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D R Leach

Publications and source records attributed to D R Leach.

45 records · Page 3Linked to original sources
PubMed1989

Molecular organization of sbcC, a gene that affects genetic recombination and the viability of DNA palindromes in Escherichia coli K-12.

The sbcC gene product of Escherichia coli interferes with the growth of a lambda red gam phage carrying a long palindrome in its DNA. This phenotype was used to identify recombinant plasmids harbouring the wild-type gene and to isolate sbcC mutant derivatives carrying Tn1000 insertions. Analysis of these plasmids located sbcC between proC and phoR at a slightly different position from that reported before (Lloyd, R.G. and Buckman, C. 1985, J. Bacteriol. 164, 836-844). Nucleotide sequencing revealed that the gene spans a DNA segment of 3.3 kb that encodes a poorly expressed protein of 118 kDa and which lies downstream of a gene of unknown function that encodes a polypeptide of 45 kDa. The amino acid sequence of SbcC contains a nucleotide binding fold similar to that in RecB and other recombination proteins.

Amino Acid Sequence↗
PubMed1989

Slow replication of palindrome-containing DNA.

Lambda red gam phage carrying a 571 base-pair palindrome are unviable in wild-type Escherichia coli hosts. By using de-methylation to study the fate of DNA strands introduced into E. coli, we have demonstrated that a decrease in the yield of palindrome-containing molecules with two newly synthesized strands can occur without any concomitant loss of replicated molecules containing input strands. This implies that the palindrome-containing DNA is not being destroyed even as a consequence of replication, but rather that its replication rate is reduced. These results demonstrate that a palindrome can mediate unviability without directing cleavage of its carrier replicon.

Base Composition↗
PubMed1989

Lymphocyte alterations in zinc-deficient calves with lethal trait A46.

Lymphocyte numbers and activities were evaluated at 2, 4, 8 and 12 weeks of age in two calves with lethal trait A46 (A46), a genetic disorder affecting intestinal zinc absorption. Plasma zinc concentrations declined to subnormal by 3 weeks of age, after which anorexia, diarrhea, alopecia and hyperkeratosis occurred. Lymphocyte response to phytohemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) stimulation was variably reduced. CD4+ T-lymphocytes were subnormal on at least one observation period following onset of zinc deficiency, and relative numbers of B lymphocytes were decreased at 8 weeks. Secondary antibody responses to bacteriophage phi X 174 were significantly reduced. The results demonstrate that calves homozygous for the A46 trait have normal numbers of functional lymphocyte subpopulations at birth, and that the activity of their lymphocytes is altered once the calves become zinc deficient.

Animals↗
PubMed1988

Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome.

Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived. However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable. In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry [Collins, Cold Spring Harbor Symp. Quant. Biol. 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326]. We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli. This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.

Bacteriophage lambda↗
PubMedDate not supplied

Viability of lambda phages carrying a perfect palindrome in the absence of recombination nucleases.

In Escherichia coli in vitro constructions of perfect palindromes larger than 30 base pairs (bp) long have in general been unstable. A perfect palindrome has the unique possibility of forming a cruciform structure, and it is this feature which probably results in its instability. Negative supercoiling favours the formation of the cruciform conformation, which in turn causes the molecule to relax. This relaxation may render replicons containing large perfect palindromes inviable. An alternative hypothesis for inviability has been that the cruciform interferes with replication by favouring strand switching by polymerase I. Here we show that the simultaneous absence of two recombination nucleases, the recBC product, exonuclease V, and the sbcB product, exonuclease I, confers viability on a derivative of phage lambda carrying a perfect palindrome of inverted repeat length 1,600 bases. This observation suggests a third hypothesis--that nucleolytic cleavage of the cruciform is responsible for the inviability of the phage. Such an activity has been shown in vitro for T4 exonuclease VII.

Bacteriophage lambda↗