Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine

We have studied two X-ray-sensitive mutants xrs 5 and xrs 6 (derived from the CHO-K1 cell line), known to be defective in repair of double-strand breaks, for cell killing and frequency of the chromosomal aberrations induced by X-irradiation. The survival experiments showed that mutants are very sensitive to X-rays, the D0, for the wild-type CHO-K1 was 6-fold higher than D0 value for the mutants. The modal number of chromosomes (2 n = 23) and the frequency of spontaneously occurring chromosomal aberrations were similar in all 3 cell lines. X-Irradiation of synchronized mutant cells in G1-phase significantly induced both chromosome- and chromatid-type of aberrations. The frequency of aberrations in xrs mutants was 12-fold more than in the wild-type CHO-K1 cells. X-Irradiation of G2-phase cells also yielded higher frequency of aberrations in the mutants, namely 7-8-fold in xrs 5 and about 3.5-fold in xrs 6 compared to the wild-type CHO-K1 cells. There was a good correlation between relative inability to repair of DNA double-strand breaks and induction of aberrations. The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase on the frequency of X-ray-induced chromosomal aberrations in these 3 cell lines was also studied. 3AB potentiated the frequency of aberrations in G1 and G2 in all the cell types. In the mutants, 3AB had a potentiating effect on the frequency of X-ray-induced chromosomal aberrations only at low doses. X-Ray-induced G2 arrest and its release by caffeine was studied by cytofluorometric methods. The relative speed with which irradiated S-G2 cells progressed into mitosis in the presence of caffeine was CHO-K1 greater than xrs 5 greater than xrs 6. Caffeine could counteract G2 delay induced by X-rays in CHO-K1 and xrs 5 but not in xrs 6. Large differences in potentiation by caffeine were observed among these cells subjected to X-rays and caffeine post-treatment for different durations. These responses and possible reasons for the increased radiosensitivity of xrs mutants are discussed and compared to ataxia telangiectasia (A-T) cells and a radiosensitive mutant mouse lymphoma cell line.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase

The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of DNA topoisomerase II

Induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) was studied in wild-type Chinese hamster ovary (CHO-K1) cells and its 2 X-ray-sensitive mutants xrs 5 and xrs 6 (known to be deficient in repair of DNA double-strand breaks (DSBs] by restriction endonucleases (REs) and inhibitors of DNA topoisomerase II known to induce DNA strand breaks. Five different types of REs, namely CfoI, EcoRI, HpaII (which induce cohesive DSBs), HaeIII and AluI (which induce blunt DSBs) were employed. REs that induce blunt-end DNA DSBs were found to be more efficient in inducing chromosomal aberrations than those inducing cohesive breaks. xrs 5 and xrs 6 mutants responded with higher sensitivity (50-100% increase in the frequency of aberrations per aberrant cell) to these REs than wild-type CHO-K1 cells. All these REs were also tested for their ability to induce SCEs. The frequency of SCEs increased in wild-type as well as mutant CHO cells, the induced frequency being about 2-fold higher in xrs mutants than in the wild-type cells. We also studied the effect of inhibitors of DNA topoisomerase II, namely 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposid (VP 16), at different stages of the cell cycle of these 3 types of cells. Both drugs increased the frequency of chromosomal aberrations in G2 cells. The mutants showed increased sensitivity to m-AMSA and VP 16, xrs 6 cells being 10- and 2-fold more sensitive than wild-type CHO-K1 cells respectively, and xrs 5 responding with 2-fold higher sensitivity than xrs 6 cells. G1 treatment of CHO cells with m-AMSA increased both chromosome- and chromatid-type aberrations, xrs mutants being about 3-fold more sensitive than CHO-K1 cells. The frequency of SCEs increased also after treatment of exponentially growing and S-phase CHO cells with m-AMSA and the higher sensitivity of xrs mutants (2-fold) was evident. The S-phase appeared to be a specific stage which is most prone for the induction of SCEs by m-AMSA. The results indicate that DNA DSBs induced by REs and inhibitors of DNA topoisomerase II correlate closely with induced chromosomal aberrations and SCEs in these cell lines, indicating that DSBs are responsible for the production of these 2 genetic endpoints.     

Effect of radiosensitivity gene irradiation and genotype on yeast cytoduction]

Effects of gamma- and UV-irradiation of one parent on progenies of yeast crosses are studied. In the crosses of the type: p+ a alc4 X p-alpha ade2 (alc4--nuclear respiration deficiency) zygotic colonies (white) and haploid p+ cytoductant colonies (red) were scored. The irradiation of p+ parent increases the cytoductant frequency up to 17%. When both parents are radiation-sensitive (xrs1, allelic to rad54), the frequency of cytoduction reaches 90%. Crosses p+xrs1 X p-xrs+ or p+xrs+ X p-xrs1 are similar to crosses of wild types. The same results were observed in the case of xrs4 mutants as well as for UV-irradiation. In the progenies of crosses irradiated p+ a alc4 (xrs+ or xrs1) X non-irradiated p-alpha ade2 ura (xrs+ or xrs1) the genotype of red colonies was analysed: in most cases (82.3%-99.8%) they were p+ alpha ade2 ura, that was true haploid cytoductants. It is concluded that irradiation induces particular damages of yeast nuclei leading to a block of normal karyogamy and thus to a cytoductant formation. The highest frequency of cytoduction was observed in crosses of radiosensitive mutants.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Gene recombination in X-ray-sensitive hamster cells.

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.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Reparation after the action of 8-methoxypsoralen and light (lambda=365 nm) on radiosensitive mutants of Saccharomyces cerevisiae yeasts]

The method of repeated irradiation allowed to study kinetics of excision of mono-adducts induced by 8-methoxypsoralen (8-MOP) plus light (lambda=365 nm) in DNA of UV-sensitive mutants rad4 and rad15 and X-ray sensitive mutants rad54, xrs2, xrs4. The survival of the mutant rad4 was not practically increased after incubation in complete liquid medium for 3 hours at 28 degrees C before the repeated irradiation. These data suggest that the mutant rad4 is characterized by nearly complete absence of the mono-adduct excision. The survival of mutants rad15 and rad54 in the same environment was increased less effectively than the survival of the control radioresistant strain, but the mutants xrs2 and xrs4 did not differ from the control strain. Possible causes of differences in survival between radiosensitive strains are discussed. The increased sensitivity of the excision defective strain (rad4) and of the postreplicative recombination defective strains (xrs2, xrs4, rad54) to the lethal effect of 8-MOP plus light (lambda=365 nm) suggests that two systems of reparation take part in the removal of photoproducts induced by 8-MOP in DNA of yeast cells.     

Effect of split doses of radiation on mutation frequency in rodent cell lines

Mutation frequencies in mouse cells and Chinese hamster cells were measured following single or split doses of UV light or X-rays. Split doses separated by a few to over 24h induced no more ouabain- or 6-thioguanine-resistanc mutants than did comparable single doses. These data lend no support to the possible existence of an inducible error-prone repair system in rodent cells.     

High chromosomal sensitivity of Chinese hamster xrs 5 cells to restriction endonuclease induced DNA double-strand breaks

The cytogenetic effects of restriction endonucleases (RE) and X-rays were examined in the radiosensitive mutant Chinese hamster cell line xrs 5 and its normal parental line CHO K1. Cells were permeabilized with Sendai virus and exposed to Pvu II and Eco RV which induce blunt-ended double-strand breaks (dsb) in the DNA of cells, or Bam H1 and Eco R1 which induce cohesive-ended dsb with a four-base overlap. Treated cells were then assayed for the presence of metaphase chromosomal aberrations by sampling at multiple fixation times and in experiments where cells were exposed to graded series of RE concentrations. Exposure to X-rays or RE causing blunt-ended dsb was found to be between two and three times more effective in xrs 5 than in CHO K1 cells. We interpret this higher chromosomal sensitivity of xrs 5 cells as reflecting the reported defect in dsb repair in xrs 5. Both xrs 5 and CHO K1 cells yielded less aberrations after exposure to Bam H1 or Eco R1 than after exposure to Pvu II or Eco RV, confirming our previous results and demonstrating that cohesive-ended dsb are less damaging than blunt-ended dsb. Multiple fixation time experiments showed that the higher sensitivity of xrs 5 was evident at several different sampling times after treatment. Similarly the low yield of aberrations after exposure of cells to Bam H1 was evident at all sampling times. Overdispersion of chromosomal aberrations was observed in samples exposed to RE. This is thought to be due to a non-uniform permeabilization of the cell population to RE. Our results indicate that RE-induced dsb are handled by cells in a similar way to those arising during X-ray exposure.     

Genetic analysis of mitomycin-C-sensitive mutants of a Chinese hamster ovary cell line

5 mutants of a Chinese hamster ovary (CHO) cell line, which exhibit similar levels of sensitivity to killing by mitomycin C, have been analysed genetically to determine whether they represent one or more genetic complementation groups. Hybrids were constructed by fusing cells carrying either the neo or the Ecogpt marker and selecting in medium containing G418 and mycophenolic acid. Selectable markers were introduced into the cells by DNA transfection using pSV5-neo or pSV5-gpt, which represents a quick and convenient method for generating resistant derivatives. Hybrids generated by crosses between any one mutant and the parental cell line exhibited near wild-type resistance to mitomycin C, indicating that the mutants are phenotypically recessive. Self-cross hybrids for all 5 mutants had D37 values for killing by mitomycin C of between 20 and 30 ng/ml. The values obtained for crosses between different mutants were 60-105 ng/ml, with the exception of 1 pairing which gave a value of 33 ng/ml. These results indicate that that the mutants represent at least 4 different genetic complementation groups, suggesting that cellular resistance to mitomycin C is mediated via a number of different mechanisms.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Genetic and biochemical studies of phosphomannose isomerase deficient mutants ofSaccharomyces cerevisiae

Summary Three mannose-negative mutants ofSaccharomyces cerevisiae have been isolated. These mutants showed growth inhibition when mannose was added to a growth medium containing glycerol or fructose. Crosses between wild type mutants showed segregation of 2⁺/2⁻. Crosses between the mutants themselves showed that they were closely linked. Two mutants (XM3 and D2) showed characteristics of allelic structural alteration of phosphomannoseisomerase. Mutant D4 had a deficiency of phosphomannoseisomerase activity, but with a normal thermostability. Revertants from D4 had a normal thermostability.     

Mutants of Arabidopsis Thaliana Hypersensitive to DNA-Damaging Treatments

A simple screening method was developed for the isolation of Arabidopsis thaliana mutants hypersensitive to X-ray irradiation. The root meristem was used as the target for irradiation with sublethal doses of X rays, while protection of the shoot meristem by a lead cover allowed the rescue of hypersensitive individuals. We isolated nine independent X-ray-hypersensitive mutants from 7000 M2 seedlings. Analysis of three chosen mutants (xrs4, xrs9 and xrs11) showed that alterations in single recessive alleles are responsible for their phenotypes. The mutations are not allelic but linked and map to chromosome 4, suggesting mutations in novel genes as compared to previously mapped mutant alleles. Importantly, hypersensitivity to X rays was found to correlate with hypersensitivity to the DNA-alkylating agent mitomycin C, which provokes interstrand crosslinks, and/or to methyl methanesulfonate, which is known as a radiomimetic chemical. These novel phenotypes suggest that the mutants described here are altered in the repair of DNA damage, most probably by recombinational repair.     

DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining

Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 heterodimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3′-overhangs that abut a 5′-overhang or blunt end. While the sequences of the 3′-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.     

Genetic effects of sulfur-35 decay in Saccharomyces cerevisiae cells. V. Comparative study of the lethal and mutagenic effectiveness of the decay of 35S and 32P incorporated into cells of the radiosensitive mutant xrs2]

The lethal effect of 35S and 32P decays on cells of yeast radiation-sensitive mutant xrs2 was studied. The mutant is 7 times more sensitive than the wild type to transmutation of both isotopes. The survival curve for xrs2 was exponential. In spite of the lethal effect, mutant cells are not more mutable than the wild type under decays of both isotopes (the number of mutations in ade1 and ade2 genes was counted), xrs2 and wild type strains differ in kinds of mutations induced by the decay of incorporated 35S in ade2 locus. Namely, there are 82% of base substitutions and 18% of other types mutations induced in xrs2 strain despite 97% and 3% respectively for the wild type strain. Also it was shown that complete and mosaic mutants, induced by the the 35S decay in xrs2 strain, differ in a pattern of interallelic complementation.     

Studies on mammalian mutants defective in rejoining double-strand breaks in DNA

Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E. coli and the yeast, Saccharomyces cerevisiae. More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described. These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups. The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms. Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed. All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation. This is similar to the rad52 mutants of S. cerevisiae but contrasts to the majority of the E. coli mutants with defective dsb rejoining. Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs. This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained). Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism. The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants.     

Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.     

Differentiation is not restored in hybrids between independent variants of a rat hepatoma

Crosses have been undertaken between cells of three independent clones of dedifferentiated rat hepatoma variants to investigate whether "complementation" leading to restoration of the original differentiation would occur. Hybrids were examined between ten days and two months after fusion for the presence of intracellular albumin and for their ability to proliferate in glucose-free medium where survival requires activity of the liver-specific gluconeogenic enzymes. In none of the three possible crosses involving the three variants was evidence of reexpression of hepatic functions obtained.