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Biomedical subjects

P Sankar

Publications and source records attributed to P Sankar.

27 records · Page 2Linked to original sources
PubMed1989

Genomically linked cellular protein databases derived from two-dimensional polyacrylamide gel electrophoresis.

In its most useful form a cellular protein database should be genomically based, because it is the genome which determines both the total number of proteins a cell can make and the particular ones that will be made under any given condition. Such a database should trace each protein back to its structural gene, and should account for every structural gene of a cell. Recent advances in molecular biology greatly facilitate the construction of such gene-protein databases. The mapping of genes of unidentified proteins resolved from total cell extracts on two-dimensional gels can now be accomplished by largely biochemical methods, without the necessity of isolating mutants or performing genetic crosses. Other techniques permit one to search gels for the product of any newly discovered gene (or open reading frame) suspected of encoding a protein. Consequently, gene-protein indices can be built independently and simultaneously from either direction--deducing the genetic map from the protein pattern, or finding the protein pattern from information encoded in the genome. A database of this sort is being constructed for the bacterium, Escherichia coli. Given the current pace of DNA nucleotide sequencing, the development of total gene-protein indices for a variety of cells can be anticipated in the near future.

Amino Acids↗
PubMed1988

Biochemical and genetic analysis of hydrogen metabolism in Escherichia coli: the hydB gene.

Production of active hydrogenase by Escherichia coli requires several gene products. One of the essential genes, hydB, is encoded by a DNA fragment of approximately 1.0 kilobase. The hydB gene produced a protein with an apparent molecular weight of 32,000. The hydB gene was transcribed only under anaerobic conditions. Oxygen and nitrate repressed transcription of this gene. hydB gene transcription also required sigma 60, the product of the rpoN gene.

Bacterial Proteins↗
PubMed1988

Gene-product relationships of fhlA and fdv genes of Escherichia coli.

Synthesis of formate dehydrogenase coupled to formate hydrogenlyase activity in Escherichia coli was found to require the product of the fhlA gene. Transcription of fdhF, the gene coding for the 80-kilodalton (kDa) selenopeptide of formate dehydrogenase, was not detected in an fhlA genetic background. Mutations in the fhlA gene also abolished production of the hydrogenase activity associated with formate hydrogenlyase activity. The fhlA gene resides next to the hydB gene at 59 min in the E. coli chromosome, and the two genes are transcribed in opposite directions. The fhlA gene codes for a 78-kDa protein. A neighboring gene, fdv, codes for an 82-kDa protein, and the physiological role of this gene product is unknown, although a role in H2 metabolism can be detected.

Aldehyde Oxidoreductases↗
PubMed1988

Hydrogen metabolism in Escherichia coli: biochemical and genetic evidence for a hydF gene.

A new gene whose product is essential for production of all three hydrogenase isoenzymes in Escherichia coli has been identified. This gene, termed hydF, mapped at 59 min in the E. coli chromosome and resided next to the hydB gene. The map order of these genes was hydE, hydF, hydB, fhlA, and fdv. The hydF gene was transcribed from its own promoter and coded for a protein with an apparent molecular weight of 43,000 to 44,000. Expression of the hydF operon was enhanced by anaerobic growth conditions. Partial products of the hydF gene were capable of supporting various levels of hydrogenase activity in a hydF mutant in the presence of the fhlA gene product, also produced from multicopy plasmids. In the presence of a second mutation in an unidentified, unlinked gene, hydrogenase activity in a hydF mutant was restored by plasmids which carried incomplete hydF and hydB+ genes. These results suggest that the products of hydF and fhlA interact with each other and with yet one other gene product.

Cloning, Molecular↗
PubMed1985

Studies on clonal heterogeneity in two spontaneously metastasizing mammary carcinomas of recent origin.

We have studied the clonal heterogeneity of 2 spontaneously metastasizing mammary carcinomas which recently arose spontaneously in C3H/He female retired breeders. Cells of early (2nd to 5th) transplant generations of these tumors were cloned by a combination of semi-solid agarose colony formation and limiting dilution techniques. Growth characteristics of the various clones in vitro and their tumorigenicity in vivo were evaluated. Subsequently, the role of host immunity and of interclonal interactions in regulating growth of the different clones in vivo were analyzed. We found that, whereas all 16 clones isolated from one tumor (T-58) grew rapidly in vivo and in vitro, 10 clones isolated from the second tumor (MT-2) showed a wide disparity in their growth rates in vivo. Taken together, these clones could generally be divided into 3 categories: (1) rapidly growing lines which grew in vivo at rates similar to or higher than those of the parental line; (2) slow-growing lines which grew more slowly than the parental line; and (3) non-growers which failed to produce tumors in vivo with doses of up to 5 X 10(6) cells injected either s.c. or i.v. but grew in vitro at rates comparable to the parental line. No correlation could be established between the various growth potentials exhibited by these tumor lines and tumor cell morphology in vitro and in vivo, as determined by light and electron microscopy. Sublethal irradiation (550-650 R) of young animals prior to tumor inoculation, or before inoculation of tumor cells into old, low NK syngeneic mice, failed to modify the growth of slow-or non-growing lines in vivo, indicating that host cellular defense mechanisms against the clones, if existent, were not mediated by NK or radiosensitive B or T cells. When clonal interactions were studied by the simultaneous injection of different clones in vivo at different s.c. sites, we found that a slow-growing line failed to modify the growth rate of a rapidly growing line, but accelerated the growth of a second slow-growing line injected simultaneously on the contralateral side, and that this enhancement of tumor growth was radioresistant. A mixture of these 2 lines also grew more rapidly than the individual lines alone. Our findings suggest that phenotypic variations in tumorigenicity can be found in clonal lines derived from spontaneous primary tumors and that these variations are not related to cell cycle properties as measured in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism.

A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli. Mutant strains isolated by this procedure can be divided into two major classes. Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor. Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities. All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase. The mutation in class I mutants mapped near 65 min of the E. coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome. The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl. These results indicate that there are two hyd operons and one hup operon in the E. coli chromosome. The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell.

Bacteriophages↗
PubMed1985

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.

Cloning, Molecular↗