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W S Kelley

Publications and source records attributed to W S Kelley.

15 recordsLinked to original sources
PubMed1983

Genetic characterization of early amber mutations in the Escherichia coli polA gene and purification of the amber peptides.

The polA1 mutation of Escherichia coli K12 and two further mutations, resA1 and resA2, characterized in E. coli B have been shown to produce enzymatically active nonsense (amber) peptides. These enzymes can be purified to virtual homogeneity by use of the lambda polA transducing phage system. The peptides are immunologically related and react weakly but specifically with antibody to whole DNA polymerase I. In their purified form the peptides are less heat-labile than the whole enzyme or the Klenow fragment produced by proteolysis. Physiological studies indicate that all three alleles are compatible with a number of different streptomycin resistance mutations (rpsL alleles) in a variety of genetic backgrounds. There is, however, clear evidence for slight amounts of "read-through" of these mutations under these conditions. DNA sequence studies have indicated the exact nucleotides that have been mutated to produce the amber alleles. The resA1 and resA2 alleles appear to be independent isolates of the same mutation both resulting in CAG (Gln) leads to TAG (amber) at amino acid residue 298. The polA1 mutation results in TGC (Trp) leads to TAG (amber) at amino acid residue 342. The significance of these findings is discussed with reference to the structure of the whole enzyme as shown by the DNA sequence data of Joyce et al. (1982) and protein chemistry of Brown et al. (1982).

Antigens, Bacterial↗
PubMed1982

Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I.

We report the nucleotide sequence of 3.2 kilobase pair region of the Escherichia coli polA gene, comprising the coding region for DNA polymerase I with about 400 base pairs of flanking sequence. The amino acid sequence for DNA polymerase I derived from our DNA sequence is largely consistent with previous protein chemical data. In the following paper, Brown et al. (Brown, W. E., Stump, K. H., and Kelley, W. S. (1982) J. Biol. Chem. 257, 1965-1972) present additional protein chemistry experiments that further confirm our sequence. Mild proteolysis of DNA polymerase I is known to produce two enzymatically active fragments (Brutlag, D., Atkinson, M. R., Setlow, P., and Kornberg, A. (1969) Biochem. Biophys. Res. Commun. 37, 982-989; Klenow, H., and Henningsen, I. (1970) Proc. Natl. Acad. Sci. U. S. A. 74, 5632-5636). We have located the site of this cleavage between residues 323 and 324 of the 928 amino acid polymerase molecule. By sequence comparison of the polA1 and wild type alleles, we have identified the polA1 mutation as a change from Trp (TGG) to amber (TAG) at residue 342.

Amino Acid Sequence↗
PubMed1982

Escherichia coli DNA polymerase I. Sequence characterization and secondary structure prediction.

The primary sequence of DNA polymerase I from Escherichia coli K12 as derived from the DNA sequence (Joyce, C. M., Kelley, W. S., and Grindley, N. D. F. (1982) J. Biol. Chem. 257, 1958-1964) has been verified. Protein sequencing through eight cycles of the Klenow large fragment yields a unique sequence corresponding to residues 324 to 331 from the translated DNA sequence and defines the subtilisin cleavage site for formation of the large and small fragments as Thr323-Val324. Site-specific cleavage of whole enzyme and large fragment at cysteines and sizing of the resulting fragments verify the location of the two cysteines at residues 262 and 907 as assigned by the DNA sequencing. Isolation of tryptic peptides derived from DNA polymerase I yielded unique peptides whose composition exactly corresponded to theoretical tryptic peptides derived from the translated DNA sequence. Identification of the expected carboxyl-terminal tryptic peptide and carboxypeptidase digestion of whole enzyme and large fragment confirm histidine-928 as the carboxylterminus. A secondary structure prediction is made using the available primary sequence data. The model contains 43% alpha helix, 17% beta-structure, 58 beta-turns, and several interesting super-secondary structure elements.

Amino Acid Sequence↗
PubMed1980

Mapping of the polA locus of Escherichia coli K12: genetic fine structure of the cistron.

The close linkage of the glnA gene with polA was exploited to construct a fine structure map of polA by means of generalized transduction with phage P1. Nine different polA- alleles were mapped by recombinational crosses. The results indicate a gene order consistent with previous observations (KELLEY and GRINDLEY 1976a; MURRAY and KELLEY 1979). Three mutations, polA5, polA6 and polA12 map within the "carboxy-terminal" or "large-fragment" portion of the gene in unambiguous order. Four alleles, known to affect the "amino-terminal" portion of the gene, polA107, polA214, polA480ex and polA4113, appear to be closely linked with certain ambiguities in their exact order. All four of these mutations are known to alter the 5' leads to 3' exonuclease activity of DNA polymerase I and three of them result in the conditional lethal polA- phenotype. The polA1 nonsense mutation maps between these two groups in a position consistent with its known effect, production of an amber fragment that includes the 5' leads to 3' exonuclease. The final allele, resA1, is another nonsense mutation that maps at the extreme "amino-terminus" of the cistron.----A number of control experiments were conducted to determine the effects of polA- mutations on the P1-mediated recombinational event. These experiments indicated that abortive transduction occurs quite frequently, but the formation of abortive transductants and segregation of unselected transduced markers among daughter progeny is like that observed by other investigators. There was no evidence that any individual polA- allele behaved in an exceptional fashion during recombination.

Alleles↗
PubMed1979

A rapid procedure for isolation of large quantities of Escherichia coli DNA polymerase I utilizing a lambdapolA transducing phage.

DNA polymerase I produced by infection of Escherichia coli K12 with the specialized transducing phage lambdapolA has been purified by a simplified procedure and shown to be identical with the enzyme produced by uninfected E. coli in all aspects which have been examined. The abundance of the enzyme in infected cells and the ease with which it may be purified will simplify the study of the enzyme's physical and chemical characteristics. In addition, the enzyme is now much more readily available for use as an analytical tool in nucleic acid sequence and structure studies.

Amino Acids↗
PubMed1979

Characterization of lambdapolA transducing phages; effective expression of the E. coli polA gene.

lambdapolA phages carrying the polA gene in either orientation were isolated and characterised by genetic tests and by assay of the polA gene product after infection of E. coli or induction of lysogens. Lytic infection gave consistently better amplification of DNA polymerase I than that obtained by induction of a lysogen. Optimal amplification of DNA polymerase I was not achieved from the PL promoter of cro-phages, but some advantages accrued when the polA gene was oriented for transcription from the PL promoter of a cro+ phage. lambdapolA phages in which the polA allele was from E. coli strain C600 provided better amplification than phages with the polA allele from E. coli ED8659. Induction of a lambdapolA1 cI857 Qam Sam prophage gave levels of DNA polymerase I approaching 100 times that found in the non-lysogenic Pol+ host. Genetics studies with the lambdapolA phages confirmed the previously postulated orientation of the polA gene within the E. coli genome.

Bacteriophage lambda↗
PubMed1977

Isolation and characterization of a lambdapolA transducing phage.

A plaque-forming lambdapolA phage was isolated from a population of transducing phage made in vitro from Escherichia coli DNA and a phage vector digested with restriction endonuclease HindIII. Amber mutations, in genes whose products are necessary for late protein synthesis (Q) and cell lysis (S), were crossed into the lambdapolA phage. Infection of either polA+ or polA- bacteria with this phage, under conditions permitting DNA replication but preventing phage production and lysis, elevated the levels of DNA polymerase I to between 75- and 100-fold that detected in a wild-type strain. The kinetics of enzyme production suggest that the polA gene is transcribed from its own promoter rather than from any of the well-characterized phage promoters. The fragment of E. coli DNA within the lambdapolA phage comprises approximately 5000 base pairs, sufficient to accommodate the polA gene and one, or two, coding sequences for smaller proteins.

Coliphages↗
PubMed1976

Mapping of the polA locus of Escherichia coli K12: orientation in the amino- and carboxy-termini of the cistron.

Three mutations of the polA cistron, the structural gene for DNA polymerase I of E. coli, have been ordered by three factor transductional crosses. The three mutant polymerase species have altered properties which may be ascribed to defects located in different portions of the polypeptide chain. Our data indicate that the amino terminal end is encoded by the end of the polA cistron nearer to metE and that transcription and translation proceed clockwise on the E. coli circular map towards the rha locus.

Chromosome Mapping↗
PubMed1976

Effects of different alleles of the E. coli K12 pol A gene on the replication of non-transferring plasmids.

The effects of eight different polA alleles on the replication of six different non-transferring enterobacterial plasmids have been tested. Using phage P1CM transduction, different allelic polA mutations were introduced into E. coli K12 strains carrying one of several antibiotic resistance plasmids. Plasmid stability in the transductants was examined by testing clones for drug resistance after growth under various conditions. From the results, the R factors may be divided into three different classes. One plasmid is only affected by PolA conditions which inhibit host cell growth, 3 plasmids (from the same compatibility group) are unstable under conditions in which the cells are severely deficient in DNA polymerase I and two other plasmids (compatible with each other and with the other 4) are immediately lost from such transductants and are unstable in a number of others. Furthermore, the plasmids which are most dependent on DNA polymerase I have been shown to replicate in the presence of chloramphenicol and therefore typigy a class of plasmids which includes bacteriocinogenic factors such as ColE1 and CloDF13, resistance determinant RSF1030 and the E. coli 15 minicircular plasmid.

Alleles↗