Title: Molecular cloning and restriction endonuclease mapping of the glutamate synthase gene from salt-tolerant Bradyrhizobium spp. strain WR1001
Abstract:In order to clone the glutamate synthase gene (glt) from salt-tolerant Bradyrhizobium spp. strain WR1001, a genomic cosmid library was constructed using the vector pHC79. The library was mass-conjugat...In order to clone the glutamate synthase gene (glt) from salt-tolerant Bradyrhizobium spp. strain WR1001, a genomic cosmid library was constructed using the vector pHC79. The library was mass-conjugated into Escherichia coli ET1194 (glt−) in a triparental mating, using pRK2013 as a mobilizing plasmid. Transconjugants of ET1194 which grew on minimal medium and had Glt+ phenotype were selected for the isolation of recombinant cosmids. Plasmid pHL27, which contained a 40-kb insert of WR1001 DNA and complemented ET1194 to Glt+ phenotype, was chosen to delimit the glutamate synthase gene. The region that encoded glt in cosmid pHL27 was determined by Southern blot analysis using the cloned glt gene of E. coli DH1 as a probe. An 11-kb HindIII DNA fragment from pHL27 contained the fully functional glt gene. Partial PstI and EcoRI restriction enzyme digests of cosmid pHL27 were subcloned into the vectors pBS+ and pUC8 and transformed into ET1194 to test for Glt+ phenotype. The glt coding region was defined by aligning the physical maps of the subclonesRead More