Biomedical subjects
Publications and source records attributed to I M Stepanova.
The cloned recA gene from Serratia marcescens Sb was expressed and complemented defects in the UV repair, recombination, and SOS induction of an Escherichia coli host deleted for recA. Moreover, the Serratia gene, recA (Sm), supported the same frequency of recombination per unit length of DNA as did the homologous Escherichia coli gene, recA(Ec).
RecF, recQ, ruv, recJ and recN genes of so called RecF pathway of recombination appear to be not silent on the RecBCD pathway also. These genes are responsible for the frequency of recombination exchanges per unit length of DNA. The list: recF::Kmr greater than recQ::Tn3 greater than ruv54 greater than recJ::Tn9 demonstrated the efficiency of inhibition of recombination exchanges by these mutations. The recN262 mutation gives a feeble contrary effect. It slightly increases the frequency of recombination exchanges.
The hybrid plasmid pYBP2 with bacterial (ampR), yeast (LEU2) and bacteriophage T4 (denV) genes has been constructed. The plasmid transformed Escherichia coli CSR603 uvrA recA ampS leuA phr- to ampicillin resistance, leucine independence, UV-resistance similar to the one of uvrA+ recA strain. Cell-free extracts of transformed Escherichia coli cells contain low level of ultraviolet-endonuclease activity in contrast to nontransformed cells containing no enzyme.
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Genetically deficient cells were supplied with the missing enzymes, purified from an independent source. The introduction of exogenous enzymes into the cells was effected by two independent methods: plasmolysis and liposome transformation. The latter procedure yielded a homogenous cell population which had been rescued from the defect even if the molecular weight of the enzyme amounted to 70 KD (Kilodaltons).
The effect of radioprotection of indolylalkylamines (5-methoxytryptamine) and aminothiols (cysteamine) on E. coli cells is practically absent if the cells have genetic defects in the repair systems. This means that the explanation of radioprotection by scavenging of free radicals is invalid and that specific repair mechanisms may be involved. In order to explain the radioprotective mechanism it was suggested that the radioprotectors interact with the damaged sites in DNA so that they become partly screened from repairing endonucleases. Under these conditions the reduction of incision rate results in diminished enzymatic induction of lethal double-strand breaks in DNA, this being important only in wild type cells. To prove this hypothesis an experimental procedure was developed using bacterial cells carrying plasmids (ColE1). This procedure enabled to determine the in vivo rate of enzymatic incision of gamma-sites. It was found that the protectors did not change the total amount of gamma-damages in DNA but reduced the rate of enzymatic incision.
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A marked increase in radioresistance of E. coli cells, of the wild-type repair genotype, was observed when they were exposed to gamma-radiation at 40-45 degrees C. The effect of the thermoinduced radioresistance did not depend on the growth medium and the pre-incubation temperature but disappeared completely after treatment of cells by chloramphenicol or CaCl2 or after modification of cell membranes by exogenous cholesterol. This phenomenon was not observed with UV-irradiation. It is suggested that the thermoinduced radioresistance is connected with the activation of the membrane-associated repair complex.
A study was made of the influence of the repair genotype of E. coli cells on the realization of the effect of enhanced radioresistance during gamma-irradiation at elevated temperatures (40-45 degrees C). The effect of the thermoinduced radioresistance (TIR) was diminished significantly but not abolished completely in mutant cells selectively deficient in excision or recombination repair systems (po1A1, recB21C22sbcB15, recF143 mutants). However mutations which exclude the recA gene product (recA13, recA13B21C22 or lexA3 mutants) inhibited TIR completely. The introduction of recA+ gene into recA- or lexA- mutants almost normalized TIR. On the basis of the data obtained the authors discuss the role of recA protein in activation of the membrane-associated repair complex whose efficiency depends on the temperature of gamma-irradiation.
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