GENETIC TAGGING, CLONING, AND DNA-SEQUENCE OF THE SYNECHOCOCCUS SP STRAIN PCC-7942 GENE (GND) ENCODING 6-PHOSPHOGLUCONATE DEHYDROGENASE
GENETIC TAGGING, CLONING, AND DNA-SEQUENCE OF THE SYNECHOCOCCUS SP STRAIN PCC-7942 GENE (GND) ENCODING 6-PHOSPHOGLUCONATE DEHYDROGENASE
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DOI：
10.1128/jb.172.7.4023-4031.1990
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发表时间：
1990-07-01
影响因子：
3.2
通讯作者：
WOLF, RE
中科院分区：
文献类型：
--
作者：
BROEDEL, SE;WOLF, RE
采用遗传学方法克隆了聚球藻属菌株PCC 7942（聚球藻菌株R2）gnd基因，该基因编码6-磷酸葡萄糖酸脱氢酶（6PGD）。使用大肠杆菌基因作为异源探针，通过Southern分析制备gnd基因座的限制性图谱。通过将Tn 903的npt II基因限制性位点特异性插入聚球藻菌株R2染色体DNA的pUC 19质粒文库中，对聚球藻菌株R2基因进行遗传标记。用该插入突变文库转化聚球藻菌株R2，携带gnd：：npt II基因的菌株被鉴定为对黑暗中孵育超敏感的突变体。中断的基因是从其中一个突变体中克隆出来的。将携带gnd：：npt II基因的质粒重新引入聚球藻菌株R2中，并选择卡那霉素抗性转化体。通过基因置换产生的转化体是暗敏感的并且缺失6PGD活性。质粒插入产生的转化子是抗暗的，并具有6PGD活性。然后，利用从中断基因得到的限制性图谱，从含有质粒插入物的引物中克隆野生型基因。聚球藻R2 6PGD在E.当克隆的gnd基因从载体上的lacZ启动子转录时，基因的边界和转录方向由携带从任一端进入gnd的缺失的质粒所赋予的表型确定。测定核苷酸序列。聚球藻R2 6PGD推导的氨基酸序列与大肠杆菌E. coliK-12酶。
A genetic approach was used for the cloning of the Synechococcus sp. strain PCC 7942 (Synechococcus strain R2) gnd gene which encodes 6-phosphogluconate dehydrogenase (6PGD). A restriction map of the gnd locus was prepared by Southern analysis using the Escherichia coli gene as a heterologous probe. The Synechococcus strain R2 gene was genetically tagged by restriction site-specific insertion of the nptII gene of Tn903 into a pUC19 plasmid library of Synechococcus strain R2 chromosomal DNA. Synechococcus strain R2 was transformed with this insertion mutation library, and isolates carrying the gnd::nptII gene were identified as mutants hypersensitive to incubation in the dark. The interrupted gene was cloned from one of the mutants. A plasmid carrying the gnd::nptII gene was reintroduced into Synechococcus strain R2, and kanamycin-resistant transformants were selected. Transformants arising by gene replacement were dark sensitive and missing 6PGD activity. Transformants arising by plasmid insertion were dark resistant and had 6PGD activity. The wild-type gene was then cloned from a transformant containing a plasmid insertion, making use of the restriction map derived from the interrupted gene. Synechococcus strain R2 6PGD was expressed in E. coli when the cloned gnd gene was transcribed from the lacZ promoter resident on the vector. The boundaries of the gene and the direction of transcription were determined from the phenotypes conferred by plasmids carrying deletions entering gnd from either end. The nucleotide sequence was determined. The deduced amino acid sequence of Synechococcus strain R2 6PGD has 56% homology to that of the E. coli K-12 enzyme.