Biomedical subjects
Publications and source records attributed to G R Sastry.
The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined. A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation. However, unlike its Escherichia coli equivalent, A. tumefaciens recA lacks the upstream 'SOS box', suggesting a different mechanism of regulation for this gene.
Plasmids containing hybrid genes, in which different Klebsiella pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce beta-galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nif A is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of nif promoters in A. tumefaciens are discussed.
None of the Agrobacterium tumefaciens and A. rubi strains tested produces detectable amounts of beta-galactosidase although they are capable of utilizing lactose as sole source of carbon. This opportunity was taken to investigate the expression of lac transposon Tn951 (Cornelis et al. 1978) in Agrobacterium with the ultimate goal of using this system to investigate alien gene expression. When the transposon was introduced with the help of a broad-host range plasmid, RP1, the transconjugants produced significant quantities of beta-galactosidase which was inducible by isopropyl-beta-D-thiogalactopyranoside. Tn951 was capable of restoring the Lac+ phenotype to an A. tumefaciens mutant not capable of using lactose. Cellobiose, a known inducer of aldohexopyranoside: cytochrome c oxidoreductase (which regulates the characteristic 3-ketolactose production in Agrobacterium; van Beeumen and De Ley (1968), had no effect on beta-galactosidase activity.
A large plasmid enables its host Agrobacterium tumefaciens to cause tumorous condition in a wide variety of dicotyledonous plants[see Ooms et al. Gene 14:33--50 (1981) )). The location and role of chromosomal genes in this phenomenon are not known. As the first stage in studying this aspect, a project was initiated to investigate the chromosomal genetics of the bacterium. R68.45, a P group plasmid, was chosen as a transmission agent. After a preliminary assessment it was decided to use C58 as a standard strain to carry out the mapping. The plasmid itself, as judged by the presence of antibiotic markers, appears to be stable in A. tumefaciens; its ability to promote chromosomal mobilisation, however, remains only in 60--80% transconjugants. Good Agrobacterium donors are capable of transferring chromosomal genes at a frequency varying between 10(-5) to 10(-6) per recipient. The recombinants are stable even under non-selective conditions. A linear linkage map consisting of 16 markers was built using coinheritance frequencies obtained from 21 four-point crosses.
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We describe the cloning of an ntrC gene of Agrobacterium tumefaciens C58 by interspecific complementation of an Escherichia coli ntrC mutant. Restriction mapping and Southern blot analysis of the complementing clone identified a 1.7-kb EcoRI-PvuII DNA fragment whose sequence was determined. Analysis of this sequence revealed coding regions corresponding to a complete ntrC gene and the C-terminal region of an ntrB gene. Amino acid sequence comparisons of A. tumefaciens NTRC protein with NTRC sequences from Rhizobium meliloti, Bradyrhizobium sp. (Parasponia), Klebsiella pneumoniae, E. coli, and Salmonella typhimurium show strong sequence conservation supporting DNA hybridization data, demonstrating strong evolutionary homology among ntrC genes of Rhizobiaceae. The C58 NTRC protein has been identified, by 35S-labeling, in a T7 RNA polymerase (pT7-7) expression vector system.