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Inactivation of λ 11 c and its purified DNA by UV irradiation, γ-rays of 137 Cs (in conditions of indirect action), nitrous acid, hydroxylamine and N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) was studied. The biological activity of isolated phage DNA was measured by the calcium transfection procedure. 14 different recipient strains of Escherichia coli K12 were used, including mutants deficient in excision and recombination repair ( uvrA6, uvrB5, uvrC34, polA1, recA13, recC38, recD34, recA13B21C22, recA56uvrA6, exrA and recB21C22sbcB15 ). Whole phage was more resistant to the action of γ-rays than was isolated DNA. On the other hand, the chemical agents HNO 2 and MNNG inactivated phage much faster than isolated DNA. Of all mutations of the host cell only polA1 considerably increased the sensitivity of phage DNA to UV irradiation, γ-rays and MNNG. The mutations uvr − affected the inactivation kinetics under UV action. In all other cases the genotype of the host cell was indifferent for the inactivation kinetics of phage DNA, even if it belonged to recombination deficient mutant λ red3 int6 (in which only UV and γ inactivation was studied). Possible reasons for the low efficiency of the host-cell repair toward the damage caused to λ DNA by different agents are discussed.

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.—Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, exceptfor the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information nd shhowed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I; VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.—Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possiblc to sequence markers which normally are barely linked.

Experiments were conducted to inquire whether the radioressitance observed in an irradiated laboratory population (ROI) of Drosophila melanogaster might be associated in some way with recombinational processes. Simultaneously, data were collected on the stage distribution of radioresistance in ROI by studying the induction of dominant lethals and X-chromosome losses in mature females at various exposure levels of X-irradiation (in eggs sampled from subsequent 12-h broods). The data show that (1) the radiation response of both populations (ROI and its control + K) is equal in the highly sensitive mature stages, (2) ROI is resistant relative to +K in the medium-sensitive stage-7 and younger oocytes collected on days 1.0 to 5.5 after exposure, and (3) the difference between the populations disappears again when the sensitivity steeply decreases on days 5.5 to 6.5. Similar brood-pattern experiments indicate that exchanges between homologous chromosomes are induced (by temperature shock or X-irradiation) in eggs sampled after day 5.5. Thus it is evident that the relative radioresistance in ROI is due to mechanisms which operate in the developing oocyte in the stages of a medium radiosensitivity between that phase in which recombination is inducible and stage-14. The observed temporal sequence of recombination and relative radioresistance in ROI supports the speculation that the latter might be associated with recombination repair. However, the natural recombination frequencies were equal in +K and ROI. Likewise, no clear evidence was obtained on differences between the two populations with respect to X-ray-induced modifications of homologous exchanges in various para- and pericentric parts of the genome.

The anti-tumor drug cis -platinum(II)diamminodichloride (PDD) induced extensive filamentation in wild-type Escherichia coli and in mutants lacking certain deoxyribonucleic acid (DNA) repair functions ( uvrA, recB, recC , and polA ); viability of repair-deficient mutants treated with PDD was significantly less than that of wild-type cells. PDD was highly toxic to lex1, lex1 uvrA6 (where its effect was cummulative), and recA13 mutants, all of which were killed without formation of filaments. 3 H-thymine incorporated into DNA of cells subsequently treated with PDD became trichloroacetic acid-soluble at rates similar to those observed after exposure to comparable doses of ultraviolet light (UV) or mitomycin C. PDD, like UV, induced extensive degradation of DNA in recA organisms. After a 30-min lag, PDD inhibited significantly the synthesis of DNA but not of ribonucleic acid or protein in E. coli . However, the relative differences between rates of DNA synthesis observed in PDD-treated and control cells decreased substantially when the duration of pulses ( 3 H-thymine) was prolonged from 2 to 5 min. These observations suggest that PDD-induced damage to DNA is reversible, possibly by defined mechanisms of excision and recombination repair.

Polar cells of female embryos heterozygous for the genetic markers st in ri were UV irradiated. The effect of UV irradiation on the meiotic recombination within the st-ri segment was analysed. In individuals that contained this chromosome segment in its normal position, UV increased the rate of meiotic recombination. If this segment were separated from the centromeric heterochromatin and translocated on the left arm of chromosome II, UV did not influence meiotic recombination to a significant extent. On the other hand, in non-irradiated controls the individuals containing the translocated segment showed an increased rate of recombination within the segment compared with individuals having the segment in its normal position. These results can be explained by Suzuki's hypothesis according to which a heterocatalytic activity of heterochromatin shows an antagonism to meiotic recombination. UV seems to interfere with this antagonism indirectly via induction of physiological damage.

Tritium applied as 3 H-orotic acid to the anthers of tomato during interphase—early prophase of meiosis increased recombination between the marker loci a and hl by approximately 50 per cent. The same amount of tritium applied as 3 H-thymidine or 3 H-water caused only small, non-significant increases. Equal parts of 3 H-orotic acid and 3 H-thymidine increased recombination by approximately 25 per cent. The 3 H-orotic acid and the 3 H-thymidine were localized in the RNA and DNA fraction respectively. The 3 H-water was not localized in nucleic acids. It is postulated that the 3 H-orotic acid is incorporated into the RNA of the synaptonemal complex during prophase of meiosis and increases recombination by localized β-irradiation.

Action of the carcinogenic chemical 4-nitroquinoline 1-oxide (4NQO) on DNA was studied in two types of UV-sensitive Bacillus subtilis mutants (Hcr− Exc− Deg− Rec+ and Hcr+ Exc+ Deg+ Rec−). The former type of mutant was sensitive to 4NQO and also to its reduced metabolite 4-hydroxyaminoquinoline 1-oxide (4HAQO), but the latter type of mutant was not. The results indicate that the in vivo 4NQO damage to DNA was more susceptible to excision repair than to recombination repair. DNA extracted from 4NQO-treated cells was host-reactivated in the parental strain cells as measured by its transforming activity, but not in the UV-sensitive (Hcr−) cells. The host cell reactivation was not found on free DNA treated with 4HAQO in vitro, indicating that 4HAQO is not the sole active form among metabolites of 4NQO. Induction of DNA single-strand breaks in 4NQO-treated cells was independent of the cellular 4NQO sensitivity. Partial repair of the breaks was found only in the parental strain.

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut + ) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC . In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA + transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC .

THE DNA-binding protein in meiotic cells of Lilium1 has a very high binding affinity for single stranded DNA and also the unusual property of catalysing the renaturation of thermally denatured lily DNA at room temperature. Significantly, these and other in vitro properties are very similar to those of the “gene 32-protein” which is essential to genetic recombination in T4 bacteriophage2 and the possibility that this protein may have a function in meiotic recombination of Lilium led us to a more extensive study of its behaviour3.

Mutants ad-9 of Schizosaccharomyces pombe are characterised by a double requirement for adenine and histidine. The heteroallelic diploids synthesised have shown no complementation. These diploids are subject to mitotic recombinations, whose frequency can be increased by X-rays and UV-light. The recombinations are in fact conversions, which have been analysed by haploidisation of prototrophic diploids under the effect of m-fluorophenylalanine. We have detected no polarity. Contrary to what occurs at the ad-3 locus of Saccharomyces cerevisiae, L -histidine has no effect on the frequency of mitotic recombination. Quantitative data concerning the frequency of mitotic conversion as a function of X-rays or UV-light dose enabled us to construct a map of sites at the ad-9 locus. The order found is the same as that deduced from meiotic recombination.

The action of UV on genetic recombination in Drosophila melanogaster oogonia In this investigation, results concerning the action of UV radiation on the genetic recombination in females of Drosophila melanogaster are presented. Utilizing the technique of transplantation introduced by Ephrussi and Beadle ovaries which had been irradiated in vitro were injected into the body cavity of larvae. This technique was used since it allowed good penetration of UV rays through the gonad. The Latarjet' dosimeter was used to measure the amount of exposure. Four experiments were performed and seven different exposure levels were used. Recombination was studied between (a) genes b pr located near the centromere on chromosome II (Expt. I, exposure: 820 ergs/mm 2 ); (b) genes al, dp, c, px also on chromosome II (Expt. II, exposure: 1500 ergs/mm 2 ); (c) genes y, cv, f on the X chromosome, (Expt. III, exposures: 1000, 1500, 2000, and 3000 ergs/mm 2 ; (d) closely linked genes th, st, and cp located adjacent to the centromere on the third chromosome (Expt. IV, exposures: 500, 1500, and 2500 ergs/mm 2 ). The data show that: (1) UV increased the meiotic recombination frequency; (2) UV irradiation produced some anomalies in the segregation of the genes under study. Investigation of these anomalies indicated that disjunction of each pair of alleles in the irradiated sample was not different from the control. However, comparisons between the individual progenies were very heterogenous. The extent of this heterogeneity varied with the position of the gene along the chromosome. The regions where the heterogeneity was maximum were called ‘hot regions’. When the segregation of two genes was investigated, it appeared that the recombination frequency was increased. This increase was particularly important for closely linked genes located near the centromere. Evidence from this study indicated that somatic recombinations were induced in oogonia, however, these recombinations were not as symmetrical as crossing over. They resembled a process similar to conversion observed in fungi. In fact, an absence of correlation was observed between the frequencies of reciprocal cross-over classes and no systematic association existed between a cross-over and a non cross-over class. The maximum dose of effectiveness was located around 1500 ergs/mm 2 . The nature of the delayed mechanism through which meiotic recombination is affected by UV remains a point of speculation. The finding that some somatic recombinations resemble conversions suggests that (a) UV induces lesions in the DNA and (b) the cell presumably contains an efficient mechanism for repairing these lesions. During this process, the non reciprocal recombinations are thought to result from an error in the rejoining of DNA fragments.

In order to irradiate oogenic primordial germ cells with 2537 A UV light of slight penetrating ability, polar cap cells were exposed. Heterozygous markers on the third chromosome enabled us to measure the crossing-over among progeny from treated cells. Two distinct effects of UV can be observed; one is on mitotic recombination and the other on meiotic recombination. 1. (1) Induction of mitotic recombinations. (a) Progenies from three single females having clusters of cross-overs were found. These clusters were related to UV-induced mitotic recombinations during the first division following irradiation time. (b) Mitotic recombinations were induced in the centromeric region. 2. (2) Increasing of meiotic recombination frequencies. (a) Although some variability exists, crossing-over among descendants from the UV-treated flies was consistetnly higher than spontaneous crossing-over. (b) These UV-induced meiotic cross-over were predominantly found in the centromeric region. (c) This effect is photoreactivable. (d) Although premeiotic cells were exposed to the UV light, meiotic cells were affected which may indicate a delay in the time of action. The most likely hypothesis is that UV produces some physiological damage. This disturbing of metabolic activity might be the primary influence on meiotic crossing-over.

Meiotic cells of Lilium contain a protein that can be identified by its very high affinity for single-stranded DNA. The protein has not been found in either proliferating or nonproliferating somatic tissues. It is present only during the prophase stages in meiotic cells. This protein is localized in nuclei and has been isolated in association with lipid. Its properties are similar to those of “gene 32-protein” of T4 phage. It thus has the property of catalyzing the renaturation of DNA, and in its presence 60% of denatured lily DNA is converted to a double-stranded form at 25°C in 2 hr. The significance of this protein to meiotic recombination is considered in relation to other biochemical properties of meiotic cells.

Optimization of direct DNA delivery into tobacco ((Nicotiana tabacum var. Xanthi) male gametophytes was devised together with development of an efficient transient expression system to study gene expression under controlled conditions. Use of a GFP gene driven by strong promoter and enhancer sequences allowed an efficient non-lethal transient gene expression assay with an overall transient gene expression frequency of > 4% for uninucleate microspores and between 10 and 20% for binucleate pollen. The technique demonstrated its suitability for analysis of developmental stage-specific gene expression. The assay allowed observation of real-time transgene expression during microspore maturation proving useful for in vitro pollen selection. We have also used this protocol to determine the recombination potential of tobacco male gametic cells by assessing the frequency of extra-chromosomal homologous recombination events after co-delivery of two loss-of-function GFP genes. No increase of extrachromosomal recombination was observed in assays for transient transformation. Key words: Biolistic, GFP, Microspore, Tobacco, Nicotiana tabacum, Transformation D.O.I. 10.3329/ptcb.v19i1.4078 Plant Tissue Cult. & Biotech. 19(1): 9-23, 2009 (June)

Non-lethal doses of gamma-irradiation (5 krad) increased meiotic recombination in Coprinus lagopus when treatments were given at the beginning of karyogamy. The division stage at this time was judged to be late leptotene and the duration of the sensitive period was assessed to be 3–4 h. In C. lagopus the radiation-sensitive stage is distinct from the cold-sensitive stage (pachytene). The additive effect of irradiation at early karyogamy followed by cold treatment in pachytene suggested that the two factors influenced different steps in the recombination process. On the other hand, irradiation followed by heat treatment did not significantly alter recombination frequency as compared to single treatments. It was surmised that radiation and high temperature act on the same factor(s) or at the same steps to bring about a similar net result. It was suggested that irradiation at leptotene may cause single-strand breaks in DNA which eventually participate in exchange.

When cultures of Bacillus subtilis were treated with 4-nitroquinoline-1-oxide (4NQO), this compound complexed with their DNA. Strains with a Rec + phenotype recovered from this treatment, whereas Hcr − , Rec − mutants did not. Those strains able to recover were not sensitized to the action of 4NQO by caffeine (CAF), but addition of chloramphenicol (CAP) to the post-treatment growth medium prevented recovery. A measurable level of protein synthesis was maintained by the Rec + strains during the post-treatment incubation, but was absent in Hcr − , Rec − cultures. Treatment of any B. subtilis culture with 4NQO stopped DNA synthesis, and recovery only followed when synthesis was reinitiated. The 4NQO-DNA complex was retained longer by the Hcr − , Rec − mutants. DNA synthesis was never reinitiated in these mutants, and consequently they failed to recover. Complexation of 4NQO with DNA was shown in vitro , and could be correlated with the loss of transforming activity. The data show that DNA is the primary target of 4NQO action in B. subtilis , and that recovery is likely to be through the recombination repair mechanism demonstrated in Escherichia coli .

Bromouracil (BU)-induced mutagenesis of λC17 am o8 phage, in relation to the recombination systems of phage ( red ) or bacteria ( rec ), was studied. The mutations investigated were am → am + . For efficient BU-induced mutagenesis, red or rec A genes as well as bacterial lex gene functions, known to be involved in UV-induced mutagenesis, were required. This suggests a common mechanism or some common step(s) in UV- and BU-induced mutagenesis. Moreover, a several-fold increase was observed in the number of mutants induced by BU in the excision-repair-deficient strain ( uvr A), implying that incorporated BU induces some premutational lesions that are recognized and repaired by excision-repair enzymes. A hypothesis on the possible mechanism of BU-induced mutagenesis is proposed, which assumes a common mechanism for UV- and BU-induced mutagenesis, involving recombination repair processes. Incorporation of a tautomeric or ionized form of BU is considered only as a premutational change in DNA activating the dark-repair mechanisms in cells. The observation that BU enhances the frequency of recombination in λ phages also supports teh idea that recombination processes are involved in BU-induced mutagenesis.

The antibiotic, mitomycin C (MC), induces non-homologous chromosomal interchanges in gonial cells of Drosophila males and females. In both males and females, the interchanges were recovered in a non-random distribution within the gonial broods of individual families. MC also increased the frequency of crossing-over in Drosophila females especially in the centromeric regions of gonial cells, yet did not induce crossing-over in males. The MC-induced increase in crossing-over in females was not a result of gonially induced events that were multiplied mitotically in stem cells as was the case with MC-induced interchanges. MC was also mutagenic in both sexes especially in those cells which had completed the first meiotic prophase. Since MC can either mono- or bifunctionally alkylate DNA, it was proposed that excision of monofunctionally alkylated bases would give rise to single-strand “nicks”, whereas excision of two bases involved in a bifunctionally alkylated crosslink would generate double-strand “cuts”. By this model, single-strand nicks could act as the precondition for crossing-over and would produce crossovers when the necessary factors for spontaneous meiotic crossing-over are present. Double-strand “cuts”, on the other hand, could generate translocation-type interchanges independently of the crossover apparatus in both males and females. A test of this prediction was made with the monofunctional alkylating agent ethyl methanesulfonate (EMS). Results were consistent with the model and revealed that gonial cells just preceding the stage at which crossing-over occurs, are most sensitive to EMS.

The UV response of four K12 strains of E. coli that differ in their repair characteristics was studied as a function of their stage of growth. The four strains used were AB1157 that possesses the full complement of repair genes, the excision-deficient AB-1886 ( uvr A -6), the recombination-deficient AB2463 ( recA -13) and the excision- and recombination-deficient AB2480 ( recA -13, uvrA -6). The UV sensitivity-growth phase profile of the double mutant showed a decrease in early exponential phase and then an increase to the original level by the start of stationary phase that remained unchanged up to 48 h. This decreased sensitivity was found to be associated with an increase in the shoulder of the full UV survivor curve. Deviations from the AB2480 profile shown by other strains reflect the influence of repair mechanisms on the UV-induced damage in cells, and indicate the influence that physiological changes occurring during different phases of the growth cycle have on the efficiencies of the repair systems. Since AB1886 showed a similar profile to AB2480, it appears that postreplicatory recombination repair is unaffected by the intracellular changes. However, the recombination-negative AB2463 showed a marked increase in UV sensitivity throughout exponential growth followed by an increase in resistance that continued up to 48 h. It is postulated that this increased sensitivity in rapidly dividing cells results from a decreased repair efficiency due to a decrease in time before DNA replication takes place and also to a temporary decrease in numbers of repair enzyme molecules within the cells. The enzyme concentration increases in stationary phase and accounts for the enhanced recovery noted during this stage of growth. Results of experiments on pre-irradiation and postirradiation liquid holding of cells support this view. Studies with AB1157 indicate the interrelationship of excision and recombination, working sequentially, at different stages of the growth cycle. A comparison of the AB1157 results with those from single mutants indicate that while both processes are necessary for maximum cellular recovery, in most cases recombination can compensate for decreased excision repair efficiency. However, when excision efficiency falls below a certain limit, recombination can no longer fully cope with the partially repaired cells and an increased sensitivity results. Possible reasons for the interaction of these two dark repair processes are discussed.

When arabinose-grown Escherichia coli B/r is ultraviolet (UV) irradiated in the logarithmic phase of growth, the dose inactivation curve for both colony formation and deoxyribonucleic acid (DNA) synthesis (based on the relative rates of synthesis) is exponential in nature. When protein synthesis is inhibited before UV-irradiation, both inactivation curves have a large shoulder. Pre-irradiation inhibition of protein synthesis increases considerably the colony-forming ability of a UV-irradiated Hcr − and Rec − strain of E. coli B/r. However, with the repair-deficient strains, both the shoulder and slope of the survival curve are affected. We investigated the effect of UV irradiation on DNA synthesis in Hcr − bacteria and found that pre-irradiation inhibition of protein synthesis increases UV resistance of DNA replication in this strain also. The results suggest that inhibition of protein synthesis before irradiation increases UV resistance in E. coli B/r by a mechanism which is independent of both the excision and recombination repair systems.

Amber mutants of bacteriophage can be reverted by ionizing radiation to pseudo wild type particles, i.e. particles able to propagate in a suppressorless host. Since the DNA of phi X 174 is single stranded mutation cannot be due to errors in repair of radiation damage by the excision repair or by the recombination repair system of the host cell, unless local base pairing occurs accidentally at the site of the mutation studied. When the circular single stranded phi X 174 DNA enters the host cell, it is converted by host enzymes into a double stranded RF, which is subsequently replicated. The results presented show that expression of a radiation induced mutation does not depend on replication of RF.

Investigation of the effect of photoreactivation, excision, and recombination repair, individually and in combination, on the survival of ultraviolet-irradiated Escherichia coli K-12 mutants has led to a possible explanation of the loss of photoreactivability and of the complex changes in viability observed during liquid-holding. It is suggested that at higher ultraviolet light doses the excision repair mechanism becomes saturated due to overlapping of excised regions on opposite strands of the deoxyribonucleic acid helix. The results also provide support for the existing hypothesis that states that the shape of shouldered survival curves of ultraviolet-irradiated bacteria can be described in terms of the probability of occurrance of overlapping excised regions. Using the data obtained with repair-deficient mutants with closely related genetic makeup, we present a mathematical model that accurately predicts the shape of the observed survival curves and provides an estimate of the number of nucleotides in each fragment of deoxyribonucleic acid removed by the excision repair mechanism.

Some features of the mechanism of meiotic recombination have been investigated using temperature changes at different times prior to and during meiosis as a tool in order to alter the frequency of recombination. The response to temperature changes (from 25°C to 17°C) of ten different chromosome intervals in three linkage groups of Neurospora has been studied and compared to the response to constant incubation at either temperature (Figs. 4-7).

Diploids with different mutations in the structural gene for nitrate reductase recombine to produce measurable enzyme activity. Radiation induces recombination with high frequency, but only in cells which will survive the treatment. The results suggest that a recombination repair mechanism is induced in irradiated cells.

Crosses of Neurospora crassa, segregating for the spore colour marker asco, were exposed at various stages prior to and during meiosis to a chelating agent, a paramagnetic salt and the classic inhibitor of DNA synthesis, 2′-deoxyadenosine, respectively. The responses of the crosses in terms of second division segregation of asco were studied. All three agents, when added at two different periods, 24–36 hours and about 120 hours after fertilization, respectively, caused significant decreases in recombination frequency. The first of the two responsive periods probably coincides rather well with the premeiotic interphase and the second one with pachytene. The striking similarities of the patterns of response after the three different treatments suggest that the same underlying cellular function is affected in all cases, i.e. DNA synthesis.

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λ imm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation). Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage. To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced. By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background. The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA + function, the red + but not the int + function can substitute for recA + to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage. These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.

MITOTIC recombination can be induced in Saccharomyces cerevisiae by exposing them to ultra-violet and X-radiation1. The genetic maps so obtained closely parallel those from meiotic recombination2. One of the most useful features of mitotic recombination is that it is relatively easy to observe intracistronic recombination. It is possible that this results from a mechanism somewhat different from that leading to meiotic recombination characteristic of higher organisms. For this reason, we decided to study the effects of certain chemical mutagens on the process of mitotic recombination in the hope of being able ultimately to compare these results with those obtained in more usual systems.

Conjugal transfer of UV-damaged F-prime sex factors was investigated in connection with indirect induction. Using donors and recipients of different ability to perform pyrimidine dimer excision and/or recombination repair, it was found that: 1. Transfer of UV-irradiated F-prime DNA to the recipient cell is unaffected by the presence of pyrimidine dimers. The efficiency of transfer does not decrease even at rather high UV doses given to the donor in which, as a result of irradiation, chromosome replication is strongly inhibited. 2. Transfer of the signal responsible for indirect UV induction can be correlated with the transmission of the UV-damaged sex factor from donor to recipient. 3. In the recipient cell, the sex factor DNA does not undergo excision repair but can be repaired by recombination. It is inferred that the transferred DNA structure is not fully double-stranded. 4. In conclusion, indirect UV induction occurs in a λ-lysogenic recipient upon transmission of a UV damaged replicon which carries stabilized lesions as a consequence of its conjugal transfer.

The presence of a perfect or sexual stage in the fungus Emericellopsis salmosynnemata has made possible an investigation of the effect of meiotic recombination on yields of antibiotic. While most of the fruiting bodies produced by this organism are the result of selffertilization, conclusive evidence of cross-fertilization and recombination between two mutants was obtained. Cross-fertilization occurred rarely.

A theoretical derivation of survival curves of UV-irradiated λ phage, in different phage-bacterium systems (lacking or possessing excision and/or recombination repair) is presented. The mathematical expressions are satisfactory fitted with experimental points, and the validity and self-consistency of the model is shown.

SUMMARYAfter liquid-holding treatment in saline, cultures of yeast exposed to u.v. light showed an increased resistance to a second exposure to u.v. irradiation. This increase in resistance during a second series of u.v. exposures was correlated with an increase in the induction of intragenic recombinants. In contrast, no increase in the frequency of the intergenic recombinants or mutations to prototrophy could be detected during the second u.v. dose range. The results obtained could not be explained by the induction of meiosis during liquid holding or by changes in the timing of cell division after u.v. exposure.A model of u.v. repair in yeast is postulated in which liquid-holding treatment results in changes in the proportions of lesions repaired by excision repair and recombination repair (respectively).

SUMMARY: Pyronin Y (0.06 and 0.16 mM), coumarin (2 and 4 mM), 6,9-dimethyl 2-methyl-thiopurine (2 mM) and caffeine (8 mM), strongly inhibit Uvr repair (presumed excision repair) of u.v. lesions in Escherichia coli. With 0.16 mM-pyronin Y or 4 mM-coumarin, one thymine dimer was a lethal event in the K12 strain JC 2926 rec A-13 and one to three dimers in the B/r derivative WP-2 rec A-13 and the B exr A derivative BS-2. Neither these compounds nor 8 mM-caffeine had a significant effect on the survival of irradiated bacteria of the corresponding uvr strains WP-2 Hcr-, BS-12, and BS-8. I suggest that 8 mM-caffeine, 0.16 mM-pyronin Y, 4 mM-coumarin and 2 mM-6,9-dimethyl 2-methylthiopurine selectively block excision repair without substantially affecting recombination repair. Caffeine at 12 mM only slightly depressed the viability of a u.v.-irradiated culture of WP-2 Hcr- or BS-12, but at 16 mM there was a much greater effect, particularly at low u.v. doses (< 40 ergs/mm2). Since excision repair appeared to be effectively blocked by 8 mM-caffeine, the depression of u.v. survival of the uvr mutants by 16 mM-caffeine was presumed to be due to interference with recombination repair (Witkin & Farquharson, 1969).

In Drosophila melanogaster intragenic mitotic recombination between the two lozenge alleles, lz36 and lzy4, separated from each other by 0.14 meiotic recombination units, was observed. Among 48 725 females of the genotype w+lz36/w lzy4 which had been irradiated by a dose of 1000 r X-rays as larvae 41–47 hours after oviposition, a total of 11 faceted eye spots (not lz) were induced. All 11 spots were w+, none w. Possible reasons for the lack of the expected w, faceted spots were checked. Inversion of the X chromosome which would suppress recombination between the w and lz loci was not involved. Gene order of lz36-lzy4-kinetochore was confirmed by meiotic recombination test. Nonautonomy of lz gene action was not a factor, as tested by gynanders which showed that lzy4 and lzs were autonomous. Possibility of reverse mutation was not likely as shown by the large scale control experiments. Gene conversion is suggested as a likely mechanism for the lack of the expected w, faceted spots although the possibility of unequal crossing over induced by X-rays can not be excluded, nor can the preferential z-segregation hypothesis.

The effect on meiotic recombination of acute irradiation treatments, lasting 2 min or less, have been compared with the effect of protracted treatments lasting 24 min. Doses from 2–8 krad were used in both cases. The results support the previous finding that non-lethal doses of radiation change recombination only if administered during two short stages, one in preleptotene and the other in prophase11. The differences between the acute and protracted dose-response curves at the preleptotene ste are dependent on the duration of the sensitive stage, estimated to be about 12 min, in relation to the degree of synchrony in the cell population. At the prophase stage, the difference between the results from the two kinds of treatment arise from a true dose-rate effect, low dose rates failing to produce any change in recombination frequency. The targets at both the preleptotene and the prophase stage are thought to be concerned with DNA synthesis.

An experimental design and rationale for detecting and recovering Saccharomyces cerevisiae mutants specifically blocked in meiotic gene conversion is presented. The system utilizes an otherwise haploid strain disomic (n+1) for chromosome III which is simultaneously heterozygous for the mating-type locus and heteroallelic at leu2. The former is an essential requirement for inducing meiotic development; i.e., DNA replication and sporulation upon transfer to acetate media, while the latter provides a convenient signal for assaying recombination at the intragenic level. Of 940 clones screened qualitatively after mutagenesis with ethyl methanesulfonate, 91 presumptive mutants were isolated. These are classed arbitrarily into four groups according to the reduction in interallelic recombination observed in quantitative tests.

SUMMARYIt is argued that the primary break-points for meiotic recombination arise from the replication of regions in which DNA synthesis is delayed until meiotic prophase. The gaps would be in nucleotide chains of opposite polarity in sister chromatids. This would place a restriction on the recombination models of Holliday and of Whitehouse, so that neither is able to explain observed chromatid interference. It follows that if the primary breaks originate in the manner proposed here, the Whitehouse and Holliday models are either both wrong or both right.

SUMMARYRecombination data from crosses made at a single constant temperature of incubation were compared with those from crosses transferred to a different temperature at either the time of conidiation of protoperithecia by the strain of opposite mating-type, or after fertilization when crozier stages were first visible. Results were also compared from reciprocal crosses, from crosses made in different ways and from crosses in which protoperithecia were conidiated at different stages of maturity.Different temperature regimes during vegetative growth and proto-perithecial development had highly significant effects on subsequent meiotic recombination, while temperature differences during later premeiotic stages (between conidiation of protoperithecia and the crozier stage) had no or little effect. It was found that premeiotic controls could have as great, or greater, effects on meiotic recombination than those operating directly during meiosis. The possible adaptive significance of this is discussed.Recombination frequencies were affected by the method of making a cross (joint-inoculation of strains of opposite mating-type, or conidiation of protoperithecia), and by protoperithecial age at the time of conidiation by the opposite mating-type. Differences in recombination between reciprocal crosses were obtained and were dependent on temperature of incubation and age of protoperithecia at the time of conidiation. Recombination was not affected by different lysine concentrations in the medium. Genetic differences in premeiotic effector-production between the strains used were inferred.

Number: 5 Publisher: Nature Publishing Group; The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal–Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4–RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4–RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication–transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

The question whether recombination in any way affects transcription is unsettled. Of the three possible answers, inhibition, help, or no effect, all have been proposed.

Recombination at both intergenic and intragenic levels has been investigated in diploid yeast strains each homozygous for one allele of three different rev loci that cause reduced UV mutability. Meiotic recombination between leu1 and trp5 and between arg4-6 and arg4-17 was found to occur at control frequencies. The frequencies of UV- and X-ray induced mitotic recombination, measured for both the ade2 -centromere and arg4-6–arg4-17 regions, increased more sharply with radiation dose in rev/rev diploids than in wild type. Since rev mutations do not cause recombination deficiency, it is suggested that mitotic recombination as measured in this study, although induced by UV damage, is not correlated with UV mutagenesis.

DOES sister strand exchange occur as a normal feature during meiosis, or are meiotic crossovers restricted to non-sister chromatids? Evidence from experimental cytology shows that the contribution of sister strand exchange to meiotic recombination is insignificant1,2, or well below the values expected from chiasma frequencies3–6, indicating that sister strand exchange is generally discriminated against in meiotic recombination. Considering fragmentary knowledge from various experimental sources, I suggest how such discrimination may occur.

Four rec genes, responsible for a deficient mitotic conversion in yeast, have been studied for their effects on mitotic crossing over and in meiotic intragenic and intergenic exchanges. Mutations in rec1 do not affect either mitotic segregation of ade2 or meiotic intragenic or intergenic recombination. Homozygous rec2/rec2 diploids yield 1-2% sporulation and ten percent spore viability. They also exhibit normal frequencies of x-ray induced mitotic crossing over of ade2. Mutations in rec3 suppress sporulation, but do not affect mitotic segregation of cyh2. Diploids homozygous for rec4 show normal frequencies for x-ray induced mitotic crossing over in ade8. Such diploids exhibit 60% sporulation and 90% spore viability. Intragenic meiotic recombination between alleles 2 and 17 in arg4 is reduced about 500 times. Meiotic crossing over appears normal for the region between mating type and ade8 and their respective centromers and also in the intervals between thr1 and CUP1 and the centromere of chromosome VIII....