Studies on three mutagen-sensitive mutants of mouse L5178Y cells. II. Complementation analyses between two methyl methanesulfonate-sensitive mutants and between two 4-nitroquinoline-1-oxide-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, were isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline-1-oxide (4NQO); and Q31 cells are cross-sensitive to UV and 4NQO. MMS-, X-ray- and UV-sensitive markers in these mutants behaved recessively in hybrids between pairs of these mutants as in hybrids between L5178Y and these mutants as reported before (Shiomi et al., 1982b). Complementation analyses were carried out by forming hybrids between two MMS-sensitive mutants (MS-1 and M10) and between two 4NQO-sensitive mutants (M10 and Q31). MMS and 4NQO survivals were measured in these hybrid cells. MS-1 and M10 were found to belong to different complementation groups for MMS-sensitive phenotypes. The hybrid clones between M10 and Q31 were as sensitive to 4NQO as each of the mutants, indicating codominance of 4NQO sensitivity in these mutants. The hybrids constructed with L5178Y and three mutants were stable as to their chromosome constitution for 100 days of cultivation without selective pressure. From the segregation studies on these hybrids, it is concluded that neither the X-ray-sensitive mutation in M10 nor the UV-sensitive mutation in Q31 is located on the X chromosome.     

A UV-supersensitive mutant in the yeast Schizosaccharomyces pombe

Summary A high UV-sensitive mutant was obtained from a UV-sensitive strain of the yeast Schizosaccharomyces pombe after a mutagenic treatment. By genetic analysis, it was possible to distinguish two independent loci. The double mutant is supersensitive, that is more UV-sensitive than either of the two single mutants. This suggests that the mutations involved interfere with two repair pathways that are, at least partially, independent of each other.Some properties of the two single mutants were studied. These mutants differ notably in their response to caffeine, to liquid-holding, to exposure to visible light after UV irradiation, and in their UV-sensitive during the logarithmic growth phase.Comparison of the properties of the wild-type strain and of the different UV sensitive mutants leads to the conclusion that one repair pathway is used preferentially in the wild-type strain.Abbreviations DRF dose reduction factor - LH liquid holding     

UV- and X-ray-sensitive double mutants of mouse L5178Y cells are synergistically more sensitive to 4-nitroquinoline-1-oxide than is either of the single mutants

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.     

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in Neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similar to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produced some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in "error-prone" repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.     

Competence mutants. 3. Responses to radiations

Class 3 com(-) mutants [normal in deoxyribonucleic acid (DNA) uptake but poor in ability to transform] were investigated with regard to ultraviolet (UV) and X-ray sensitivity of colony-forming ability and with regard to their ability to be transformed by UV- and X-ray-irradiated DNA. Three mutants, com(-)40, 60, and 78, were highly UV-sensitive in colony-forming ability. None of the mutants was more sensitive than wild type to UV-irradiated transforming DNA; in fact, six of the mutants showed considerably greater resistance. Two of the mutants (com(-)40 and 60) were slightly more sensitive to X ray in colony formation, whereas most of the mutants showed some degree of sensitivity to X-ray-irradiated transforming DNA. In addition, the physical fate of X-ray-irradiated transforming DNA has been examined, and in one case (com(-)48) there was a significant drop in sedimentation value of X-ray-irradiated donor DNA after uptake by recipient cells. The com(-) mutants analyzed have been classified on the basis of their UV and X-ray sensitivities, and, where appropriate, possible biochemical lesions have been implicated.     

Studies on the ultraviolet-sensitive mutants of blue-green alga Synechocystis aquatilis Sanv.

Summary Several mutants of the unicellular blue-green alga Synechocystis aquatilis Sanv. were isolated. They differed from the wild type by the levels of sensitivity to ultraviolet (UV) irradiation. The most sensitive mutant is unable to carry out photoreactivation and shows increased resistance to mitomycin C, N-methyl-N-nitro-N-nitrosoguanidine and methyl methanesulfonate. This strain shows an enhanced rate of spontaneous and UV-induced mutagenesis. Another UV-sensitive mutant with normal level of X-ray sensitivity is characterized by a decreased mutability. The three other UV-sensitive mutants show simultaneous decrease of resistance to X-ray and alkylating agents. The existence of these cross-sensitive mutants indicates that a repair mechanism may operate in blue-green algae similar to dark repair systems of bacteria and yeast.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Isolation of UV-sensitive clones from a haploid frog cell line.

An isolation procedure has been developed which yielded five clones of haploid frog cells which are sensitive to ultraviolet light. This procedure employed a conventional mutagenesis, followed by time for phenotypic expression and then an enrichment for UV-sensitive mutants. The enrichment relies upon the uptake of bromodeoxyuridine (BrdU) by repairing cells following UV-induced damage, rendering repair-proficient cells differentially sensitive to photolysis by black light. The photolysis is potentiated by use of the bisbenzimidazole dye Hoechst 33258. The enriched population was screened for radiation-sensitive isolates resulting in 5 sensitives from 96 tested. No mutants were obtained from 300 isolates tested from a population which had not undergone enrichment.     

Isolation of UV-sensitive variants of human FL cells by a viral suicide method.

A new method (viral suicide method) for the isolation of UV-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with UV-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their UV sensitivity. Nineteen of 238 clones examined were sensitive to UV irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their UV sensitivity for 4 months in culture. UV sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (D0) are 2.9, 3.7, and 7.8 J/m2 for UVS-1, UVS-2, and the parental FL cells, respectively- They are no more sensitive than FL cells to X-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability.     

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Summary Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (radl-1, 2, 3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants radl-1, 3, 5, 6, 9 and 11 differ in their response to LH afterr the two treatments. Survival of radl-1 and rad3 increases significantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11 and 22 show marked LHR after UV but no increase of survival after DEB treatment.     

Synergistic interactions between rad mutations in yeast

UV-survival data are presented for haploid yeast strains carrying mutations in two or more rad genes. Some of these mutants are not, by themselves, very sensitive to UV-light but nevertheless show a strong synergistic interaction with other UV-sensitive mutants. It is proposed that such synergism arises when mutations block two repair pathways each acting upon a common substrate. Multiple-mutant strains have been used for exposing interactions between mutants which only become fully expressed in an “excisionless” genetic background. The results indicate that the seven genetic loci studied mediate three different types of recovery from UV-irradiation.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Repair of UV-induced damage in wild-type and mutant strains of Schizophyllum commune

The basidiomycete fungus Schizophyllum commune was found to have both photo-repair and dark-repair systems for UV-induced damage. Three UV-sensitive mutants were isolated and characterized for ability to repair UV-induced damage in light and dark, and for cross-sensitivity to caffeine and methyl methanesulfonate. Two of the mutants were damaged, to different extents, in their capacity for excision repair; one of these mutants was also probably damaged in post-replication repair. The third mutant was damaged only in post-replication repair.     

Mutagen-sensitive cell lines are obtained with a high frequency in V79 Chinese hamster cells

A replica-plating technique has been adopted for the isolation of mutagen-sensitive mutants of Chinese hamster V79 and CHO cell lines. After the mutagenic treatment (ENU) clones derived from these cell lines were replica plated into micro wells and replicas were treated with UV (254 nm), X-ray, MMC, EMC or MMS. Clonal cell lines which demonstrated mutagen sensitivity were retested by the determination of survival. Only one UV-sensitive line was obtained in 1500 clonal lines derived from CHO cells. This mutant appeared also sensitive to 4NQO and MMC. The sensitivity to UV and MMC was 2-3-fold enhanced, while the increase in sensitivity to 4NQO was 4-5-fold. In V79 cells 9 mutagen-sensitive lines were found after screening of 500 clonal lines; six of them showed increased sensitivity towards UV, two towards MMC, and one cell line was found to be X-ray sensitive. A considerable cross-sensitivity for the various agents was found among the isolated mutants. When a 2-fold increase is taken as a minimum to indicate mutagen sensitivity 6 mutants were sensitive to UV, 8 mutants were sensitive to MMC, 6 mutants were sensitive to 4NQO and 4 mutants were sensitive to X-rays. The difference in sensitivity to UV versus 4NQO makes it unlikely that 4NQO can be considered as a UV-mimetic agent. The sensitivity to MMC appears to fall into 2 classes: a class with moderate sensitivity (2-8-fold) and a class with high sensitivity (30-100-fold). The presence of similar classes is indicated for UV. Except for the two lines V-E5, V-B7 and the two lines V-H11, V-H4 all obtained mutants have a different spectrum of mutagen sensitivities which suggests that different genetic alterations underly these effects. The observed high frequency of mutagen-sensitive mutants in V79 cells, although unexpected and substantially higher than those published for CHO cells and L5178Y cells, can still be explained by the presence of functionally hemizygous loci.     

Directed mutagenesis of chromosomal genes of Escherichia coli in vivo: construction of RecA- and HtpR- mutants

The deletions in Escherichia coli chromosomal genes recA and htpR were constructed using the site-directed mutagenesis techniques. The obtained RecA- mutants are UV-sensitive and have a phenotype defective for the homologous DNA recombination. HtpR- mutant is temperature sensitive for growth and deficient in intracellular proteolysis. As a result a HtpR- mutant seems to be a preferable candidate for attempting to synthesize efficiently any alien protein in Escherichia coli cells.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Repair of potentially lethal damage in yeast induced by fast neutrons

Survival curves of 3 diploid (D7) yeast strains: one wild-type, one deficient in excision of pyrimidine dimers (UV-sensitive) and one blocked in DNA double-strand-break repair (X-ray-sensitive), were compared after irradiation with cyclotron-produced fast neutrons. It was observed that both the UV-sensitive (rad3/rad3) and the X-ray-sensitive (rad52/rad52) mutants were more sensitive to neutrons than the wild-type. The role of DNA double-strand-breaks in neutron-induced cell death was further studied by comparing the relative sensitivity of the rad52/rad52 mutant to gamma-rays and fast neutrons. A comparison of the dose modification factors revealed that the deficiency in DNA double-strand-break repair did not make the yeast cells more sensitive to neutrons than to photons, which suggests that lesions of a different type may also be produced by neutrons. Survival curves obtained upon immediate plating and after delayed plating of neutron-irradiated cells showed that all 3 yeast strains were efficient in liquid holding recovery. The role of different repair pathways in cellular recovery from neutron-induced lethal damage is discussed.     

Isolation of recombination-defective and UV-sensitive mutants of Bacillus megaterium.

Mutants of Bacillus megaterium QMB1551 sensitive to mitomycin C or methyl methanesulfonate were isolated and characterized phenotypically. Cell survival after UV-light and gamma-ray exposure was determined, as was transductional recombination. Of the mutants tested, three were sensitive to UV but remained recombination proficient. The UV-sensitive mutants were also reduced in host cell reactivation. At least three mutants had undetectable transduction frequencies, i.e., less than 0.3 to 1.3% of the parental strain frequencies, and so appear to be recombination deficient. Sensitivities of these mutant strains to UV light and gamma radiation were compared with those of parental B. megaterium as well as parental, recE4, recA1, uvrA19, and uvrB109 strains of Bacillus subtilis. In each case, the strains of B. megaterium, including the parental strains, showed a higher percentage of cell survival than B. subtilis.     

Recombination in Relation to Ultraviolet Sensitivity in CHLAMYDOMONAS REINHARDI

A mutant strain of Chlamydomonas reinhardi which is UV sensitive as a result of a single-gene chromosomal mutation has also been found to exhibit reduced recombination. In crosses homozygous for the mutant allele, a reduction in recombination frequency was demonstrated in two different linkage groups and in three different genetic backgrounds. Thus a single mutation affecting UV sensitivity also has a possible effect on recombination. Such a mutant could be analogous to the rec(-) mutants discovered in E. coli and as such be useful in the study of recombination mechanisms. Three additional UV-sensitive isolates were tested. Recombination was not altered in these mutant strains.     

Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively.