Biomedical subjects

E Bardwell

Publications and source records attributed to E Bardwell.

3 recordsLinked to original sources

PubMed1977

Genetic effects of photoadducts and photocross-links in the DNA of phage lambda exposed to 360 nm light and tri-methylpsoralen or khellin.

The furocoumarin, 4,5',8-trimethylpsoralen, sensitizes cells and viruses to 360 nm light, producing cross-links and monoadducts in their DNA. The furanochromone khellin is a less effective sensitizing agent than psoralen, but has been found to induce cross-links and adducts in DNA also. The number of cross-links increases as the square of the time of exposure to light. We found that greater fluences were required for khellin than for psoralen, possibly because of the less favorable angle of the distal unsaturated bonds for corss-linking pyrimidines in adjacent base pairs. By adjusting the time of exposure to 360 nm light, lambda phages were damaged with [3H]psoralen and [3H]khellin so as to produce equal numbers of cross-links. These exposures were found to produce 8-times more [3H]khellin than [3H]psoralen adducts in the DNA of the phages. Similar exposures were made with nonradioactive photosensitizers to determine the effectiveness of lambda phages carrying cross-links and monoadducts in producing genetic recombinants. Lambda phage-prophage genetic corsses were performed with psoralen and khellin-damaged phages under repressed conditions in which replication of the damaged DNA was blocked. It was estimated from the results that cross-links were about 20-times more effective than monoadducts for inducing recombination under repressed conditions. In tests on the survival of plaque forming ability on wild type bacteria, it was estimated that cross-links were about 15-times more effective than the adducts. The results support the conclusion that, in homoimmune crosses with psoralen-damaged lambda phages infecting wild type lysogens, more than three-quarters of the induced recombination can be attributed to cross-links rather than to monoadducts.

Binding Sites↗

PubMed1977

Initiation of genetic exchanges in lambda phage--prophage crosses.

When Escherichia coli K-12 (lambda) lysogens were infected with lambda phages, genetic exchanges between phage and prophage occurred at low frequencies (less than 0.1% between the markers P3 and P80), but at frequencies above 1% if the infecting phages were first treated with the photosensitizing agent 4,5',8-trimethylpsoralen and 360 nm light. Exchanges were induced by psoralen damage at about the same frequency in wild-type lysogens and in those carrying recB(-), recC(-), recF(-), or lexA(-), but at an intermediate frequency in a quadruple mutant carrying recB(-)recC(-)recF(-)sbcB(-). Few if any exchanges were induced in lysogens carrying uvrA(-), uvrB(-), or recA(-). The increase in the frequency of recombination was presumably due to the psoralen damage in the phage DNA molecules and the action of host cell repair and recombination enzymes. The production of crosslinks in the phage DNA by psoralen and 360 nm light was measured by sedimentation in alkali. It showed second-order kinetics indicative of a two-photon reaction. In contrast, first-order kinetics had been reported for monoadduct formation. Second-order kinetics, similar to those for crosslink production, were found for genetic exchanges in homoimmune crosses. Presumably, crosslinks, rather than monoadducts, cause most of the exchanges. Because the uvrA(-) gene product (UV-endonuclease) was required, it is likely that recombination was initiated by DNA molecules cut at crosslinks. This system has been used to show that after the crosslinked phage duplex has been cut, one or more of the subsequent steps-homologous pairing, cutting, and joining-require the recA(+) gene product.

Coliphages↗

PubMed1975

Genetic exchanges induced by structural damage in nonreplicating phage lambda DNA.

Genetic recombination between irradiated lambda phage and the unirradiated lambda prophage in homoimmune lysogens has been studied under conditions in which phage DNA replication and repair were controlled. The lambda phage were exposed to one of three treatments before infecting the lysogens: (a) 254-nm light, which produces pyrimidine dimers and other photoproducts; (b) 313-nm light with acetopheneone D, which produces thymine dimers and a different spectrum of other photoproducts; (c) 360-nm light with trimethylpsoralen, which produces monoadducts and cross-links. With both replication and excision-repair of the damaged phage DNA blocked, treatment b (acetophenone D) caused no significant increase in recombination, indicating that thymine dimers do not cause recombination if the DNA in which they are contained is not replicated. Treatment a (254 nm), producing the same total number of pyrimidine dimers, caused a marked increase in recombination. This indicates that photoproducts other than pyrimidine dimers produced by 254-nm light can cause recombination in the absence of replication. Treatment c (psoralen) caused a marked increase in recombination in wild type but not in uvrA and uvrB mutants. The frequency of recombination in two-factor crosses varied with marker separation in such a way as to suggest that cross-links can act over distances of at least 5% of the lambda genome to cause exchanges between pairs of relatively closely spaced markers. The psoralen photo cross-links and monoadducts initiate recombination only following the action of excision enzymes, which appears to release one arm of each cross-link, producing a gap with free strand ends. It may be these strand ends which induce recombination. The action at a distance of 5% of the lambda genome may reflect heteroduplex formation and the subsequent reduction to homozygosity of mismatched base pairs at genetic markers. Recombination between closely spaced markers in the P gene is reduced in strains carrying polA.

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