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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Jan;80(1):181–185. doi: 10.1073/pnas.80.1.181

Construction of a Rhizobium japonicum gene bank and use in isolation of a hydrogen uptake gene

Michael A Cantrell 1, Richard A Haugland 1, Harold J Evans 1
PMCID: PMC393335  PMID: 16593268

Abstract

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 × 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.

Keywords: nitrogen fixation, hydrogenase, cloning, cosmid pLAFR1, soybean

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Selected References

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