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Perret, X, Fellay, R, Bjourson, AJ, Cooper, JE, Brenner, S and Broughton, WJ (1994) SUBTRACTION HYBRIDIZATION AND SHOT-GUN SEQUENCING - A NEW APPROACH TO IDENTIFY SYMBIOTIC LOCI. NUCLEIC ACIDS RESEARCH, 22 (8). pp. 1335-1341. [Journal article]

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URL: http://nar.oxfordjournals.org/cgi/content/abstract/22/8/1335


Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation. Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of mast nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids. To target genes not present in the closely related strain R. fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.

Item Type:Journal article
Faculties and Schools:Faculty of Life and Health Sciences
Faculty of Life and Health Sciences > School of Biomedical Sciences
Research Institutes and Groups:Biomedical Sciences Research Institute
Biomedical Sciences Research Institute > Stratified Medicine
ID Code:1951
Deposited By: Mrs Caroline Adams
Deposited On:30 Nov 2009 09:36
Last Modified:14 Oct 2013 14:51

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