Biomedical subjects
Publications and source records attributed to W D Rupp.
Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.
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Several radiosensitizing chemicals, including a family of simple nitroimidazoles, were examined in E. coli and compared with misonidazole for toxic side-effects on endpoints such as mutagenesis, cell killing and inhibition of the synthesis of the inducible enzyme beta-galactosidase. While all the compounds were similar to misonidazole or better in radiosensitization, marked differences in the various side effects were found. There results show that for E. coli it is possible to find compounds that sensitize as well as misonidazole but which have decreased mutagenicity and fewer other side-effects. Of the compounds examined, KA121 (2,5-dinitroimidazole) is the most promising for future study because it combines good radiosensitization with low mutagenicity and toxicity.
The available data concerning postreplication repair are summarized. In Escherichia coli, recombination is implicated in this repair because the recA+ gene is necessary and because strand exchanges occur that extend over long regions. Other experiments involving phage-induced resistance also point to an interrelation between recombination and repair. In this phenomenon, gene products of lambda bacteriophage are introduced into bacteria, resulting in an increased resistance of the cells when they are subsequently exposed to X rays.
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A method was developed in which E. coli cells were irradiated with four MeV electrons and transferred to alkaline detergent within a fraction of a second. This technique minimizes the amount of repair of radiation damage before analysis without the necessity of using physical or chemical treatments to inhibit repair and alter the physiological condition of the cells. The yield of DNA strans breaks formed in covalent circular superhelical lambda DNA molecules superinfecting E. coli lysogens was about 4-fold greater when the cells were irradiated in oxygen than when they were irradiated under nitrogen anoxia. The same yields were obtained in phosphate buffer at 3 degrees and 22 degrees as well as in growth medium at 37 degrees, and the yields were not altered by the polA1 mutation. When E. coli lysogenic cells superinfected with lambda were irradiated with doses sufficient to introduce at least seven breaks in the phage DNA, the chromosomal DNA and the superinfecting phage DNA sedimented similarly in alkaline sucrose gradients, indicating that both DNAs were broken to a similar extent during irradiation. However, the yield of breaks calculated for chromosomal DNA in similar experiments was greater than the yield calculated from the first break introduced into covalent circular lambda DNA molecules. These apparently contradictory results are explicable either if the initial break in a superhelical molecule occurs with an efficiency different from that for subsequent breaks, or if the pulsed electron radiation produces a high proportion of double-strand breaks.
When gene products of lambda bacteriophage are introduced into a cell by transient induction of a lysogen, increased resistance of the cells to X rays results. This phenomenon has been called phage-induced radioresistance. Genetic studies show at least two classes of induced radioresistance. The first type depends on the products of the lambda red genes and is observed in bacteria that are mutated in the recB gene. It is thought that the lambda red products compensate for the missing RecBC nuclease in the repair of X-ray damage. An optimal effect is obtained even when the lambda red products are supplied 1 h after irradiation. The lesions that are affected by the red-dependent process are probably not deoxyribonucleic acid strand breaks because the extent of deoxyribonucleic acid strand rejoining is not altered by the red products. The second type of phage-induced radioresistance requires the gam product of lambda and is observed in wild-type and polA strains. The lambda gam+ gene produce must be present immediately after irradiation to exert its full effect. In its presence, DNA breakdown is decreased, and a greater fraction of DNA is converted back to high molecular weight. Strains carrying lex, recA, or certain other combinations of mutations do not show any detectable phage-induced radioresistance.
Three isolates of a lactose-fermenting, xylose-negative variety of Salmonella enteritidis ser. newington, identical in biochemical and serological reactions and in the antibiogram, were recovered from three patients in different areas of Connecticut in January 1974. Hydrogen sulfide production was not visible in Salmonella-Shigella agar, in triple sugar iron agar, and in Kligler iron agar but was noticed in lysine iron agar and on XLD agar, among others. The amount of fermentable carbohydrates present was found to correlate with failure to show hydrogen sulfide production (pH effect). In contrast to lactose-fermenting Salmonella strains reported by other authors, we could not elicit a direct transfer of the lac(+) character at frequencies above 10(-6). An epidemiological follow-up remained unsuccessful. Recommendations for the recognition of similar strains are presented.
E. coli cells lysogenic for the thermoinducible prophage lambdacI857 can be transiently induced by a brief heat treatment. Although this treatment does not kill the cells, some lambda products normally formed during vegetative phage development are made that can alter the response of host cells to x-irradiation by causing an increase in radioresistance. This increased resistance is particularly striking in the recombination-deficient recB-strain, which is normally much more radiosensitive than its recB(+) parent. After pulse-heating at 42 degrees , the survival curve of E. coli recB(-) lysogenized with lambdacI857 does not differ from that of the wild-type strain. Since lambda red mutants do not increase the radioresistance of recB(-) strains, both lambda red gene products, lambda exonuclease and beta-protein, are required to compensate for the missing recB product. Furthermore, phage-induced radioresistance also occurs in recB(+) lysogens even when they carry lambda red(-), but not when the lambda prophage is gam(-). Thus, in wild-type cells, phage-induced radioresistance requires some interaction between the bacterial recB gene product (exonuclease V) and the phage lambda-protein.
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Deoxyribonucleic acid (DNA) transfer from (3)H-thymine-labeled Hfr cells has been measured by determining the amount of radioactivity remaining after selective lysis of the donor cells in the mating mixture. DNA transfer was less effectively reduced by ultraviolet irradiation of excision-defective Hfr cells than was the yield of recombinants. The buoyant density of DNA transferred from unirradiated and irradiated Hfr cells was equivalent to that of double-stranded DNA. Mating-dependent DNA synthesis in the recipient has been measured by mating Hfr cells deficient in thymidine kinase with irradiated thymine-requiring F(-) cells in the presence of (3)H-thymine. The extent of such DNA synthesis approximated the amount of DNA transferred from unirradiated donors. Neither DNA transfer nor mating-dependent DNA synthesis could be reliably measured when both parents were irradiated. It is proposed that transferred Hfr DNA is replicated in the recipient and that this replication still occurs when the Hfr DNA contains dimers.
The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.
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After conjugation of E. coli, only the preformed donor DNA strand with a leading 5' end can be recovered from the recipient. DNA that was transferred to recipients from radioactive-labeled donor strains lysogenic for lambda bacteriophage was recovered as radioactive material in mature phage particles. The radioactivity was recovered in strand l when gene transfer was in the order gal-lambda-bio, and in strand r when the order of transfer was bio-lambda-gal. The assignment of the 5' end as the origin of the transferred strand follows directly from the known orientation of prophage lambda in the chromosome.