文章基本信息

标题：Construction of a Rhizobium japonicum gene bank and use in isolation of a hydrogen uptake gene
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作者：Michael A. Cantrell ; Richard A. Haugland ; Harold J. Evans 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1983
卷号：80
期号：1
页码：181-185
DOI：10.1073/pnas.80.1.181
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A gene bank of Rhizobium japonicum DNA was constructed by using the broad host range conjugative cosmid pLAFR1. Eighty-three percent of the clones in the bank contained cosmids with insert DNA averaging 22.6 kilobase pairs in length. A series of cosmids containing a hydrogen uptake (hup) gene was identified by transferring the gene bank into a H2 uptake-negative (Hup-) R. japonicum point mutant (PJ17nal) and screening tetracycline-resistant colonies for the ability to grow chemolithotrophically and to reduce methylene blue in a recently devised colony assay. Hup+ transconjugants arose at a frequency of approximately 6 x 10-3. Plasmid DNAs from II of the Hup+ transconjugants were isolated and used to transform Escherichia coli. EcoRI digests of all plasmids isolated from Hup+ transconjugants had three DNA fragments in common. Eight of the E. coli transformants containing hup gene cosmids were conjugated with PJ17nal and another Hup- point mutant, PJ18nal. All PJ17nal transconjugants were Hup+. The frequency of Hup+ transconjugants with PJ18nal was approximately 10-3. The results indicate that the hup gene cosmids may contain one gene and a portion of another.
关键词：nitrogen fixation ; hydrogenase ; cloning ; cosmid pLAFR1 ; soybean