Environmental and Molecular Mutagenesis - Legacy Tobacco ...

108 1989 EMS Abstracts
Notes
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
THE AMPHIBIAN (BAI4A pjpgNS ) LENS AS A BIOLOGICAL MONITOR OF WATERBORNE
XENOBIOTICS . J.S. Kung, C,$,Geard and B.V. Worgul, Eye Radiation and Environmental Research
Laboratory and Radiological Research Laboratory, College of Physicians and Surgeons of Columbia
University, New York, NY
309
$gpa p,jpj= shows promise as a biological monitor of environmental mutagens . As vertebrates, they
are able to enaymatically activate promutagens to their genotoxic metabolites . Being aquatic, they inhabit
waters that are polluted by domestic sewage and industrial waste containing waterbotne xenobiotics which
maY chronically concentrate in their tissues . Since the products of all kns epithelial divisions are
matntained within the lens for the entire lifespan of the animal, the lens may serve as a useful long-term
indicator of genotoxicity. Frogs were exposed through the ambient environment to Benzo(a)pYrene (BP), a
promutagen, 2 .5, 5.0, 10.0 and 20.0 µg/ml, and to Ethyl methanesulfonate (EMS), a direct-acting
clastogen, 25, 50,100 and 200 µg/ml, by partial immersion for a period of one week . Following sacrifice
of the animals, lens epithelial whole-mounts were prepared. The epithelia were stained by the Feulgen
technique to permit visualization and quantitative analysis of nuclear DNA content . An increase in the
premitouc 02 population was found with increasing concentrations of either mutagen . An inverse dose
relationship with mitoses suggests that these mutagens may exert a premitotic block in the cell cycle .
Micronuclei frequency, a measure of genotoxicity, was found to be elevated with both mutagens in a nondose-dependent
fashion. A block in the cell cycle may well account for this since micronuclei production is
a mitosis-dependent event . $= may prove to be valuable in the study of environmental mutagens since
they are easily handled, adaptable to laboratory conditions, ubiquitously distributed and amenable to short
term genotoxicity assays .
Supported by Grant EY 02648 from the ?dEI .
310
THE SUp4-o SYSTEM FOR ANALYSIS 0F MUTATIONAL SPECIFICITY IN YEAST . B .A . Kunz, J .D .
Armstrong, M . Clattke, S .E . Kohalmi and J .R .A . Mis, Microbiology Department, The
University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Knowledge of the DNA sequence alterations produced by mutagens, as well as their
frequency and location within a gene, can provide valuable clues about the DNA damage
responsible and how it is processed into mutations . To obtain detailed mutational
specificity data, we developed a shuttle vector system wherein mutations in
the tRNA suppressor gene &UP4-o of the yeast Saccharomvcas cereviaiae can be easily
analyzed to determine the DNA sequence alterations involved . In this system, the
SUP4-o gene is carried on a plasmid that mimics the behavior of yeast chromosomes . To
date, more than 2,000 SUp4-o mutants have been characterized . The spontaneous mutation
frequency is typical for chromosomal genes in yeast and all 6 possible base-pair
substitutions (single and double changes), deletions of various lengths, base-pair
insertions, duplications, transposon insertions and more complex events arise spontaneously
. Single base-pair changes have been found at 68 of the 75 exon sites, and
at 2 of the 14 intron positions, within the gene . The system can be used in the
forward mode to analyze mutational specificity or as a reversion assay to provide
additional information on site-specific mutagenesis . In addition, isogenic error-free
repair deficient derivatives have been constructed to allow assessment of the role of
DNA repair in mutagenesis . The $Up4-o system and its use will be described and
results obtained in SUP4-o will be compared with data available for mammalian shuttle
vector systems . (Supported by NSERC Canada)
311
CHARACTERIZATION OF THE YEAST rad52 MUTATOR EFFECT BY DNA SEQUENCING . B .A . Kunz, S .E .
Kohalmi, J .D . Armstrong, and M . Clattke, Microbiology Department, The University of
Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Defects in the $aD52 gene of the yeast Saccharomvices carevisiae confer a mutator
phenotype . This effect was characterized by sequencing a collection of 238 spontaneous
SUP4-o mutations arising in a gDd52 strain . The resulting mutational spectrum
was compared to that derived for an isogenlc wild-type strain (222 mutations analyzed)
. This comparison revealed that the mutator phenotype was associated with an
increase In the frequency of base-pair substitutions . All possible types of substitution
were detected but there was a decrease in the fraction of A-T -> G•C transitions
and an increase In the proportion of G-C -> C-G transversions . These changes
were large enough to cause a two-fold greater preference for substitutions at G•C
sites In the rad52 strain despite a decrease In the fraction of G-C -> T-A transversions
. Base-pair changes occurred at fever sites In the tgd5Z strain but the mutated
sites included several that vere not detected In the $OQ52 background . Only two of
the four sites most freqdently mutated In the tyM strain were also prominent In the
wild-type strain and mutation frequencies at almnst all sites common to both strains
were greater for the X{¢n derivative . Although single base-pair deletions occurred
at similar frequencies in the two strains, several classes of mutation that were
recovered in the wild-type background including multiple base-pair deletions, insertions
of the yeast transposable element Ty, and more complex changes, were not recovered
in the tgdn strain . Our results suggest that the XAM mutator effect is not
due defects in repair of abasic sites or base mismatches . (Supported by NSERC Canada)
S0869 3620