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Title: Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster

Abstract

The mutational specificity of N-ethyl-N-nitrosourea (ENU) was determined in Drosophila melanogaster using the vermilion locus as a target gene. 25 mutants (16 F{sub 1} and 9 F{sub 2} mutants) were cloned and sequenced. Only base-pair changes were observed; three of the mutants represented double base substitutions. Transition mutations were the most prominent sequence change: 61% were GC {yields} AT and 18% AT {yields} GC substitutions. Both sequence changes can be explained by the miscoding properties of the modified guanine and thymine bases. A strong bias of neighboring bases on the occurrence of the GC {yields} AT transitions or a strand preference of both types of transition mutations was not observed. The spectrum of ENU mutations in D. melanogaster includes a significant fraction (21%) of transversion mutations. The data indicate that like in other prokaryotic and eukaryotic systems also in D. melanogaster the O{sup 6}-ethylguanine adduct is the most prominent premutational lesion after ENU treatment. The strong contribution of the O{sup 6}-ethylguanine adduct to the mutagenicity of ENU possibly explains the absence of distinct differences between the type of mutations observed in the F{sub 1} and F{sub 2} mutants.

Authors:
;  [1]; ;  [2];  [3]
  1. (State Univ. of Leiden (Netherlands) J.A. Cohen Institute (Netherlands))
  2. (State Univ. of Leiden (Netherlands))
  3. (Univ. of North Carolina, Chapel Hill (USA))
Publication Date:
OSTI Identifier:
6936612
Alternate Identifier(s):
OSTI ID: 6936612
Resource Type:
Journal Article
Resource Relation:
Journal Name: Genetics; (USA); Journal Volume: 123:1
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; DROSOPHILA; MUTAGENESIS; GENE MUTATIONS; DNA SEQUENCING; NITROSOUREAS; GENETIC EFFECTS; DNA ADDUCTS; DNA BASE TRANSITIONS; GUANINE; THYMINE; ADDUCTS; AMINES; ANIMALS; AROMATICS; ARTHROPODS; AZAARENES; AZINES; BIOLOGICAL EFFECTS; DIPTERA; FLIES; FRUIT FLIES; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; INSECTS; INVERTEBRATES; MUTATIONS; NITROSO COMPOUNDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PURINES; PYRIMIDINES; STRUCTURAL CHEMICAL ANALYSIS; URACILS 560300* -- Chemicals Metabolism & Toxicology

Citation Formats

Pastink, A., Vreeken, C., Nivard, M.J.M., Vogel, E.W., and Searles, L.L. Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster. United States: N. p., 1989. Web.
Pastink, A., Vreeken, C., Nivard, M.J.M., Vogel, E.W., & Searles, L.L. Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster. United States.
Pastink, A., Vreeken, C., Nivard, M.J.M., Vogel, E.W., and Searles, L.L. Fri . "Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster". United States. doi:.
@article{osti_6936612,
title = {Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster},
author = {Pastink, A. and Vreeken, C. and Nivard, M.J.M. and Vogel, E.W. and Searles, L.L.},
abstractNote = {The mutational specificity of N-ethyl-N-nitrosourea (ENU) was determined in Drosophila melanogaster using the vermilion locus as a target gene. 25 mutants (16 F{sub 1} and 9 F{sub 2} mutants) were cloned and sequenced. Only base-pair changes were observed; three of the mutants represented double base substitutions. Transition mutations were the most prominent sequence change: 61% were GC {yields} AT and 18% AT {yields} GC substitutions. Both sequence changes can be explained by the miscoding properties of the modified guanine and thymine bases. A strong bias of neighboring bases on the occurrence of the GC {yields} AT transitions or a strand preference of both types of transition mutations was not observed. The spectrum of ENU mutations in D. melanogaster includes a significant fraction (21%) of transversion mutations. The data indicate that like in other prokaryotic and eukaryotic systems also in D. melanogaster the O{sup 6}-ethylguanine adduct is the most prominent premutational lesion after ENU treatment. The strong contribution of the O{sup 6}-ethylguanine adduct to the mutagenicity of ENU possibly explains the absence of distinct differences between the type of mutations observed in the F{sub 1} and F{sub 2} mutants.},
doi = {},
journal = {Genetics; (USA)},
number = ,
volume = 123:1,
place = {United States},
year = {Fri Sep 01 00:00:00 EDT 1989},
month = {Fri Sep 01 00:00:00 EDT 1989}
}
  • The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. The combinationmore » of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and point mutations'. Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the breakpoint with one of the repeats deleted.« less
  • As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletions map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethalsmore » was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5). 28 refs., 1 fig., 3 tabs.« less
  • Transgenic rodents harboring easily recoverable shuttle vectors offer the prospect of studying gene mutations in vivo in any tissue of interest. The value of these models in genetic toxicology could be enhanced by evaluating a cytogenetic and point for chromosomal mutations from the same treated animals. The utility of such an approach is being evaluated in our laboratory by determining the frequencies of lac I mutations and micronuclei in B{sub 6}C{sub 3}F{sub 1} Big Blue{reg_sign} mice treated with known genotoxic agents. Groups of 12-week old male mice (4/group) were administered 3 daily oral doses of BP (250 mg/kg/day), ENU 50more » mg/kg ENU on Days 1, 2, and 3, respectively. The last group was intended to optimize and probability of detecting gene mutations in various tissues from the same treated animals (to serve as potential positive control treatment in future studies). Forty eight hours after the last dose, peripheral blood was collected from the tail vein and wedge smears were prepared for the evaluation of micronucleated erythrocytes. After an expression period of 21 days (only 11 days for NDMA-treated animals due to toxicity), various tissues were collected, flash-frozen in liquid nitrogen, and stored at approximately -80{degrees}C for the analysis of lacI mutations. The incidence of micronucleated polychromatic erythrocytes were substantially elevated in all the treatment groups (25-, 29-, 12-, and 36-fold increase in groups treated with BP, ENU, NDMA, and the combination treatment, respectively). Analysis for lacI mutations in the DNA extracted from the livers indicated increases of approximately 9-, 4-, 7-, and 7-fold in groups treated with BP, ENU, NDMA, and the combination treatment, respectively (control frequency = 27 x 10{sup -6}). These data will be used for development of protocols for the simultaneous evaluation of chromosomal as well as gene mutation end points.« less