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Biomedical subjects

R J Redfield

Publications and source records attributed to R J Redfield.

25 records · Page 2Linked to original sources
PubMed1989

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.

Alleles↗
PubMed1988

Evolution of bacterial transformation: is sex with dead cells ever better than no sex at all?

Computer simulations of bacterial transformation are used to show that, under a wide range of biologically reasonable assumptions, transforming populations undergoing deleterious mutation and selection have a higher mean fitness at equilibrium than asexual populations. The source of transforming DNA, the amount of DNA taken up by each transforming cell, and the relationship between number of mutations and cell viability (the fitness function) are important factors. When the DNA source is living cells, transformation resembles meiotic sex. When the DNA source is cells killed by selection against mutations, transformation increases the average number of mutations per genome but can nevertheless increase the mean fitness of the population at equilibrium. In a model of regulated transformation, in which the most fit cells of a transforming population do not transform, transforming populations are always fitter at equilibrium than asexual populations. These results show that transformation can reduce mutation load.

Models, Biological↗
PubMed1987

Structure of cryptic lambda prophages.

When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome. The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E. coli chromosome. The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E. coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases. Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized. They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage. The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5). The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential.

Bacteriophage lambda↗
PubMedDate not supplied

Genes for breakfast: the have-your-cake-and-eat-it-too of bacterial transformation.

Bacterial transformation, in which cells take up and recombine free strands of DNA, is the simplest process thought to have evolved for genetic exchange, i.e., because of the potential benefits of producing progeny with recombinant genotypes. However, two other functions are equally plausible: acquisition of intact DNA strands to use for recombinational repair of DNA damage, and acquisition of the nutrients contained in DNA molecules. Although the recombinant progeny produced by transformation can be beneficial, the success of genes causing transformation is limited by other factors, especially by the genetic quality of DNA derived from dead cells. Our recent experiments in the naturally transformable bacteria Haemophilus influenzae and Bacillus subtilis suggest that the DNA-repair hypothesis is unlikely to be correct. In H. influenzae, transformation does not detectably increase the cells' ability to survive DNA damage. More importantly, we have found that, although competence (the ability to take up DNA) is induced by nutritional limitation in both H. influenzae and B. subtilis, it is not induced by DNA damage in either. Thus we favor the hypothesis that transformation evolved as a nutrient-uptake system, especially because unrelated DNA is abundant in the environments of many naturally transformable bacteria.

Bacteria↗
PubMedDate not supplied

Phylogenetic placement of Trichonympha.

Flagellated protists of the Class Hypermastigida have previously been classified on morphology alone, since no molecular sequences have been available. We have isolated DNA from 350 cells of the hypermastigote Trichonympha, manually collected from the hindgut of Zootermopsis angusticollis, and used this DNA as template for polymerase chain reaction amplification of the small-subunit ribosomal RNA gene. The DNA sequence of the amplified fragment is closely related to that of a previously-unidentified gut symbiont from the termite Reticulitermes flavipes, and phylogenetic analysis places both sequences as a sister group to the known trichomonads, in agreement with the morphological classification.

Animals↗