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C M Joyce

Publications and source records attributed to C M Joyce.

34 records · Page 2Linked to original sources
PubMed1986

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Binding Sites↗
PubMed1986

Effect of restraints on diagnostic approaches to abdominal pain and weight loss.

To examine the effects of restraint on the diagnostic process, 25 physicians were urged to be as economical as possible when requesting diagnostic tests for case report simulations of nine patients who had previously been hospitalized with abdominal pain and weight loss. The results of the tests correctly changed the physicians' initial diagnoses, increased their diagnostic confidence, and led to appropriate management decisions. When restrained, individual physicians needed only 3.0 (1.7 to 4.1) diagnostic procedures per patient to reach management decisions, whereas 6.1 (3.0 to 11) diagnostic procedures had actually been performed. Diagnostic approaches were so diverse, however, that the number of procedures collectively requested by randomly assembled "teams" consisting of four to five "economical" physicians equaled the number of procedures actually performed. Moreover, 20 percent of the procedures requested by "economical" physicians were invasive compared with only 7 percent of those actually performed. These findings suggest that, without uniform diagnostic approaches, "team" management may increase testing despite restraint by individual physicians. Moreover, quests for economy may increase invasive diagnostic testing.

Abdomen↗
PubMed1986

Deletion of the spf (spot 42 RNA) gene of Escherichia coli.

To investigate the function of spot 42 RNA, a small RNA of Escherichia coli, we constructed a strain in which spf, the structural gene for this RNA, is deleted. We achieved this by using a delta att phage lambda carrying a DNA fragment spanning the spf region but with a precise deletion of spf. By integration of this phage at the spf locus and by its subsequent excision, we were able to cross the spf deletion onto the bacterial chromosome. The fact that such a deletion could be obtained indicated that spf is not an essential gene. We did not observe any major defect in delta spf cells, although in one strain background the deletion caused a slight growth impairment.

Bacteriophage lambda↗
PubMed1985

Genetic mapping and DNA sequence analysis of mutations in the polA gene of Escherichia coli.

DNA polymerase I of Escherichia coli provides an excellent model for the study of template-directed enzymatic synthesis of DNA because it is a single subunit enzyme, it can be obtained in large quantities and the three-dimensional structure of the polymerizing domain (the Klenow fragment) has recently been determined (Ollis et al., 1985). One approach to assigning functions to particular portions of the structure is to correlate the altered enzymatic behavior of mutant forms of DNA polymerase I with the change in the primary sequence of the protein. Towards this end we have developed a rapid procedure for mapping any polA mutation to a region no larger than 300 base-pairs within the polA gene. Two series of polA deletion mutants with defined end-points were constructed in vitro and cloned into bacteriophage lambda. These phages can then be used to map precisely E. coli polA mutants. Twelve polA- alleles have been mapped in this way and for nine of them the nature of the mutational change has been determined by DNA sequence analysis. Two of the mutations, polA5 and polA6, which affect the enzyme-DNA interaction, provide evidence for the location of the DNA binding region on the polymerase three-dimensional structure.

Base Sequence↗
PubMed1984

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene.

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.

Bacteriophage lambda↗
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↗
PubMed1983

Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.

Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems.

Cloning, Molecular↗
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

Identification of two genes immediately downstream from the polA gene of Escherichia coli.

We have identified two genes within a 1-kilobase region immediately following the polA gene of Escherichia coli. The first, whose transcription is initiated about 150 base pairs beyond the end of the polA coding sequence, is the gene corresponding to the previously sequenced "spot 42 RNA" (B. G. Sahagan and J. E. Dahlberg, J. Mol. Biol. 131:573--592, 1979). The second, located further downstream and transcribed towards polA, is the structural gene for a 22-kilodalton polypeptide, which we have detected by using plasmid-directed protein synthesis in maxicells. Sequence analysis of this region of the E. coli genome suggests that it contains little, if any, redundant DNA.

Bacterial Proteins↗
PubMed1980

Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903.

Base Sequence↗