Genetic analysis of purple adenine (ad-3) mutants induced by methyl methanesulfonate in Neurospora crassa

The mutagenicity of methyl methanesulfonate (MMS) was studied in a genetically marked two-component heterokaryon of Neurospora crassa. Types of genetic alterations detectable in this system are (I) point mutations in the ad-3A and ad-3B loci; (2) multilocus (chromosome) deletions in the ad-3 region, and (3) recessive lethal mutations in the whole genome. Study of the inactivation kinetics of the heterokaryotic and homokaryotic conidial fractions has made it possible to distinguish between nuclear and cytoplasmic inactivation.Forward mutations in the ad-3 region induced by MMS in the heterokaryotic fraction of conidia were obtained by a direct method with the following results: (I) The overall ad-3 forward mutation frequency increases in proportion to the 1.91 power of the concentration of MMS. (2) The forward mutation frequency of point mutations at the ad-3A and ad-3B loci increases in proportion to the 1.68 power of the concentration. (3) The forward mutation frequency of chromosome deletions in the ad-3 region increases more than exponentially with increasing concentrations of MMS. (4) After treatment for 300 min with 20 mM MMS, 15.5% of the ad-3 mutations are multilocus deletions. Tests for genotype and allelic complementation of the point mutations showed that (I) the ratio between ad-3B and ad-3A mutants was 1.75, (2) 52.1% of the ad-3B mutants showed allelic complementation, with 39.2% non-polarized and 12.9% polarized complementation patterns and 47.9% noncomplementing mutants, and (3) both the ratio between point mutations in the ad-3A and ad-3B loci and the spectrum of complementation patterns among the ad-3B mutants were independent of MMS concentration.     

A genetic and functional characterization of adenine mutants induced in yeast by 1-nitroso-imidazolidone-2 and nitrous acid

Summary The mutational activity of NIL and HNO₂ is compared in a forward mutation system of Saccharomyces cerevisiae involving ad₁ and ad₂ loci. The comparison includes the distribution of ad₁ to ad₂ mutations as well as certain functional aspects, namely, the frequency of KCl remedials and intragenic complementation with the tester strains. The results show that NIL and HNO₂ mutants are similar both in the distribution of ad₁ to ad₂ and in their functional aspects. Our findings suggest, furthermore, that in a certain group of mutants—mutants complementing with the tester strains—complementation is incompatible with KCl repair.     

Mutagenic activity of ethylenimine in Neurospora crassa

The mutagenic activity of the monofunctional alkylating agent ethylenimine (EI) was tested with the adenine-3 (ad-3) system in a two-component heterokaryon of Neurospora crassa. The results of forward-mutation experiments showed that EI is a potent mutagen in N. crassa.Genetic analysis of EI-induced ad-3 mutants showed that the frequencies of leakiness, allelic complementation, and non-polarized complementation patterns are similar to those of ad-3 mutants induced by other alkylating agents. It seems, therefore, that in addition to multilocus deletions (which occur at low a frequency), EI-induced mutations probably include base-pair substitutions, frameshift mutations, and other types of intragenic alterations.     

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.     

Analysis of lethal events induced by ultraviolet in a heterokaryon of Neurospora.

Summary The relative frequencies of heterokaryons and the two kinds of homokaryons have been scored among colonies from conidia harvested from a heterokaryon and treated with UV, in order to determine which kinds of lethal mutations were induced. Recessive lethal mutations were scored directly. The pattern of surviving types indicated that recessive lethals and mitotic lethals (events destroying whole nuclei) occurred with similar frequencies. But the absolute frequency of these mutations was not sufficient to account for the observed kill, suggesting that dominant lethals and/or cytoplasmic lethals were also induced at a similar rate.     

A Cytogenetic Analysis of the Chromosomal Region Surrounding the α-Glycerophosphate Dehydrogenase Locus of DROSOPHILA MELANOGASTER

The chromosomal region surrounding the structural gene for α-glycerophosphate dehydrogenase (αGpdh, 2-20.5) of Drosophila melanogaster has been studied in detail. Forty-three EMS-induced recessive lethal mutations and five previously identified visible mutations have been localized within the 25A-27D region of chromosome 2 by deficiency mapping and in some cases by a recombination analysis. The 43 lethal mutations specify 17 lethal loci. αGpdh has been localized to a single polytene chromosome band, 25F5, and there apparently are no lethals that map to the αGpdh locus.     

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)     

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.     

Defective kernel mutants of maize. I. Genetic and lethality studies

A planting of 3,919 M₁ kernels from normal ears crossed by EMS-treated pollen produced 3,461 M₁ plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.     

Endogenous purine metabolism in the conidia of wild type and certain adenine mutants of neurospora crassa I. The nature of the reserve pools and pool utilization during adenine starvation

Conidia of four adenine auxotrophs (ad 9, ad 3B, ad 8 and ad 4 of Neurospora crassa differ in their ability to germinate on adenine-deficient medium. A large percentage of the ad 9 and ad 3B mutant conidia germinate while those of ad 8 and ad 4 mutant do not. No correlation was found between the size of the conidial purine reserves and the conidial ability to germinate. In all the strains the major fraction of the conidial purine reserved pools was inosine. The ad 8 and ad 4 mutants are blocked after IMP formation in the adenine biosynthetic pathway and therefore cannot use the stored inosine for germination. Pool-utilization studies indicated that in all strains investigated some of the purine reserved were lost from the conidia during incubation. In the most readily germinating strain, ad 9, only small amounts of the purine pool were lost from the conidia and a large portion of the reserve pool was used for nucleic acid synthesis. The nature of the purine reserves present in the conidia, and the ability of the strains to prevent loss of the stored purines from the conidia appear to be among the factors influencing the conidial germination of the adenine mutants of N. crassa.     

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.     

Efficient isolation of ion beam-induced mutants for homoeologous loci in common wheat and comparison of the contributions of Glu-1 loci to gluten functionality

Key message

Ion beam mutations can be efficiently isolated and deployed for functional comparison of homoeologous loci in polyploid plants, and Glu - 1 loci differ substantially in their contribution to wheat gluten functionality.

Abstract

To efficiently conduct genetic analysis, it is beneficial to have multiple types of mutants for the genes under investigation. Here, we demonstrate that ion beam-induced deletion mutants can be efficiently isolated for comparing the function of homoeologous loci of common wheat (Triticum aestivum). Through fragment analysis of PCR products from M₂ plants, ion beam mutants lacking homoeologous Glu-A1, Glu-B1 or Glu-D1 loci, which encode high molecular weight glutenin subunits (HMW-GSs) and affect gluten functionality and end-use quality of common wheat, could be isolated simultaneously. Three deletion lines missing Glu-A1, Glu-B1 or Glu-D1 were developed from the original mutants, with the Glu-1 genomic regions deleted in these lines estimated using newly developed DNA markers. Apart from lacking the target HMW-GSs, the three lines all showed decreased accumulation of low molecular weight glutenin subunits (LMW-GSs) and increased amounts of gliadins. Based on the test data of five gluten and glutenin macropolymer (GMP) parameters obtained with grain samples harvested from two environments, we conclude that the genetic effects of Glu-1 loci on gluten functionality can be ranked as Glu-D1 > Glu-B1 > Glu-A1. Furthermore, it is suggested that Glu-1 loci contribute to gluten functionality both directly (by promoting the formation of GMP) and indirectly (through keeping the balance among HMW-GSs, LMW-GSs and gliadins). Finally, the efficient isolation of ion beam mutations for functional comparison of homoeologous loci in polyploid plants and the usefulness of Glu-1 deletion lines for further studying the contribution of Glu-1 loci to gluten functionality are discussed.     

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.     

Maternal effect embryonic lethal mutants identified in the 37D2-38A1 region of chromosome 2 ofDrosophila melanogaster

The genome region represented byDeficiency (2L) E55 inDrosophila melanogaster has been implicated in maternal gene activity, based on high levels of F₁ embryonic mortality in hemizygousDf(2L) E55/+female x+/+male matings. Extensive mutagenesis screens were conducted to detect any maternal embryonic lethal loci in theDf (2L) E55, 37D₂-38A₁ cytological region. Consequently, seven maternal effect embryonic lethal loci have been identified. Five of them altered the ventral embryonic cuticle pattern in various ways. In addition to these, a new ovarian tumour mutant has also been identified in the same region. This demonstrates that maternal haplo insufficiency of theDf (2L) E55/+females, leading to embryonic mortality, is due to theen bloc removal of the wild type alleles of these mutants, whose activity is required in the female for ovarian organisation and embryonic development.     

CAENORHABDITIS ELEGANS Deficiency Mapping

Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.     

Temperature-sensitive plant mutants isolated in vitro

Summary Conditional-lethal, temperature-sensitive plant mutants have been isolated using a simple protoplast cloning method. The leaf protoplasts used were obtained from sterile, haploid shoot cultures of Nicotiana plumbaginifolia. Recessive mutations are described at three loci: ts1, ts2 and ts3. The mutations are lethal when either tissue cultures or plants are incubated at 33°C but not at 26°C.     

Alignment of the genetic and physical maps in the dpy-5 bli-4 (I) region of C. elegans by the serial cosmid rescue of lethal mutations

Lethal mutations in the 0.5 map unit region between dpy-5 and bli-4 on chromosome I in Caenorhabditis elegans were serially rescued using cosmid-containing transgenic strains. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described previously. Germline transformation with cosmid DNA was used to create 25 transgenic strains bearing heritable extrachromosomal arrays. These arrays were used as small duplications for the fine-scale mapping of essential genes, via the rescue of lethal mutations. Lethal mutations in 13 essential genes have been phenotypically rescued, allowing the alignment of the genetic and physical maps in this region. Extrachromosomal arrays were found to be transmitted 2- to 7-fold less frequently in oocytes than in hermaphrodite sperm for 12 of the 16 arrays that were examined. Three of these strains showed a subsequent 4- to 13-fold increase in array stability in oocytes. This phenomenon may be influenced by cosmid sequences. Early mitotic loss of the arrays was observed in all 17 transgenic strains examined, suggesting that loss of the array can occur at any time during development when cell divisions are occurring. As a result of this work, 13 of the essential loci positioned between dpy-5 and bli-4 are anchored to the physical map, thereby providing links between the physical and genetic maps on average every 85 kb.     

Genetic characterization of ad-3 mutants induced by chemical carcinogens, I-phenyl-3-monomethyltriazene and I-phenyl-3,3-dimethyltriazene, in Neurospora crassa

180 ad-3 mutants of Neurospora crassa induced by 1-phenyl-3-monomethyl-triazene (PMMT) and 56 ad-3 mutants induced by 1-phenyl-3,3-dimethyltriazene (PDMT) were characterized by dikaryon, trikaryon and complementation tests. Results show that the spectrum of genetic alterations induced by PMMT is different from that of PDMT. This suggests that enzymatic dealkylation of PDMT to PMMT does not occur within Neuropsora crassa conidia, and that the mechanism of mutation induction of PDMT in N. crassa is different from that of PMMT. Hydrolytic breakdown products or its intact molecule or some other converted forms might be responsible for the mutagenic activity of PDMT.Mutation induction of PMMT in N. crassa appears to be via alkylation of DNA by carbonium ions produced by this compound, the same mechanism proposed for its carcinogenic activity. The frequencies of leakiness, allelic complementation and nonpolarized complementation patterns among PMMT-induced ad-3 mutants are similar to those of ad-3 mutants induced by other potent chemical carcinogens, such as MNNG and the aflatoxins.     

Genetic Analysis of Recessive Lethal Mutations Induced by the Influenza Virus in the X Chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M_{1 + 2}. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophilasalivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stages of Drosophila.Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10–3C10-11.     

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.