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The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.

The induction of mitotic recombination in the CDC8 locus was studied in a diploid strain heteroallelic for cdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strains cdc8-1/cdc8-1 and cdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction of cdc+ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle-G1, S and G2. It was also found that UV-induced gene reversion can occur during the S and G2 stages, but not during the G1 stage of the cell cycle.

gamma-induced reciprocal mitotic recombination and gene conversion have been studied under conditions inhibiting "rapid" postirradiation recovery of diploid yeast Saccharomyces cerevisiae. It turned out that, if the first postirradiation cell division occurs at higher KCl concentrations ("rapid" postirradiation recovery is inhibited), the frequency of mitotic reciprocal recombination within the gene ADE2-centromere region decreases. Keeping of irradiated cells (in the G1 phase of the cell division cycle) in water at 28 degrees C prior to plating on the selective agar containing 1.5 M KCl leads to smaller frequency of gene conversion lys2-25/lys2-22----Lys+, as compared with that for the cells immediately plated on the selective agar. Correlation has been found between the coefficient of gene conversion frequency decrease, due to postirradiation keeping in water, and "rapid" recovery efficiency. Interpretation of the data is based on the hypothesis that recombination repair of DNA double-strand breaks induced by ionizing radiation is responsible for "rapid" postirradiation recovery.

Cultures of yeast progressing from the exponential to the stationary phase of growth show increased resistance to the lethal effects of the chemical mutagens nitrous acid, ethyl methane sulphonate and mitomycin C and increased sensitivity to the lethal effects of UV light. Induced mitotic intragenic recombination produced by gene conversion also shows variation in its response to the growth phase after mutagen treatment. Higher frequencies of recombination per surviving cell were found after nitrous acid and ethyl methane sulphonate treatment of stationary phase cells whereas identical frequencies were produced by UV and mitomycin C treatment in both growth phases. The results were consistent with the hypothesis that the more nitrous acid and ethyl methane sulphonate resistant stationary phase cells were more active in postreplication repair. The sensitivity of exponential phase cells to nitrous acid and ethyl methane sulphonate may result from both increased mutagen uptake and reduced postreplication repair activity. In contrast, irrespective of growth phase all cells surviving UV and mitomycin C treatment appear to have undergone identical levels of post-replication repair.

From Escherichia coli cells we purified '8'-shaped dimeric molecules in which two circular DNA molecules of bacteriophage lambda were joined at a homologous site. Some of them had a complex junction which we interpreted as being two closely spaced Holliday structures because of (i) superhelicity of the molecule, (ii) the sedimentation rate of the molecule in sucrose gradients, and (iii) the appearance in the electron microscope. Other 'figure-eights's' had two separate homologous junctions, presumably two Holliday bridges. A possible role for these 'double Holliday structures' in UV-stimulated recA-dependent recombination in vivo is discussed.

Intracellular lambda DNA (from EDTA-sensitive tandem duplication phages) was extracted from infected rec+ bacteria and scored for infectivity and recombination (loss of duplication) by transfection of recA recB spheroplasts and subsequent assay for EDTA resistance. When phage development was blocked by repressor or by antibiotics (chloramphenicol and/or rifampin), the apparent recombination frequency was about 0.1% above the background value for recA infections. Prior irradiation of the phage greatly stimulated recombination; the frequency was 20% when UV fluence was 140 J/m2. Repair (recovery of infectivity) and recombination of irradiated phage DNA proceeded readily in the presence of chloramphenicol and rifampin. Inhibitors of DNA gyrase (coumermycin and oxolinic acid) blocked repair and reduced recombination. UV-stimulated recombination was very low in recA but nearly normal in recB cells: repair was reduced in both mutant strains. The recombination remained high as phage/cell ratios less than unity.

The temporal frequencies of different stages of prophase I were determined cytologically in Sordaria brevicollis (Olive and Fantini) as the basis for ascertaining the degree of synchrony in meiosis in this ascomycete. Croziers, karyogamy-zygotene and pachytene asci were shown to be in significant majorities at three distinct periods of the meiotic cycle. The response of recombination frequency to ionizing radiation was examined for the entire meiotic cycle. Three radiosensitive periods were determined. This response, which correlated temporally with each of the three peaks in ascal frequency, is interpreted as showing that the meiotic cycle of this organism is divided into periods of recombination commitment (radiation reduced frequencies) during the pre-meiotic S phase and recombination consummation (radiation induced frequencies) during zygotene and pachytene. The results are discussed in the context of the time at which recombination is consummated in eukaryotes such as yeast and Drosophila.

Lethal action of gamma-rays on derivatives of the wild-type strain AB1157 and of two radiation-resistant mutants (Gamr444 and Gamr445) containing additional mutations dnaA46, recB21, recF143, recA56, recA430, lexA3, lexA102 or lexA3 recAo98, was studied. When the mean number of genomes per cell was reduced by means of pre-incubation at 43 degrees C, radioresistance of the strains AB1157 dnaA46 and Gamr445 dnaA46 was not changed, and that of the strain Gamr444 dnaA46 was reduced to the level of the Gamr445 dnaA46 strain. Introduction of additional mutations recB21, recA56 or lexA3 (lexA102) into the genome of the strains Gamr444 or Gamr445 made them as radiosensitive as the corresponding variants of AB1157. Additional mutations recF143 or recA430 (lexB30) significantly decreased the radioresistance of Gamr444 and Gamr445 mutants, although did not level them to corresponding derivatives of AB1157. Operator-constitutive mutation recAo98 enhanced radioresistance of all lexA3 derivatives tested but not to the level of the corresponding lexA+ strains. The role of recombinational repair and the inducible SOS system in enhanced radioresistance of Gamr mutants is discussed. The data of post-irradiation DNA degradation in various derivatives of the strains AB1157 and Gamr suggest that Gamr mutants have a constitutive inhibitor of degradation which does coincide with RecA protein.

The repair of U.V. damages to DNA in B. subtilis cultures competent for genetic transformation has been studied. The comparison of survival curves for competent and non competent fractions shows that: i) excision repair is more effective in competent than in non competent bacteria; ii) recombination repair is more effective in non competent than in competent bacteria. These facts support the hypothesis that metabolic conditions and, very likely, DNA replication play a role in the regulation of the efficiency of the two different mechanisms of repair.

Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.

F-like plasmids (FBl, FBl drd, F-lac+) were transferred to the recipient strains E. coli, K-12 with different recombination ability. Then the sensitivity of these strains to ultra-violet irradiation and also the stability of the mentioned plasmids under conditions of bacteria treatment with elimination agents was determined. The presence of any F-like plasmids under study failed to influence the ultra-violet sensitivity of the bacterial cells. On the other hand, the research results of spontaneous and inducec elimination of these plasmids indicated considerable differences in relation to the elimination agents. It is supposed that the stability of individual F-like plasmids in the cells largely depended on the genetic differences in rec A gene in the bacterial cells containing them.

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.

Lethal, mutagenic and recombonogenic action of the solar radiation on the model microorganisms--phage T4, bacteria Escherichia coli and ascomycet Aspergillus nidulans--has been studied. A considerable lethal effect of the solar radiation on phage T4 and E. coli was found. An increasing of mutation frequency in E. coli and A. nidulans by sunlight was also revealed. Recombinogenic action of solar radiation has been demonstrated in the experiments with diploid A. nidulans strains. It was shown that the excision and postreplication repair systems took part in recovery of damages induced by sunlight. An important role of ultra-violet region (280-320 nm) solar radiation in induction of lethal and mutagenic effects was demonstrated for all investigated microorganisms.

The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor 'cross' sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.

The lethal effect of 8-methoxypsoralen (8-MOP) plus 365 nm light has been studied in haploid radiosensitive strains of Saccharomyces cerevisiae. The diploid of wild type and the diploid homozygous for the rad2 mutation (this mutation blocks the excision of UV-induced pyrimidine dimers) were more resistant to the lethal effect of 8-MOP plus 365 nm light than the haploid of wild type and rad2 haploid, respectively. The diploid homozygous for rad54 mutation (the mutation blocks the repair of double-strand breaks in DNA) was more sensitive than haploid rad54. The method of repeated irradiation allowed to study the capacity of radiosensitive diploids to remove monoadducts induced by 8-MOP in DNA. This process was very effective in diploids of wild type and in the rad54 rad54 diploid, while the rad2 rad2 diploid was characterized by nearly complete absence of monoadduct excision. The study of mitotic crossing over and mitotic segregation in yeast diploids, containing a pair of complementing alleles of the ade2 gene (red/pink) has shown a very high recombinogenic effect of 8-MOP plus 365 nm light. The rad2 mutation slightly increased the frequency of mitotic segregation and mitotic crossing over. The rad54 mutation decreased the frequency of mitotic segregation and entirely suppressed mitotic crossing over. The method of repeated irradiation showed that the cross-links, but not monoadducts, are the main cause of high recombinogenic effect of 8-MOP plus 365 nm light. The possible participation of different repair systems in recombinational processes induced by 8-MOP in yeast cells is discussed.

The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed.

It has been shown previously that the radiation sensitivity of LexA strains of Escherichia coli K-12 can be suppressed by thermosensitive mutations (designated tsl) that are closely linked to the lexA locus. These are thought to be intragenic suppressors that reduce the activity of the diffusible product that gives rise to the LexA- phenotype (Mount et al., 1973). When a recA mutation is crossed into a suppressed tsl- strain, the extreme radiation sensitivity usually conferred by a recA mutation is considerably reduced without any detectable change in genetic recombination deficiency. Suppression of UV sensitivity depends upon the activity of the uvrA+ product. We propose that at least part of the radiation sensitivity of a recA- strain is due to a DNA repair defect that is different from inability to perform genetic exchanges and depends upon the presence of the lexA+ product. We hypothesize that the lexA+ product is a repressor of the synthesis of repair enzymes. In recA+ cells with DNA lesions, repressor is inactivated leading to enzyme induction but this does not occur in recA- cells. tsl mutations inactivate repressor leading to constitute enzyme synthesis and bypassing the need for recA+ product to inactivate the lexA+ product.

A new model of mammalian cell killing by ionizing radiation is presented. This model, termed the critical DNA target size model, postulates that DNA double-strand breakage is the critical radiation-induced lesion and that the dose-response for such breakage can be non-linear due to the action of a saturable chemical repair process. DNA double-strand breakage occurring within critical targets (proto-oncogene- or common fragile site-associated sequences) is postulated to initiate recombination events with undamaged sequences, leading to chromosomal aberrations. The subsequent loss of acentric fragments at mitosis is postulated to prevent the continuity of the genome and to produce cell death by the induction of chromatin structural changes. Experimental evidence contrary to other radiation action models is examined, and the hypotheses of the model are justified.

The effect of inversions on mitotic recombination outside the inversion was studied in inversion-heterozygotes. Seven euchromatic inversions of the X-chromosome, with breakpoints within the interval between two cell markers, were chosen. The size of the inverted region and the distance from the proximal breakpoint to the proximal cell marker varied. Mitotic recombination was X-ray induced in larvae and clones scored in the tergites of emerged adults. The frequency of recombinants between both cell markers and the frequency of recombinants proximal to the proximal cell marker was used to estimate the effect of interference in pairing caused by the inversions. Such an effect only occurs in small chromosome intervals. This indicates that homologous sequences are tightly paired in the interphase nuclei of somatic cells. This conclusion is derived from data based on X-ray induced mitotic recombination. The possibility of extending this conclusion to non-irradiated cells is discussed.

Recombination of bacteriophage phi X174 was effectively promoted when the Red function of lambda was supplied by either co-infection with lambda or induction of lambda lysogens. Mutations in red alpha and red beta genes of lambda abolished recombination nearly completely, whereas a mutation in gam gene reduced it only slightly. The Red-promoted recombination of phi X174 occurred in recA, recB, and polA mutants as well as in wild-type strains of Escherichia coli. It was further stimulated when phi X174 mutants were irradiated with UV light before infection.

The three major features of meiosis are first synapsis, then exchange, and finally, disjunction of homologous chromosomes; these phenomena occur before pachytene, during pachytene, and after pachytene respectively. The effects of meiotic mutants, or other perturbations, either endogenous or exogenous, on the meiotic process may be assigned tentatively to one of these intervals, based on the earliest discernible abnormality. Thus mutants exhibiting abnormal disjunction and normal exchange affect post-pachytene functions; mutants exhibiting abnormal disjunction and exchange but with ultrastructurally normal appearing synaptonemal complex affect pachytene functions; and mutants with abnormal disjunction, exchange, and synaptonemal complex affect prepachytene functions. This rationale is applied to the temporal seriation of effects of meiotic mutants and chromosomal abnormalities on the meiotic programme.

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.

We have tested the mutant Chinese hamster cell line xrs-5, which is sensitive to ionizing radiation, for the ability to carry out homologous recombination. In an in vivo assay to detect recombination between two transfected plasmids carrying non-complementing mutants in the neomycin resistance gene, xrs-5 showed a 6-fold reduction in recombination frequency when compared to the parental cell line K1. Extracts prepared from nuclei of the mutant were also tested for their ability to catalyze homologous recombination between the same two plasmids in vitro. Extracts from xrs-5 were found to mediate recombination in this assay at frequencies not significantly different from those obtained with extracts from the parental cell line.

The frequency of genetic recombination (with allowance for the fertility system action) has been studied in crosses of 7 different Uvs mutants with two Uvs+ strains (ant+ and ant-) and one specially constructed Uvs strain. It is shown that mutation Uvs, inducing the highest level of sensitivity to UV-light, belongs to the rec-type mutations.

The premutational changes induced by X-irradiation (3 kr or 1 kr) in X-chromosomes of female gametes gave rise to mitotic recombination between irradiated female X-chromosomes and non-irradiated male X-chromosomes in early cleavage nuclei of F1 daughters. Most of the recombinants were non-mosaic females. It can be concluded from the analysis of their phenotypes as well as their offsprings that all nuclei of somatic and germinal tissues of these females were descendants of one from the four first cleavage nuclei (the recombinant one). There were differences in rates and in patterns of recombination between experiments with different stocks. The recombination induced by treatment of female parents usually occurred in the proximal part of X-chromosomes.