X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. V. Irreparable mutants of genotype ad-3A ad-3B, ad-3A ad-3B nic-2, and ad-3B nic-2 result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations

More extensive complementation tests than those performed initially (Webber and de Serres, 1965) on a series of 832 X-ray-induced specific-locus mutations in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa (de Serres, 1989a) showed that unexpectedly high frequencies of specific-locus mutations in the ad-3 region have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than that expected on the basis of target theory and classical models of chromosome structure during interphase (de Serres, 1989a). Genetic fine structure analyses, by means of homology tests with tester strains carrying genetic markers in the ad-3 and immediately adjacent regions, have been performed to map the presumed multiple-locus mutations. In a previous paper (de Serres, 1989c), X-ray-induced irreparable ad-3 mutants of the following genotypes and numbers (ad-3A or ad-3B were analyzed, and the high frequency of multiple-locus mutations was confirmed. In the present paper, X-ray-induced irreparable ad-3 mutants of the following genotypes and numbers (ad-3A ad-3B, ad-3A ad-3B nic-2, and ad-3B nic-2 have also been subjected to the same genetic fine structure analysis. These experiments, in the previous (de Serres, 1989c) and present papers, were designed to determine the extent of the functional inactivation in the ad-3 and immediately adjacent genetic regions in individual mutants classified as presumptive multilocus deletions or multiple-locus mutations.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. IV. Irreparable mutants of genotype ad-3A and ad-3B result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations

The induction of specific-locus mutations in the ad-3 region of Neurospora crassa after X-irradiation was studied in a two-component heterokaryon to determine: (1) the ratio of reparable ad-3 mutants (presumed gene/point mutations, designated ad-3R) to irreparable ad-3 mutants (presumed multilocus deletions, designated ad-3IR), and (2) the induction kinetics of each class (Webber and de Serres, 1965). More extensive genetic tests made subsequently (de Serres, 1989a) on the 832 X-ray-induced specific-locus mutations recovered in those experiments showed that unexpected high frequencies of reparable and irreparable ad-3 mutants are actually multiple-locus mutants that have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions (designated ad-3R + RLCL or ad-3IR + RLCL). The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory (where the frequency of the double mutant is expected to be the product of the frequencies of each single mutant) and classical models of chromosome structure during interphase (de Serres, 1989a). In the present paper, a random sample of 832 X-ray-induced ad-3 mutants of genotype ad-3A or ad-3B that are irreparable have been subjected to more extensive genetic fine-structure analysis. These experiments were designed to determine the extent of the functional inactivation in individual mutants in the ad-3 and immediately adjacent genetic regions in mutants classified as presumptive multilocus deletions or multiple-locus mutations. These experiments have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3A or ad-3B map as a series of overlapping multilocus deletions. Among the 29 irreparable mutants of genotype ad-3A, there are 16 different subgroups of complementation patterns; and among the 63 irreparable mutants of genotype ad-3B, there are also 16 different subgroups. In addition, mutants classified as presumptive multiple-locus mutants result from a variety of separate, but closely linked, sites of genetic damage.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray-induced specific locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. III. Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions by means of complementation tests

Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions was made by means of complementation tests on all possible pairwise combinations of 50 X-ray-induced irreparable adenine-3 mutants (designated ad-3IR). All mutants were induced in either heterokaryon 11 or heterokaryon 12 of Neurospora crassa, 2-component heterokaryons heterozygous for mutants at the 3 closely linked loci ad-3A and ad-3B and nic-2 (nicotinamide-requiring) located about 5.0 map units distal to ad-3B. The complementation tests involved mutants of the following genotypes: 15 ad-3A, 27 ad-3B, 7 ad-3A ad-3B nic-2 and 1 ad-3B nic-2. To facilitate mapping, 5 additional strains (each consisting of a gene/point mutation at the ad-3A or ad-3B locus and a separate site of closely linked recessive lethal damage in the immediately adjacent regions [designated ad-3R + RLCL]) were also included. The data from these complementation tests showed that the majority (46/50) of X-ray-induced irreparable ad-3 mutants mapped as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B. The remaining mutants (4/50) were found to result from a series of closely linked, but separate, mutations (designated multilocus mutations) of the type ad-3IR + RLCL, different from those found in previous studies (de Serres, 1968; de Serres and Brockman, 1968). The data from the present complementation tests have expanded the process of genetic fine structure mapping of the ad-3 and immediately adjacent regions (de Serres, 1968) and defined the presence of the following 11 genetic loci: (a) 4 loci (with either known [i.e. col-1t] or unknown [i.e. unknA]) function proximal to ad-3A: unknA, unknB, col-1t, and col-2t, (b) 4 loci in the 'X' region: unknC, unknD, unknE, and unknF, (c) 2 loci distal to ad-3B: unknG, col-3t, and (d) 1 locus distal to nic-2: unknH.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. X. Heterozygous effects of multilocus deletion mutations of genotype ad-3A or ad-3B.

Previous studies on X-ray-induced irreparable adenine-3 mutations (designated [ad-3]IR), induced in heterokaryon 12 of Neurospora crassa, demonstrated that they were not recessive and exhibited heterozygous effects in terms of markedly reduced linear growth rates (de Serres, 1965). Complementation tests with a series of tester strains carrying multilocus deletion mutations in the ad-3 and immediately adjacent genetic regions demonstrated that X-ray-induced irreparable mutations map, in the main part, as a series of overlapping multilocus deletion mutations that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B (de Serres, 1968, 1989). Further studies (de Serres and Miller, 1988) have shown that the heterozygous effects of multilocus deletion mutations in the ad-3 region can be modified genetically and biochemically. In the present paper, the heterozygous effects of X-ray-induced multilocus deletion mutations of genotype ad-3A or ad-3B, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989), have been determined. These data show that 57.7% (15/26) of X-ray-induced multilocus deletion mutations covering the ad-3A locus have heterozygous effects, in terms of reduced linear growth rates, in forced dikaryons with a gene/point mutant at the ad-3B locus and 80.0% (20/25) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 35.1% (20/57) of X-ray-induced multilocus deletion mutations covering the ad-3B locus have heterozygous effects in forced dikaryons with a gene/point mutant at the ad-3A locus, and 100.0% (35/35) in forced dikaryons with a multilocus deletion mutation covering the ad-3A locus. These results demonstrate that the dominant or recessive characteristics of X-ray-induced specific-locus mutations resulting from multilocus deletion mutations are allele specific.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. VI. Induction kinetics of gene/point mutations, multilocus deletions and multiple-locus mutations

Genetic fine-structure analysis of X-ray-induced specific-locus mutants in the ad-3 region of two-component heterokaryons of Neurospora crassa has shown that gene/point mutations, multilocus deletions and multiple-locus mutations are induced. When the dose-response curves for these classes of ad-3 mutants were plotted, it was demonstrated that X-ray-induced gene/point mutations (ad-3R) increased linearly with X-ray dose and X-ray-induced multilocus deletions increased as the square of the X-ray dose. However, all classes of multiple-locus mutations, which would be expected to result from 3 to 8 hits on the basis of target theory (Lea, 1955), were found to increase as the square of the dose. Target theory assumes that the DNA of individual chromosomes is distributed randomly throughout the interphase nucleus. A model of eukaryotic interphase chromosome structure in which the DNA of individual chromosomes presents a nonrandom target to X-rays [Pinkel et al., Proc. Natl. Acad. Sci. (U.S.A.) 83 (1986), 2934-2938] provides a possible explanation for the high frequency and dose-squared induction kinetics of the multiple-locus mutants induced by X-rays in the ad-3 region.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. XI. Heterozygous effects of gene/point mutations of genotype ad-3A or ad-3B.

Previous studies on X-ray-induced irreparable adenine-3 mutants (designated ad-3IR), induced in heterokaryon 12 of Neurospora crassa, showed that they were not recessive, and that they demonstrated heterozygous effects in terms of markedly reduced linear growth rates as compared with a wild-type control (de Serres, 1965, 1988). Homology tests on X-ray-induced irreparable mutants showed that they map, in the main part, as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential, function) between ad-3A and ad-3B (de Serres, 1969, 1989a). Studies on a larger sample of X-ray-induced multilocus deletion mutations of genotype (ad-3A)IR or (ad-3B)IR (de Serres et al., 1992) demonstrated that heterozygous effects are allele specific and that there was no correlation with genotype, radiation dose or complementation map position. Furthermore, the heterozygous effects of multilocus deletions in the ad-3 region can be modified genetically and biochemically (de Serres and Miller, 1988). In the present paper, the heterozygous effects of X-ray-induced gene/point mutations of genotype ad-3AR or ad-3BR, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989a), were determined. The studies presented in this paper show that 8.1% (3/37) of X-ray-induced ad-3AR mutations exhibit heterozygous effects in terms of reduced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus, and 10.8% (4/37) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 24.3% (9/37) of ad-3AR mutations exhibit heterozygous effects in terms of enhanced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus. Similar studies with X-ray-induced ad-3BR mutations showed that 54.9% (28/51) exhibit heterozygous effects in terms of reduced growth rates in forced dikaryons with a gene/point mutant at the ad-3A locus and 100.0% (48/48) in forced dikaryons with a multilocus deletion covering the ad-3A locus. These studies have also shown that about a 13-fold higher percentage of X-ray-induced multiple-locus mutations of genotype ad-3AR + RLCL have heterozygous effects resulting in reduced growth rates than X-ray-induced single-locus mutations of genotype ad-3AR. The overall data base on X-ray-induced ad-3 gene/point mutations in the present studies demonstrates that heterozygous effects are allele specific, genotype specific, and locus specific.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. VIII. Dose-dependence of the overall spectrum

There is considerable controversy in the literature concerning the nature of X-ray-induced specific-locus mutations in various experimental organisms. To investigate this problem in Neurospora crassa a series of experiments (Webber and de Serres, 1965) was performed to study the induction-kinetics of X-ray-induced mutation in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12). Subsequent genetic analyses (de Serres, 1989a,b,c, 1990a), on a series of 832 mutants recovered in these experiments, have shown that 3 different classes of ad-3 mutants were recovered, namely gene/point mutations, multilocus deletions and multiple-site mutations. Complementation studies with a series of genetic markers that define 21 genetic loci in the ad-3 and immediately adjacent genetic regions have shown that ad-3 mutants classified as multilocus deletions result from the inactivation of a series of loci in the ad-3 and immediately adjacent regions of Linkage Group I, whereas multiple-locus mutations result from combinations of gene/point mutations and multilocus deletions. Analysis of the induction kinetics of these 3 different classes, after completion of the genetic characterization of all mutants (de Serres, 1990b) demonstrated that gene/point mutations increase linearly with X-ray dose, whereas multilocus deletions and multiple-site mutations increase as the square of X-ray dose. Further analysis of allelic complementation among the gene/point mutations at the ad-3B locus (de Serres, 1990c), demonstrated that the spectrum of complementation patterns was dose-dependent: complementing mutants with nonpolarized patterns decreased and noncomplementing mutations increased with increasing X-ray dose. There was little or no change with dose in the frequency of mutants with polarized patterns. In the present report, data from studies published previously have been utilized, along with additional data from the original X-ray experiments (12-5, 12-6, 12-7, and 12-10; see Webber and de Serres, 1965) to develop composite complementation maps of the X-ray-induced specific-locus mutations in the ad-3 and immediately adjacent regions as a function of X-ray dose. This analysis of the overall spectrum of X-ray-induced specific-locus mutations in the ad-3 region demonstrated marked dose-dependence and provides an explanation for the discrepancies in the literature with regard to specific-locus studies in different experimental organisms.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa, IX. Mutational spectra as a function of X-ray dose.

In previous studies, X-ray-induced specific-locus mutations in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa were combined with a series of tester strains carrying markers in the ad-3 and immediately adjacent regions to map mutants that were presumed multilocus deletions (de Serres, 1989c, 1990a). Two new classes of X-ray-induced mutations were recovered: multiple-locus mutations consisting of gene/point mutations at the ad-3A or ad-3B locus with a closely linked recessive lethal mutation, or multilocus deletions covering the ad-3A, ad-3B and/or nic-2 loci with a closely linked recessive lethal mutation (designated ad-3R + RLCL and [ad-3]IR + RLCL, respectively). Thus, the ad-3 specific-locus assay can detect damage occurring at the ad-3A and the ad-3B loci, as well as at a minimum of 19 other loci in the immediately adjacent regions. The original overall spectrum of ad-3 mutations can be resolved, by genetic analysis, into a series of 30 subclasses. In the present paper, the data from the genetic analysis of 832 X-ray-induced mutants recovered from a series of 4 experiments (Webber and de Serres, 1965) have been presented in terms of Mutational Spectra organized as a function of X-ray dose. Comparison of these Spectra demonstrates the shift from high percentages of gene/point mutations (with a high percentage of mutants at the ad-3B locus showing allelic complementation) at low doses, to low percentages of gene/point mutations (with a low percentage of ad-3B mutants showing allelic complementation) and high percentages of multilocus deletion mutations and multiple-locus mutations (of genotype ad-3R + RLCL or [ad-3]IR + RLCL) at high doses. These Mutational Spectra demonstrate the marked dose-dependence of X-ray-induced specific-locus mutations in a eukaryotic organism.     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. VII. Genetic lesions resulting in gene/point mutations at the ad-3B locus have different dose-response relationships

Genetic characterization of X-ray-induced ad-3 mutants, induced in a two-component heterokaryon (H-12) of Neurospora crassa, has been performed to determine genotype, patterns of allelic complementation, and leakiness, and to distinguish gene/point mutations from multilocus deletions and multiple locus mutations (de Serres, 1989c, 1990a). The array of genotypes in the classes and subclasses in the spectrum of ad-3 mutants induced by a mutagenic agent constitute its mutagenicity profile; for X-rays this profile consists of 29 different genotypes. In the present paper, the data on gene/point mutations induced at the ad-3B locus (designated ad-3BR mutations) have been tabulated as a function of X-ray dose to determine the dose-dependent relationships of complementing and noncomplementing mutants. This analysis has shown that the overall percentages of mutants showing allelic complementation and the percentages of complementing mutants with nonpolarized patterns (both leaky and nonleaky) and noncomplementing mutants were dose-dependent; the former class decreased with increasing X-ray dose, and the latter class increased with increasing X-ray dose. The percentages of complementing mutants with polarized patterns were X-ray dose-independent. In addition, an unexpectedly high frequency of mutants with nonpolarized complementation patterns are discontinuous and probably result from multiple-site mutations.     

Comparison of the spectra of genetic damage in formaldehyde-induced ad-3 mutations between DNA repair-proficient and -deficient heterokaryons of Neurospora crassa.

The mutagenic effects of formaldehyde (FA) have been compared in DNA repair-proficient (heterokaryon 12) and DNA repair-deficient (heterokaryon 59) two-component heterokaryons of Neurospora crassa. The data from forward-mutation experiments were used to compare the spectra of FA-induced specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region and on the FA-induced inactivation of heterokaryotic conidia. Previous studies have demonstrated that specific-locus mutations at these two loci result from five major genotypic classes, namely two classes of gene/point mutations (ad-3A(R) and ad-3B(R)), and three classes of multilocus deletion mutations ([ad-3A](IR), [ad-3B](IR), and [ad-3A ad-3B](IR)). Genetic analysis of ad-3 mutants recovered from both heterokaryons after FA treatment demonstrates that predominantly gene/point mutations were found in H-12 (93.2% ad-3(R), 6.8% [ad-3](IR)) and a significantly higher frequency of multilocus deletion mutations in H-59 (62.8% ad-3(R), 37.0% [ad-3](IR)). The data from our experiments with FA on H-12 demonstrate and confirm the data from other assays that FA is a weak mutagen in this DNA repair-proficient strain. However, the data from our experiments with the DNA repair-deficient strain H-59 demonstrate that comparable concentrations of FA cause more pronounced inactivation of heterokaryotic conidia and, at the highest concentration tested, about a 35-fold higher frequency of ad-3 mutations. In addition, FA induced a 5.4-fold higher frequency of ad-3 mutations resulting from multilocus deletion mutation in H-59 than in H-12. Based on our earlier studies with X-ray-induced multilocus deletion mutations, it is this class of FA-induced ad-3 mutations that might be most expected to show deleterious heterozygous effects. The implications of the present data base from our experiments with Neurospora are that the mutagenic (and possibly the carcinogenic) effect of FA exposure might well vary in different human population subgroups.     

2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus deletion mutations, and (5) multiple-locus ad-3R mutations with closely linked recessive lethal mutations.     

Hydroxylamine-induced purple adenine (ad-3) mutants in Neurospora crassa. I. Characterization of mutants by genetic tests

The genetic effects of hydroxylamine (HA) on Neurospora crassa were studied in an effort to understand the difference between the results obtained on very simple prokaryotic systems and those obtained with mammalian systems. A 2-component heterokaryon was used to study the inactivation of conidia and the induction of recessive lethal mutations at specific loci and over the entire genome. The heterokaryon is heterozygous for 2 closely linked loci, ad-₃A and ad-₃B, in the ad-₃ region. Specific locus mutations can result from either point mutation or chromosome deletion. The results were as follows: (1) Both homokaryotic and heterokaryotic conidia had multi-hit survival curves, and there was no difference between the survival levels of the two as a function of treatment time. (2) The frequency of recessive lethal mutations in the ad-₃ region increased as the square of treatment time.     

X-ray-induced mutations affecting the level of the enzyme alcohol dehydrogenase in Drosophila melanogaster: frequency and genetic analysis of null-enzyme mutants.

The frequency of X-ray-induced (null-enzyme) mutations at the alcohol dehydrogenase locus in Drosophila melanogaster was measured. The rate of recovery of chromosomes that fail to direct the synthesis of a functional Adh protein is 3 x 10(-8) per R for chromosomes that do not include large chromosome rearrangements. However, this analysis excludes a larger number of chromosomes that are "null-enzyme mutations" because thye are deleted for the region of the Adh locus. The dose of X-rays required to induce a frequency of non-deletion null-enzyme mutants equal to the spontaneous frequency is about 73 rad calculated from the data reported in this communication.     

Azaguanine-resistant mutants induced by several mutagens in a neurospora heterokaryon.

This paper reports the development of a new system for the detection of forward mutations in a heterokaryon of Neurospora crassa. This system, designed to detect a wide variety of genetic alterations, is based upon the detection of 8-azaguanine-resistant mutants in an adenine-requiring heterokaryon carrying the aza-3 gene. Induction of mutations in the aza-3 gene was detected after treatment of conidia with ultraviolet light, X-rays, ethyl methanesulfonate (EMS), or the acridine mustard, ICR-170. The pattern of appearance of mutant colonies and various factors in mutant selection are discussed.     

Model and test in a fungus of the probability that beneficial mutations survive drift

Determining the probability of fixation of beneficial mutations is critically important for building predictive models of adaptive evolution. Despite considerable theoretical work, models of fixation probability have stood untested for nearly a century. However, recent advances in experimental and theoretical techniques permit the development of models with testable predictions. We developed a new model for the probability of surviving genetic drift, a major component of fixation probability, for novel beneficial mutations in the fungus Aspergillus nidulans, based on the life-history characteristics of its colony growth on a solid surface. We tested the model by measuring the probability of surviving drift in 11 adapted strains introduced into wild-type populations of different densities. We found that the probability of surviving drift increased with mutant invasion fitness, and decreased with wild-type density, as expected. The model accurately predicted the survival probability for the majority of mutants, yielding one of the first direct tests of the extinction probability of beneficial mutations.     

Qualitative differences in the spectra of genetic damage in 2-aminopurine-induced ad-3 mutants between nucleotide excision-repair-proficient and -deficient strains of Neurospora crassa.

The mutagenic effects of 2-aminopurine (2AP) have been compared in the adenine-3 (ad-3) region of two-component heterokaryons of Neurospora crassa: nucleotide excision repair-proficient (uvs-2+/uvs-2+) heterokaryon 12 (H-12) and nucleotide excision repair-deficient (uvs-2/uvs-2) heterokaryon 59 (H-59). This forward-mutation, morphological and biochemical, specific-locus assay system permits the recovery of ad-3A and/or ad-3B mutants in 3 major classes: gene/point mutations, multilocus deletion mutations, and unknowns, and 3 different subclasses of multiple-locus mutations. Previous studies (Brockman et al., Mutation Res., 218 (1989) 1-11) showed that 2AP treatment of growing cultures of H-12 and H-59 gave no difference between ad-3 forward-mutation frequencies over a wide range of 2AP concentrations in each strain. In the present experiments, genetic analyses of ad-3 mutants recovered from these experiments has demonstrated qualitative differences between the spectra of the 3 main classes of ad-3 mutations. In H-12, 84.2% (203/241) resulted from gene/point mutation, 11.6% (28/241) from multilocus deletion mutation, and 4.1% (10/241) were unknowns. In contrast, in H-59, 43.0% (99/230) resulted from gene/point mutation, 55.7% (128/230) from multilocus deletion mutation, and 1.3% (3/230) were unknowns. In addition, quantitative differences were also found between the spectra of ad-3 mutations in 1 subclass of multiple-locus mutations, but not 2 additional subclasses. The first subclass consisted of 1.7% (4/241) and 9.6% (22/230) gene/point mutations with a closely linked recessive lethal mutation, in H-12 and H-59, respectively. The second two subclasses consisted of (a) 0.4% (1/241) and 0.4% (1/230) multilocus deletion mutations with a closely linked recessive lethal mutation, and (b) 13.3% (32/241) and 15.2% (35/230) gene/point mutations with a separate recessive lethal mutation elsewhere in the genome, in H-12 and H-59, respectively. Data from studies by others have shown that 2AP inhibits adenosine deaminase, resulting in nucleotide precursor pool inbalance, and that 2AP can saturate the mismatch repair system. As a consequence of either effect of 2AP, the spectrum of 2AP-induced mutation could include frameshift mutations and chromosome aberrations such as multilocus deletions in addition to base-pair substitutions. The defect in DNA repair due to the uvs-2 allele, which has been shown to be a deficiency in pyrimidine dimer excision (Worthy and Epler, 1974), most probably has some other excision-repair deficiency (Macleod and Stadler, 1986; Baker et al., 1991).(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4⁺ and REV5⁺ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.     

The nature of X-ray-induced mutations after recovery in excision repair-deficient (mus-201) Drosophila females

This paper describes the genetic analysis of X-ray-induced mutations at several visible loci (yellow, white, Notch, vermilion and forked) located on the X-chromosome of Drosophila melanogaster after recovery in excision repair-deficient condition (mus-201). A total of 118 mutations observed in 83636 F1 females were analyzed. The white mutations in particular have been investigated at the molecular level. The results show that: (1) the frequency of recovered whole-body mutations is similar or slightly lower in repair-deficient than in repair-proficient condition (respectively 1.5 x 10(-4)/locus/15 Gy and 2.3 x 10(-4)/locus/15 Gy); (2) the frequency of observed mosaic mutations is significantly higher in the repair-deficient condition than in the proficient condition (respectively 2.7 x 10(-4)/locus/15 Gy and 0.9 x 10(-4)/locus/15 Gy); (3) the analysis of F2 male lethal mutations and the cytological analysis of the recovered mutations in the excision repair-deficient condition indicate a decrease in mutations associated with gross chromosomal aberrations (including multilocus deletions); (4) at the molecular level, the spectrum of recovered intragenic mutations is similar after excision-deficient and -proficient repair. These results indicate that excision repair is involved in X-ray-induced DNA damage that is repaired efficiently in the normal repair condition, but bypassed in the excision repair-deficient condition, leading to mosaic mutations. In addition, lesions that apparently cannot be bypassed by DNA replication lead to a decrease in the fraction of mutations due to gross chromosomal aberrations among the whole-body mutations.