containing each gene were introduced into known bacterial proline auxotrophs, and the ability to restore proline prototrophy was assessed. Interspecies complementation demonstrated that the S. cerevisiae PRO1 gene encoded gamma-glutamyl kinase, PRO2 encoded gamma-glutamyl phosphate reductase, and PRO3 encoded delta 1-pyrroline-5-carboxylate reductase. The presence of the PRO3 gene on a high-copy-number plasmid in S. cerevisiae caused a 20-fold overproduction of delta 1-pyrroline-5-carboxylate reductase. The PRO2 gene mapped on chromosome XV tightly linked to cdc66, and the PRO3 gene was located on the right arm of chromosome V between HIS1 and the centromere.
通过对酿酒酵母的pro1、pro2和pro3（脯氨酸需求型）菌株进行功能互补，分离出了PRO1、PRO2和PRO3基因。从YEp24（2微米）和YCp50（CEN）质粒中的酿酒酵母基因组文库中分离出具有重叠插入片段的独立克隆。通过基因破坏确定每个基因的身份，Southern杂交和遗传分析证实已克隆到真正的基因。将含有每个基因的质粒导入已知的细菌脯氨酸营养缺陷型菌株，并评估恢复脯氨酸原养型的能力。种间互补表明，酿酒酵母PRO1基因编码γ-谷氨酰激酶，PRO2基因编码γ-谷氨酰磷酸还原酶，PRO3基因编码δ1-吡咯啉-5-羧酸还原酶。酿酒酵母中高拷贝数质粒上PRO3基因的存在导致δ1-吡咯啉-5-羧酸还原酶过量产生20倍。PRO2基因定位于第十五号染色体上，与cdc66紧密连锁，PRO3基因位于第五号染色体右臂上，在HIS1和着丝粒之间。
