Frederick J. de Serres

Specific-locus mutations induced in eukaryotes (especially mammalian cells) by radiation and chemicals: a perspective.

In the course of discovering the first mutagen (X-rays) just over 60 years ago, Herman J. Muller asked whether X-rays induced single-gene mutations and/or chromosomal (multiple-gene) mutations. To a large extent, his question has set the agenda for mutagenesis research ever since. We explore historically the answers to this question, with special emphasis on recent developments in the field of mammalian cell mutagenesis. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens induce both gene and chromosomal mutations; however, only certain genetic systems permit the recovery and analysis of both classes of mutations. Few chemical mutagens induce only gene mutations in mammalian cells; instead, most mutagens appear to induce both classes of mutations, with chromosomal mutations (especially multilocus deletions) predominating at high doses. These results have implications regarding the mechanisms of mutagenesis, the role of chromosomal mutations in carcinogenesis and hereditary disease, and the type of data required for risk assessment of physical and chemical mutagens/carcinogens.

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

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

In situ evaluation of biological hazards of environmental pollutants

Introductory Remarks.- In Situ Evaluation of Biological Hazards of Environmental Pollutants.- Industrial and Regulatory Needs.- Toxicological Research and Related Programs Under Superfund.- Biological Assessment and Ecological Risk Assessment: New Tools for Clean-up Decisions at Hazardous Waste Sites.- Field Studies: Aquatic Systems.- Bioassessment Methods for Determining the Hazards of Dredged Material Disposal in the Marine Environment.- Use of Herring Embryos for In Situ and In Vitro Monitoring of Maring Pollution.- New Methods for On-Site Biological Monitoring of Effluent Water Quality.- Micronucleus Test Using Peripheral Red Blood Cells of Amphibian Larvae for Detection of Genotoxic Agents in Freshwater Pollution.- Predicting the Ecological Significance of Exposure to Genotoxic Substances in Aquatic Organisms.- Field Studies: Terrestrial Systems.- Flow Cytometry as a Technique to Monitor the Effects of Environmental Genotoxins on Wildlife Populations.- Developing Comprehensive Field Studies to Identify Subchronic and Chronic Effects of Chemicals on Terrestrial Ecosystems. Ecosystem Health VI.- Earthworm Immunoassays for Evaluating Biological Effects of Exposure to Hazardous Materials.- Sentinel Surveillance Systems.- Analytical Epidemiology in Pet Populations for Environmental Risk Assessment.- Sentinel Animals (Dogs) as Predictors of Childhood Exposure to Environmental Lead Contamination: Observations on Preliminary Results.- Fish Neoplasms Found at High Prevalence in Polluted Waters.- Biological Markers in Animal Sentinels: Laboratory Studies Improve Interpretation of Field Data.- DNA-Carcinogen Adducts in Fish as a Tool for Measuring the Effective Biological Dose of Aquatic Carcinogens.- IN SITU Monitoring of Environmental Clastogens Using Tradescantia-Micronucleus Bioassay.- The Use of Drosophila Melanogaster for In Situ Biomonitoring.- Chemical Etiology of Endemic Diseases: A Global Perspective.- Integration of Data for Effective Problem Solving and Assessment.- Chemical Analysis for Assessment and Evaluation of Environmental Pollutants: Fact or Artifact.- In Situ Toxicological Monitoring: Use in Quantifying Ecological Effects of Toxic Waste.- Integrated Chemical, Pathological and Immunological Studies to Assess Environmental Contamination.- Use of Wildlife for On-Site Evaluation of Bioavailability and Ecotoxicity of Toxic Substances Found in Hazardous Waste Sites.- Relevancy and Future.- Capabilities and Limitations of Approaches to In Situ Ecological Evaluation.

Genetic consequences of nucleotide pool imbalance

Voon-Loong Chan and H. Jeok Ho Department of Microbiology Fitzgerald Building The University of Toronto Toronto, Ontario Canada M5S lA8 A class of arabinosyladenine-resistant baby hamster kidney (BHK) cell mutants, isolated in our laboratory, shows cross-resistance to deoxyadenosine, alteration of adenosine kinase, elevation of spontaneous mutation rate, and extreme sensitivity to adenosine. One of these adenosine sensitive mutants, ara-slOd, was isolated spontaneously and studies with Ador revertants suggest the involvement of a single pleiotropic mutation. The enhanced adenosine toxicity in ara-slOd cells can be attributed to pyrimidine nucleotide starvation and to at least one other mechanism, which is associated with a ZOO-fold elevation of IMP, 3-5 fold elevation of ATP, GTP, S-adenosylmethionine (AdoMet) and methylthioadenosine (MeSAdo).

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

Abstract 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 ( RL CL ) 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 -3 A [29] or ad -3 B [63]) 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 -3 A ad -3 B [90], ad -3 A ad -3 B nic -2 [10]) and ad -3 B nic -2 [4]) 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 individaul mutants classified as presumptive multilocus deletions or multiple-locus mutations. The data in the present paper have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad -3 A ad -3 B , ad -3 A ad -3 B nic -2, and ad -3 B nic -2 map as a series of overlapping multilocus deletions. Among the mutants of genotype ad -3 A ad -3 B , there were 34 different subgroups of complementation patterns; among those of genotype ad -3 A ad -3 B nic -2, there were 5 different subgroups; and among those of genotype ad -3 B nic -2, there were 4 different subgroups. In addition, genetic fine structure analysis has shown that some of the mutants classified, initially, as multilocus deletions ( ad -3 IR ), are actually multiple-locus mutations: multilocus deletions with closely linked, and separate , sites of recessive lethal damage with a wide variety of genotypes. Combining data from the present experiments with previously published data (de Serres, 1989a,c), the frequency of multiple-locus mutations among X-ray-induced gene/point mutations and multilocus deletions in the ad-3 region is 6.2% ( 51 828 ).

X-Ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. II: More extensive genetic tests reveal an unexpectedly high frequency of multiple-locus mutations

Abstract More extensive genetic tests have been performed on a series of 832 X-ray-induced specific-locus mutations in the ad -3 region of a 2-component heterokaryon (H-12) of Neurospora crassa , reported earlier (Webber and de Serres 1965). Using a new tester strains and techniques for performing large-scale genetic tests (heterokaryon, dikaryon and trikaryon) to characterize ad -3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of X-ray-induced ad -3 mutants. These new data show that unexpectedly high frequencies of both single-locus (gene/point) mutations and multilocus deletions in the ad -3 region have additional, but separate, sites of resessive lethal ( RL CL ) 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 expected on the basis of target theory and classical models of chromosome structure during interphase. Current models of interphase chromosome structure in higher eukaryotes as revealed by chromosome “painting” offer a possible explanation of the Neurospora data.

Individual susceptibility to genotoxic agents in the human population

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Characteristics of spontaneous and induced specific-locus mutation in the ad-3 region of neurospora crassa: Utilization in genetic risk assessment

Data from experiments on the induction of specific‐locus mutations in model systems are utilized in genetic risk assessment to estimate potential adverse effects in the human population. In such assessments with radiation or chemical mutagens, the following information is required: (1) spontaneous and induced forward‐mutation frequencies, (2) dose‐response curves for the overall induction of specific‐locus mutations, (3) genetic characterization of spontaneous and induced mutations, and (4) dose‐response curves for the different genotypic classes. Specific‐locus assays in most eukaryote assay systems provide only portions of the information required for genetic risk assessment. In recognition of the need for a more comprehensive data base, a model system was developed for specific‐locus studies in Neurospora crassa. The adenine‐3 (ad‐3) specific‐locus assay was modeled after the 2 gene, morphological specific‐locus assay in the dilute‐short‐ear region of the mouse, and it detects forward‐mutations at two closely linked loci: ad‐3A and ad‐3B. The ad‐3 assay system has provided precise dose‐response curves not only for inactivation, but also the overall induction of ad‐3 mutations. The utilization of this assay in experiments with radiation or chemical mutagens has provided a data base on the induction and genetic characterization of specific‐locus mutations that is unique among eukaryotic organisms. In this assay, gene/point mutations, multilocus deletion mutations, and 3 different classes of multiple‐locus mutations can be identified. The latter consist of specific‐locus mutations associated with recessive lethal mutations located either closely linked to the ad‐3 region or elsewhere in the genome. The overall data base on the heterozygous effects of X‐ray‐induced ad‐3 mutations demonstrates that such effects are allele specific, genotype specific, and locus specific. There are probably a variety of mechanisms by which the heterozygous effects of individual allelic mutations at different genetic loci can be affected. In conclusion, unless the frequencies of all of the different classes of induced specific‐locus mutations are determined, and utilized in genetic risk assessment exercises, the risk of human exposure to environmental mutagens may be grossly underestimated.

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

Abstract 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 mutants 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. VI. Induction kinetics of gene/point mutations, multilocus deletions and multiple-locus mutations.

Abstract 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.