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B R Sharma

Publications and source records attributed to B R Sharma.

At least 55 records · Page 3Linked to original sources
PubMed1993

XbaI and BlnI genomic cleavage maps of Escherichia coli K-12 strain MG1655 and comparative analysis of other strains.

Complete XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 are presented, along with a comparison of the physical map of MG1655 with that of five other K-12 strains. We have mapped 35 XbaI cleavage sites generating 35 fragments ranging in size from 8 kb to 432 kb using methods similar to those used previously for the NotI and SfiI maps of MG1655. The applicability of the MG1655 map to other strains of E. coli K-12 was assessed by comparing the NotI, SfiI and XbaI digestion patterns of EMG2, W1485, W3110, AB1157 and MC4100 with those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the differences between the strains on the basis of simple insertions, deletions and, in one case, an inversion. Insertions and deletions ranging in size from 1 kb to 86 kb are suggested by this model. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains. The various features localized in these strains were used to place 9 of the 17 BlnI fragments on the E. coli physical map. The remaining fragments were placed by hybridization experiments similar to those used for the NotI, SfiI and XbaI maps. In this way, the complete BlnI map was constructed. The cleavage sites for XbaI and BlnI were assigned coordinates based on EcoMap6 developed by Rudd et al. The XbaI and BlnI maps of MG1655 presented here, when combined with the NotI (22 sites) and SfiI (31 sites) maps of MG1655 previously published, bring the total number of mapped rare restriction sites in MG1655 to 105. The strain comparison analysis shows that this map is readily adaptable for use with other K-12 strains.

Chromosomes, Bacterial↗
PubMed1992

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications.

Base Sequence↗
PubMed1990

Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites.

Epidemiologic evaluation of enterococci has been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared chromosomal restriction endonuclease digestion patterns of 27 isolates of Enterococcus faecalis from three different locations by using pulsed-field electrophoresis of large chromosomal fragments (14 to 1,000 kilobases). All but two isolates generated a clear, evaluable pattern with a single lysis and digestion, and the remaining two were visualized when a larger quantity of bacteria was used. All isolates from different locations generated different restriction patterns, as did most isolates within a single location; there was also evidence of spread of strains between individuals in each location. The ease with which this analysis can be performed, together with the clarity and polymorphism seen in the patterns, suggests that this technique will be very useful for epidemiological evaluations of nosocomial enterococcal infections.

DNA Restriction Enzymes↗
PubMed1985

Predominance of tyrosine phosphorylation of insulin receptors during the initial response of intact cells to insulin.

Anti-phosphotyrosine antibody and anti-insulin receptor antibody were used to study insulin-stimulated phosphorylation of the beta-subunit of the insulin receptor in [32P]orthophosphate-labeled Fao hepatoma cells. Without insulin, the receptor contained both phosphoserine and phosphothreonine and could be immunoprecipitated with anti-receptor antibody but not with the anti-phosphotyrosine antibody. After incubation of these cells with insulin, both antibodies immunoprecipitated the phosphorylated receptor. The beta-subunit of the receptor precipitated with anti-phosphotyrosine antibody from cells stimulated with insulin (100 nM) for 1 min contained predominantly phosphotyrosine, whereas, after 10 min with insulin, the amounts of phosphotyrosine and phosphoserine were nearly equal. These results suggest that insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the beta-subunit. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibody followed by precipitation of the remaining proteins with anti-receptor antibody suggests that insulin receptors which contain phosphoserine in the basal state are tyrosine phosphorylated more slowly than the dephosphorylated receptors or not at all after the addition of insulin. The beta-subunit of the insulin receptor was the major phosphorylated protein precipitated by the anti-phosphotyrosine antibody from insulin-stimulated Fao cells. These results confirm our notion that insulin initially stimulated tyrosine autophosphorylation and subsequently serine phosphorylation of the insulin receptor in intact cells and suggests that this sequence of reactions occurs faster on receptors that are dephosphorylated before the incubation with insulin.

Animals↗
PubMed1985

Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies.

The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.

Animals↗
PubMed1984

Relationship between the subunit structure of insulin receptor and its competence to bind insulin and undergo phosphorylation.

Insulin receptor partially purified from human placenta by chromatography on immobilized wheat germ agglutinin was subjected to affinity cross linking to determine the relationship between the subunit structure of the multiple forms of the insulin receptor and their competence to bind insulin and undergo autophosphorylation. It was demonstrated that, whereas the 340-kDa intact receptor undergoes autophosphorylation, the 290- and 320-kDa insulin binding forms of the receptor do not. Phosphorylation at tyrosyl residues in the intact receptor was verified using a new facile method for determination of phosphorylated amino acids. The competence of the phosphorylated 340-kDa protein to bind insulin was demonstrated using a double-probe labeling protocol wherein receptor phosphorylated with [gamma-32P]ATP was cross-linked with disuccinimidyl suberate (DSS) in the presence of N epsilon B29-biotinylinsulin. The observation that succinylavidin, by virtue of its interaction with biotinyl residues, decreased the electrophoretic mobility of receptor radiochemically labeled with 32P indicated that the phosphorylated 340-kDa protein was competent to bind insulin. This result is compelling evidence that the 340-kDa phosphorylated species is insulin receptor itself, rather than a closely associated contaminant. Treatment of the receptor with the crosslinking agent DSS produced (after reduction and denaturation) alpha-dimer, beta-dimer, and a smaller amount of tetramer. This observation is consistent with a symmetrical, tetrameric, alpha 2 beta 2 structure for insulin receptor from human placenta, and excludes previously proposed alternative structures containing one alpha and one beta chain.

Adenosine Triphosphate↗