Molecular and classical genetic analyses of his-3 mutants of Neurospora crassa. I. Tests for allelic complementation and specific revertibility

A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hydroxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971).     

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.     

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.     

Mutagenic potency and specificity of procarbazine in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa.

Procarbazine (Natulan) was tested for its mutagenic potency and specificity in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In these experiments, procarbazine was a weak mutagen when present in growing cultures but nonmutagenic when conidial suspensions (nongrowing conidia) were treated. A total of 208 ad-3 mutants recovered after exposure of growing cultures of H-12 to 1 mg of procarbazine/ml, and 2 ad-3 mutants of spontaneous origin, were characterized genetically. These tests distinguish among gene/point mutations (ad-3R) at the ad-3A or ad-3B locus, multilocus deletion mutations ([ad-3]IR) covering one or more loci in the ad-3 and immediately adjacent regions, and 3 different classes of multiple-locus mutations: gene/point ad-3 mutations with a recessive lethal mutation elsewhere in the genome (ad-3R + RL), gene/point mutations with a closely linked recessive lethal mutation (ad-3R + RLCL), and multilocus deletion mutations with a closely linked recessive lethal mutation ([ad-3]IR + RLCL). All of the procarbazine-induced ad-3 mutants resulted from gene/point mutations; 92.2% (200/217) resulted from gene/point mutations at the ad-3A or ad-3B locus, and 3.7% (8/217) resulted from gene/point mutations with a recessive lethal mutation elsewhere in the genome. Identical percentages (15.4% [20/130] and 15.4% [12/78]) of the sigma ad-3BR and sigma ad-3AR mutants were leaky, and a high percentage (71.5% [93/130]) of the sigma ad-3BR mutants had nonpolarized complementation patterns. These results indicate that procarbazine-induced ad-3 mutants of Neurospora crassa are composed solely of gene/point mutations (ad-3R) that resulted, predominantly or exclusively, from base-pair substitutions. The Neurospora specific-locus data on procarbazine-induced ad-3 mutants are compared with data from similar experiments with the mouse using the morphological specific-locus assay; marked similarities were found between the mutagenic effects of procarbazine in the 2 specific-locus assays.     

Mutation Induction by Difunctional Alkylating Agents in NEUROSPORA CRASSA

The genetic characterization of ad-3 mutants of Neurospora crassa induced by two carcinogenic difunctional akylating agents, 1,2,4,5-diepoxypentane (DEP) and 1,2,7,8-diepoxyoctane (DEO), has shown that point mutations at the ad-3B locus have similar complementation patterns. In addition to the induction of point mutations, DEP induces a low frequency (7.5%) of multilocus deletions, whereas DEO induces an extremely high frequency (42.0%). The distribution of the different classes of ad-3 mutants and the frequency of multilocus deletion mutants among DEP-induced mutants are not significantly different from those induced by the monofunctional alkylating agents EI, EMS and ICR-177 at comparable forward-mutation frequencies. Moreover, the frequencies of DEP-induced ad-3B mutants showing allelic completion or having nonpolarized complementation patterns are similar to those of ad-3B mutants induced by monofunctional agents. It is suggested, therefore, that the mechanism of mutation-induction by DEP in N. crassa is similar to that of monofunctional alkylating agents. Mutation-induction by DEO probably results both from the mechanism of action of monofunctional alkylating agents and from inter-strand cross-linkage of the DNA molecular by the two functional epoxy groups.     

The Suppression of AD-3B Mutants by Supersuppressors in NEUROSPORA CRASSA

The action of eight suppersuppressors has been tested on 76 ad-3B mutants which were induced by base-pair transition mutagens. Thirteen mutants were found to be suppressible by at least one ssu gene. Most of the suppressible mutants were found to belong to a class in which one would expect to find nonsense mutants (polar complementing or noncomplementing classes, with AT at the mutant site). However, suppression was observed in other classes, including those containing presumed missense mutants. The specificity of the suppressors and the criteria for molecular classification of the mutants are discussed.     

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.     

Mutagenic activities of aflatoxin B 1 and G 1 in Neurospora crassa

Summary The mutagenicity and mutagenic specificity of aflatoxin B₁ and G₁ were studied with the adenine-3 (ad-3) test system of Neurospora crassa. Aflatoxin B₁ and G₁ failed to show mutagenicity in resting conidia, but both agents were mutagenic in growing vegetative cultures. The frequencies of ad-3 mutants induced by aflatoxin B₁ and G₁ (40g/ml) were 70.7x10^-6 survivors and 9.6x10^-6 survivors, respectively. Since aflatoxin B₁ gave a 177-fold increase over the spontaneous mutation frequency it is a rather potent mutagen, whereas aflatoxin G₁ gave only a 24-fold increase and so is only moderately mutagenic.Genetic analyses of ad-3 mutants induced by aflatoxin B₁ and G₁ indicate that both agents induce a low frequency of multilocus deletions. The spectra of point mutations at the ad-3A and ad-3B loci induced by aflatoxin B₁ and G₁ are not distinguishable from each other. Hence both agents probably induce the same relative frequencies of genetic alterations. The frequencies of leakiness, allelic complementation, and classes of complementation patterns among the ad-3 mutants induced by both agents are higher than the frequencies among ICR-170-induced mutants and somewhat lower than those among NA- and AP-induced mutants. The results of reversion tests with NA, MNNG, and ICR-170 indicate that in addition to multilocus deletion, aflatoxin B₁-induced ad-3 mutants consist of frameshifts, base-pair transitions, and possibly other types of intragenic alterations.     

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.     

Genetic effects of N-methyl-N''-nitro-N-nitrosoguanidine in Neurospora crassa

Summary The mutagenicity of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was studied with a genetically marked, balanced heterokaryon of Neurospora crassa. Types of genetic alterations detectable in this system are (a) point mutations in the ad-3 A and ad-3 B loci, (b) multilocus (chromosome) deletions in the ad-3 region, and (c) recessive lethal mutations in the whole genome. Study of the inactivation kinetics of the heterokaryotic and homokaryotic conidial fractions makes it possible to distinguish between nuclear and cytoplasmic inactivation.Forward mutations in the ad-3 region induced by MNNG in the heterokaryotic fraction of conidia were obtained by a direct method, with the following results: (1) forward-mutation frequency increases as the square of the time of treatment, (2) MNNG is an extremely efficient mutagen, e. g., the frequency of mutation in the ad-3 region (2 loci) was 0.14% after 240 min treatment with 25 M MNNG at pH 7.0 with 73.4% survival, (3) at least 98.1% of the MNNG-induced ad-3 mutants are point mutations, (4) tests for genotype and allelic complementation showed that (a) the frequency of genotypes was ad-3 A=19.7%, ad-3 B=80.3% and ad-3 A ad-3 B=0.0%, and (b) 81.8% of the ad-3 B mutants have allelic complementation with 79.9% nonpolarized and 1.9% polarized complementation patterns and 18.2% noncomplementing mutants, and (5) the ration between mutations in the ad-3 A and ad-3 B loci and spectrum of complementation patterns among the ad-3 B mutants was independent of dose. Comparison of the spectrum of the complementation patterns among ad-3 B mutants induced by MNNG with the spectrum among ad-3 B mutants induced by 2-aminopurine, nitrous acid, hydroxylamine, and the acridine mustard derivative ICR-170 suggests that the majority of the MNNG-induced mutants have guanine-cytosine at the mutant site.Abbreviations used in this paper GC guanine-cytosine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - HA hydroxylamine - AT adenine-thymine - MMS methyl methanesulfonate - 2AP 2-aminopurine - ICR-170 acridine mustard derivative-(2-methoxy-6-chloro-9-[(ethyl-2-chloroethyl) amino propylamino] acridine dihydrochloride) - NA nitrous acid Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

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)     

Classical and molecular genetic analyses of his-3 mutants of Neurospora crassa. II. Southern blot analyses and molecular mechanisms of mutagenicity

Previous studies (Overton et al., Mutation Res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3B mutants. In the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. The restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-OR23-1A and were consistent with expectations based on previous data suggesting that they resulted from single base-pair alterations (Overton et al., Mutation Res., 1989). His-3 mutants with altered banding patterns were only found among those with polarized complementation patterns or noncomplementing mutants. One of the mutants with a polarized complementation pattern, 1-189-83, and another noncomplementing mutant, 1-189-85, are associated with genetic alterations proximal to the his-3 locus. In one other mutant, 1-226-565 (with a polarized complementation pattern), an insertion of approx. 2 kb has occurred in the proximal region of the his-3 locus. Two other mutants, 1-155-270 and 1-155-276 (both noncomplementing), contained a large insertion of approx. 12.8 kb in the proximal region of the his-3 locus.     

Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium.

An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis.     

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.     

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 nature of spontaneous mutations during vegetative growth in Schizosaccharomyc pombe

Summary Fifty five purple spontaneous mutants of the haploid fission yeast Schizosaccharomyces pombe have been isolated and distributed, by genetic analyses, between two loci (ad-6 and ad-7) which control two independent steps in the purine biosynthesis.The molecular nature of these mutants has been determined by both their physiological behaviour (leakiness, temperature-sensitivity, pH-sensitivity) and their interallelic complementation ability.It is concluded that during the mitotic cycle the large majority of spontaneous mutations are base-pair substitutions.Publication n. 6 from the Laboratorio di Mutagenesi e Differenziamento del Consiglio Nazionale delle Ricerche (C.N.R.), Pisa, Italy.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Nonessential Sequences, Genes, and the Polytene Chromosome Bands of DROSOPHILA MELANOGASTER

From earlier work, there appears to be an underlying one-to-one correspondence of polytene chromosome bands and complementation groups within a sizeable, continuous X-chromosome segment, 3A1-3C7 ( Judd, Shen and Kaufman 1972; Lefevre and Green 1972). However, most of the data supporting this one-to-one relation of bands and genes were gathered from mutants that upset vital functional units, thus leading to lethality. Among this series of mutants, only four loci, zeste, white, roughest and verticals, have no known lethal alleles. If phenotypic changes less drastic than lethality result from the loss of other chromosomal segments, they probably would not have been recognized in the earlier studies.-We report here some chromosomal sequences localized in 3A, 3B, and 3C whose loss effects no lethal change in the development of the animal. A portion of the 3A3-3A4 region can be disrupted in a nonlethal fashion, yet this sequence does not seem to be a part of either the zeste locus or l(1)zw1, which are known to be located in these bands. Two more complementation groups have been discovered that have no lethal alleles and map to 3B4-3B6; a third falls within 3B1-2. The loss of a sequence in 3C2-3 is tolerated without any genetically observable effect. Between 3C7 and the boundary of 3D there is at least one more sequence that behaves in this manner.-The discovery of these units, which are not allelic to any of the loci previously known, makes it clear that division 3B contains more genes (i.e., complementation groups) than polytene chromosome bands, while portions of 3A and 3C seem to have no functional significance. Accordingly many polytene chromosome bands may be composites of several complementing functional units. This investigation also indicates that there are chromosomal segments that are seemingly dispensible and thus function in a manner that is difficult or impossible to define with available methods.     

Kinetic analysis of genetic complementation in heterokaryons of propionyl CoA carboxylase-deficient human fibroblasts.

We studied genetic complementation of propionyl CoA carboxylase (PCC) deficiency in cultures of polyethylene glycol (PEG)-induced heterokaryons, using mutant fibroblast lines assigned to five mutant classes, designated bio, pcc A, pcc B, pcc C, and pcc BC. By measuring PCC activity directly in extracts of fused cells or indirectly in intact cells by [1-14C]propionate utilization, we confirmed the nonlinear nature of the PCC deficiency complementation map described by Gravel et al. [1]. When we studied the kinetics of complementation, we detected three distinct patterns using the [1-14C]propionate utilization assay. When either pcc A or pcc C lines were fused to bio cells, 14C-fixation increased to half of the maximally restored values within 4 hrs. In pcc A x pcc C crosses or in pcc A x pcc B crosses, however, complementation was much slower. In fusions between pcc B and pcc C cells, a third pattern was elicited; complementation was incomplete, maximum restoration of PCC activity begin less than 20% of that observed in other complementing crosses. From these data and previous biochemical evidence, we suggest (1) that the bio and pcc mutations affect different genes; (2) that complementation between pcc A and either pcc B, pcc C, or pcc BC lines is intergenic and involves subunit exchange and synthesis of new PCC molecules; and (3) that complementation between pcc B and pcc C mutants is interallelic.