Ultraviolet-inactivation of conidia from heterokaryons of Neurospora crassa containing uv-sensitive mutations.

The effect of three UV-sensitive mutations of Neurospora crassa, upr-I, uvs-4 and uvs-6, on the ultraviolet-inactivation of conidia from two-component heterokaryons was investigated. In two-component heterokaryons with wild-type sensitivity to radiation inactivation, all three conidial fractions exhibited similar ultraviolet-inactivation curves. Each UV-sensitive mutation studied uniquely modified the ultraviolet-inactivation curves of conidia from two-component heterokaryons. In heterokaryons heterokaryotic for upr-I, the upr-I mutation was recessive and the repair function determined by the wild type allele was functional to some degree in homokaryotic upr-I conidia. All three conidial fractions of heterokaryons containing upr-I in both components showed increased sensitivity to ultraviolet light. The uvs-4 mutation was recessive and resulted in conidia with increased UV-sensitivity only when included in both components of a heterokaryon. Homokaryotic uvs-4 conidia, which arose from heterokaryons containing both uvs-4 and wild-type components, exhibited wild-type survival. Therefore, as with upr-I, there was a carryover the repair capability to conidia which were genetically UV-sensitive. The uvs-6 mutation, when included in one component of a two-component heterokaryon, resulted in increased UV-sensitivity of both heterokaryotic and homokaryotic uvs-6 conidia. When both components contained uvs-6, the UV-sensitivity of all three conidial fractions was increased and all showed similar inactivation curves. Thus, as with upr-I and uvs-4, there was a carryover of the wild-type repair capability to genetically uvs-6 conidia. Heterokaryon tests for complementation between two non-allelic UV-sensitive mutations showed that in heterokaryotic conidia, complete complementation occurred between upr-I and uvs-4.     

Mutagenesis at the ad-3A and ad-3B loci haploid UV-sensitive strains of Neurospora crassa. III. Comparison of dose-response curves for inactivation and mutation induced by gamma-rays

Gamma-Ray-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 6 different UV-sensitive strains and a standard wild-type strain. The 6 strains show varying degrees of sensitivity to gamma-ray-induced inactivation, with the relative sensitivity at 37% survival being uvs-6 greater than upr-1 greater than uvs-2 greater than uvs-3 greater than wild-type greater than uvs-5 greater than uvs-4. Studies on the induction of ad-3 mutants by gamma-rays show that when the dose-response curve (expressed in terms of ad-3 mutants among the surviving colonies) of the UV-sensitive strains are compared with wild-type, the 2 excision-repair-deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, uvs-3 exhibits reduced ad-3 mutant frequencies whereas both uvs-4 and uvs-5 show lower mutant frequencies than wild-type.     

UV-induced recessive lethals in uvs strains of Neurospora which are deficient in UV mutagenesis

The frequencies of spontaneous and UV-induced recessive lethal mutations were compared for UV-sensitive and wild-type heterokaryons of Neurospora crassa. These heterokaryons were homokaryotic either for one of two alleles of uvs-3, or for uvs-6 or uvs+. For uvs-3, which is known to have mutator effects, spontaneous recessive lethals were found to be 4-6 times more frequent than observed in uvs+. After correction for clonal distribution of spontaneous mutants, an observed 2-fold increase for uvs-6 was not statistically significant and may have been due to chance occurrence of a few large clones of mutants. Treatment with low doses of UV (50-200 J/m2) produced very similar overall rates of increase for recessive lethals in uvs and uvs+ heterokaryons. This means, that in contrast to results obtained when mutation to ad-3 was measured, both uvs-3 alleles showed highly significant increases for recessive lethals when treated with UV. It is proposed that certain types of UV damage may be processed into recessive lethal mutations by an alternate mechanism from that responsible for viable mutations.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. II. Comparison of dose-response curves for inactivation and mutation induced by UV

UV-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 7 different UV-sensitive strains and a standard wild-type strain. The 7 strains show varying degrees of sensitivity to UV-induced inactivation, with the relative sensitivity being: uvs-2 greater than uvs-3 greater than uvs-4 greater than uvs-6 greater than upr-1 greater uvs-5 greater than uvs-1. Studies on the induction of ad-3 mutants by UV show that the 2 excision-repair deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, while uvs-4 and uvs-5 exhibit reduced ad-3 mutant frequencies, and uvs-3 completely eliminates UV mutagenesis. The ad-3 mutation-induction curves obtained with uvs-1 or uvs-6 are not significantly different from that found with the wild-type strain.     

Epistatic Grouping of Repair-Deficient Mutants in Neurospora: Comparative Analysis of Two uvs-3 Alleles, uvs-6 and Their mus Double Mutant Strains

The nuclease halo mutant, nuh-4, of Neurospora crassa was identified conclusively as an allele of uvs-3, a gene involved in error-prone DNA repair. Like uvs-3, nuh-4 showed spontaneous mutator effects, and any previous contradictory findings were found to be due to newly arisen mutants. In normal strains the two alleles are noncomplementing and indistinguishable for sensitivity to UV and methyl methanesulfonate (MMS). Like uvs-3, nuh-4 lacked secretion of the extracellular enzyme, DNase A, a Ca⁺⁺-dependent strand-nonspecific endonuclease which was found to be phosphate repressible. However, nuh-4 differed from uvs-3 in showing much higher conidial viability and lower sensitivity to ionizing radiation and mitomycin C.——Epistatic relationships of the two uvs-3 alleles with seven other MMS-sensitive mutants were determined and compared with those of the highly X-ray-sensitive mutant, uvs-6. Three epistatic groups were found, based on survival of double mutant strains relative to that of their component single mutant strains after treatment with MMS. Both, uvs-3 and nuh-4, were epistatic to mus-9 which also is a mutator. None of the three produced viable double mutants in crosses to uvs-6. On the other hand, uvs-6, but not the uvs-3 alleles, was found to be epistatic to mus-7 and mus-10. The excision-defective uvs-2 and mus-8 both showed synergism with the uvs-3 alleles and with uvs-6, forming a third, separate epistatic group.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

Effect of the homokaryotic state of the uvs-2 allele in Neurospora crassa on formaldehyde-induced killing and ad-3 mutation

Formaldehyde was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 3 two-component heterokaryons (dikaryons) of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on formaldehyde-induced killing and the induction of ad-3 mutants. These dikaryons were homokaryotic for uvs-2+ (H-12), homokaryotic for usv-2 (H-59), and heterokaryotic for uvs-2 (H-71). Formaldehyde induced killing and ad-3 mutants in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a 9-fold increase in killing and a 40-fold increase in the induction of ad-3 mutants. This increased sensitivity to formaldehyde-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is similar to that noted by others in Escherichia coli. Salmonella typhimurium and Saccharomyces cerevisiae. The dikaryon heterokaryotic for uvs-2 (H-71) has the same sensitivity to formaldehyde-induced ad-3 mutation as H-12, indicating that uvs-2 is recessive to uvs-2+.     

The ability of ionizing radiations of different LET to induce chromosomal deletions in Aspergillus nidulans.

Conidia, derived from a strain of Aspergillus nidulans known to carry a specific chromosomal duplication, were irradiated. The duplicated segment had genetic markers, which, when eliminated from the genome, allowed the easy detection of deletion mutants. Survival curves derived following 15 MeV electron and gamma-ray irradiation were characterised by the presence of an appreciable shoulder, whilst 50 kvp X-rays gave a much smaller shoulder. Irradiation with beta-particles and alpha-particles gave rise to exponential survival curves. The RBE values for these radiations, based on the D37 value were for gamma-rays, 1.0, 15 MeV electrons 1.0, 50 kvp X-rays 1.9, beta-particles 2.1 and alpha-particles 3.4. With the exception of gamma-rays the radiations described were compared with respect to their ability to induce chromosomal deletions. When the number of deletants amongst survivors was plotted against dose, a linear relationship was found for electrons, X-rays and beta-particles. The response recorded for alpha-particles was essentially linear but with a biphasic component. The RBE values for the radiations, based on a value of unity for 15 MeV electrons were as follows: X-rays 1.3, beta-particles 0.8, alpha-particles above 7.5 krad 2.3 and below 7.5 krad 3.5. When these same data were re-plotted with number of deletants amongst survivors against log survival, electrons appeared the most efficient radiation at producing deletants amongst survivors, with an "m value" of 283 X 10(-5). Tritiated water was least efficient, the corresponding value being 182 X 10(-5). The number of deletants per 10(4) conidia plated, when plotted against dose yielded a curve which increased to a peak and then decreased linearly for all radiations. The peaks for electrons, X-rays and alpha-particles each had a value of about 14 deletants per 10(4) conidia plated and the peaks roughly corresponded with the point at which the survival curve became exponential and was clearly indicative of the accumulation of sub-lethal damage. However, for beta-particles the peak had a value of 7 deletants per 10(4) conidia plated. A non-DNA target has been implicated for cellular death following beta-particle irradiation.     

Rescue of marker phenotypes: effects of BUdR sensitization, hypoxia and high LET.

The survival curve of colony-forming ability of Chinese hamster wg3h cells has been compared with the dose-response curve for the expression of an active thymidine kinase (TK) gene from these cells. The TK+ phenotype was measured by hybrid colony formation after fusion of wg3h (TK+) cells with Chinese hamster A23 (TK-) cells. The TK+ survival data fitted a multi-target curve up to 3 krad of 137 Cs irradiation, when a highly resistant fraction of hybrid colonies was seen at about 1 per cent survival. The Do of TK+ survival for the multi-target region was 3.1-4.0 times greater, than that of wg3h survival, even when the Do for cell survival varied between 136 and 545 rad by 14 MeV neutrons and hypoxia respectively. This parallel modification of cell and TK+ sensitivities suggests that the lesions causing cell inactivation are of the same type as those that cause marker inactivation. Using 14 MeV neutron data the approximate target size for TK inactivation was calculated to be 0.54-0.91 per cent of the DNA content of the cell (or about one-fifth to one-tenth of a chromosome). The data support the idea that marker inactivation results primarily from damage occurring outside the marker gene. BUdR labelling of wg3h cells before irradiation caused slight toxicity (30 per cent reduction in plating efficiency) and a twofold increase in cell sensitivity. However, the sensitivity of the TK+ phenotype increases by only 30 per cent. The increased cell sensitivity thus appeared to result from synergism between increased sensitivity of DNA to strand breakage and metabolic toxicity, the latter being largely overcome by fusion with normal cells.     

Induced Repair of Genetic Damage in Neurospora

Repair of genetic damage in Neurospora has been studied using a procedure in which one strain is exposed to a potentially lethal dose of UV before being joined in a heterokaryon with an undamaged strain. We have monitored the ability of the second strain to rescue the first. The extent of rescue is greatly enhanced when the rescuing strain has itself received a small, nonlethal dose of UV, thus demonstrating an inducible repair system.--The experiment was modified by substituting X rays or nitrous acid for UV as either the damaging agent or the inducing agent. In every combination, induced rescue was observed.--Three repair-deficient mutants (uvs-2, uvs-3 and uvs-6) were substituted for wild type (uvs+) as the rescuing component to find out whether any of them lacked the inducible repair system. Both uvs-2 and uvs-6 demonstrated inducible repair; uvs-3 showed none, but gave a high level of repair without induction, suggesting that it is a regulation (derepressed) mutant of an inducible repair system.     

Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA.

UV survival curves of adenovirus 2 using fused, complementing xeroderma pigmentosum (XP) fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusion involved strains in the same complementation group. Extrapolation of this component indicated that at zero dose 3% of the viral plaque-forming units had infected cells capable of normal repair. These results suggest that 3% of the cells were complementing heterokaryons, a value similar to that actually observed by autoradiographic analysis of UV-induced unscheduled DNA synthesis. Thus, heterokaryons formed from XP fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells.     

An Apparent Connection between Histidine, Recombination, and Repair in Neurospora

Two mutants of Neurospora crassa, uvs-3 and mei-3, share four properties--UV sensitivity, inhibition by histidine, meiotic blockage when homozygous, and increased duplication instability (due to mitotic crossing over, to deletions or to both). The present paper shows that a third nonallelic mutant, uvs-6, exhibits the same four properties.--Also, the instability of duplications in the absence of any UV-sensitive mutant is increased by the presence of histidine in the growth medium.     

Properties of a UV-sensitive Neurospora strain defective in pyrimidine dimer excision

The UV-sensitive Neurospora strain uvs-2 is known to resemble the excision-defective uvr mutants of E. coli K12 in being both excision-defective and highly UV mutable. As shown in this report, the uvs-2 strain also resembles the uvr mutants in its ability to remain photoreactivable when held in the dark for 2 h between UV-irradiation and photoreactivating light exposure, and in its maintenance of the same spontaneous deletion rate as wild type strains.Unlike the E. coli uvr mutants, however, this strain is sensitive to ionizing radiation and shows an increase in survival when held for 2 h in distilled water before plating (liquid-holding recovery [LHR]). The strain is three times more sensitive to X-rays than the wild type strain. It is also sensitive to nitrosoguanidine (MNNG). Sensitivity to UV, X-rays and MNNG appears to be under the control of a single gene.These properties suggest that the repair defect in the Neurospora uvs-2 mutant is different from those of the uvr mutants of E. coli K12.     

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.     

Lack of genetic and cytogenetic effects in mice fed on irradiated wheat.

Experiments were conducted to test the mutagenicity of wheat irradiated with 20 and 200 krad gamma-rays by feeding male and female mice for various periods starting from weaning time. The results obtained from dominant lethal tests, specific locus mutation test, studies on chromosome rearrangements in males and gonadal cell survival studies indicated no positive evidence for genetic and cytogenetic effects.     

Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state

MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. VI. Genetic characterization of ad-3 mutants provides evidence for qualitative differences in the spectrum of genetic alterations between wild-type and nucleotide excision-repair-deficient strains

Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 (Worthy and Epler, 1973) had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, gamma-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively (de Serres, 1980; Schüpbach and de Serres, 1981; Inoue et al., 1981a, b) but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair.     

The Neurospora crassa UVS-3 epistasis group encodes homologues of the ATR/ATRIP checkpoint control system

The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.     

HETEROKARYOSIS AND PARASEXUALITY IN THE FUNGUS ASCOCHYTA IMPERFECTA

The object of this investigation was to discover whether heterokaryosis and parasexuality occur in the imperfect fungus Ascochyta imperfecta. Both phenomena have been observed. The wild type of A. imperfecta grows on a minimal medium containing only salts plus a carbon source. Auxotrophic and morphological mutants have been isolated after treatment with ultraviolet light. When 2 different mutant auxotrophs are inoculated together onto minimal medium, colonies are consistently formed. These colonies might be due, a priori, to back-mutation, diploidy, syntrophism or heterokaryosis. Back-mutation and diploidy have been eliminated, since no back-mutant nuclei have been isolated from any heterokaryon, and since the frequency of diploid nuclei is very low. The combination is primarily syntrophic (only 2% heterokaryotic hyphal tips) when the nicotinamide mutant is one component. The combination is primarily heterokaryotic (over 50% heterokaryotic hyphal tips) when both components are auxotrophs for amino acids. From the heterokaryotic hyphal tips, the 2 unaltered nuclear components have been isolated. Heterozygous diploid nuclei (4.2 X 10^−-7 per haploid nucleus) can be isolated from heterokaryons by plating, onto minimal medium, the primarily uninucleate conidia from a heterokaryon of 2 auxotrophs. The resulting colonies are isolated as potential diploids. Three properties of these isolates establish their diploid nature: (1) the isolates are wild type for nutrition and morphology; (2) their conidial length is uniformly greater than that of the haploids (1.21 times); (3) the isolates produce segregants with nonparental combinations of the marker genes. The diploid isolates are much more stable than heterokaryons. The recombinants from the diploids are still diploid, since (1) their conidial length falls in the diploid range, and (2) one of the recombinants has segregated a second-order recombinant. Many of the expected classes of recombinants have not been detected.     

Normal inhibition of DNA synthesis following gamma-irradiation of radiosensitive cell lines from patients with Down's syndrome and Alzheimer's disease

Inhibition of DNA synthesis was studied in gamma-irradiated lymphoblastoid cells from patients with Alzheimer's disease and Down's syndrome. A normal biphasic pattern of inhibition was observed over a dose range of 0-4 krad of gamma-rays in all of the cell lines. 3 out of 4 Down's and all the Alzheimer's cell lines were shown to be hypersensitive to ionizing radiation based on induced chromosomal aberrations. Increased G2 phase delay, comparable to that occurring in ataxia-telangiectasia cells, was observed for some of the cell lines, after exposure to gamma-rays. Contrary to other data in the literature these results demonstrate that radioresistant DNA synthesis is not an intrinsic feature of all disorders characterized by radiosensitivity.