Environmental and Molecular Mutagenesis - Legacy Tobacco ...

176 1989 EMS Abstracts _ 510
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
Notes --~~ gEQUENC'F.-iEtifALeR'M OF MUTATION INDUCED IN THE lac/ GENE OF Escherichla colr
BY 2-AMINOPtJRINE . W .E . SCHY. AND B .W. GLICKMAN, Dept. of Biology, York
University. 471N) Keelt*i, Toronto. Ontario, Canada M3J 1P3
We have founzl`tl ~ r.t vses of mutational spectra to be a useful approach In
elucidating mechanisms of inutation induction . To this end, we have characterized at the
DNA sequence Ievel . '~-o-tminopurine (2-AP) induced mutations that lie within the first 180
bp of the /acl gene (1-~ mutants) of Eschericlria cole. A saturated culture of NR 3835
(wtld-t)•pe) cells was diluted in 25(1 Ed (if LB/2AP (600 q/ml) so that each well of a 96well
mtcrotitrr dish contained less than 1(N) cells. Following overnight growth at 37oC
and washing. appropriate dilutions were plated directly onto plates containing LB to
determine survtval . and plates containing pbenyI .It .D-galactopyranoside (Pgal) as the sole
carhim source tt) select Iacl' n~utants. One Incl- mutant was seleeted from each well .
Mutants were mapped and I' mutants were selected for sequence analysis . Of the 42
mutants at 14 sites that have been seyuenced thus far, all are transition mutations .
Approximately 2-fold ax many mutants were G :C => A:T than A :T => G:C transitions ; .
howevrr. when hotspot sitrs werr excluded from the analysis, transition mutation in
eithcr direction apprars te~ hr ryually likely . With res(+ect to site specificity, we noted a
pruminrnt hotspot at pc~sitiun e3 (G :C ==• A :T). This site Is surrounded by 5 neighboring
G :C hase pairs . which may serve to stabilize a 2•AP :C mismatch . This regional sequence
specificity is consistent with previous observations associating hotspot sites with adjacent
Cr :C' huse pairs .
511
DEOXYNUCLEOTIDE TRIPHOSPHATE POOLS AND THE SPECIFICITY OF MUTATION FOR LACI IN E . COLI .
W .D . Sedwick, M .L . Veigl, S . Schneiter, and S . Mollis, Dept . of Medicine,Case Western
Reserve Univ . Cleveland, OH 44106
The mutational specificity of trimethoprim (TMP) for the Lacl gene of E . coli was
investigated . Mutants (500 each) were isolated for analysis from cultures which were
treated 4 hours with 5 uM trimethoprim (TMP) or TMP + 100 uM thymidine (TdR), followed
by a 4 hour recovery period which allowed resolution of the filanmentation seen only in
the cultures treated with TMP alone . The frequency of mutation in lcI increased 10and
2-fold under the two conditions, respectively . All isolated mutants were probed
for occurrence of mutation at two of the most common sites represented in the spontaneous
spectrum, the frameshift "hot spot" where the thrice repeated sequence, CTtk ;, is
frequently duplicated or deleted, and the +6 position in the operator, the site of a
recurrent transition of T-A -> C-G . Frame shift "hot spot" mutants represented only 20%
with TMP and 43% with TMP-TdR vs >66% in the spontaneous background (Shaaper et al, JMB,
189 :273, 1986) . The high frequency mutation in the operator represented 22% and 18% of
the total mutants in the TMP and TMP-TdR sets, respectively, vs 3% in the spontaneous
collection . About 60 mutants mapped to the first 200 base pairs of 1aal were carried on
for DNA sequence analysis from both TMP and TMP-TdR treated cultures . An overall
increase in single base nonsense mutations at mber_ and ochre sites was not observed, but
the predominant isolates from TNP-treated cultures were T-A -> C-G transitions clustered
at several different sites . A high frequency of A-T -> C-G and G-C -> C-G transversions
was also observed . Preliminary analysis of the TMP-TdR mutants supports a similar
pattern of base substitution mutations . The results of these experiments will be
presented with correlative DNA triphosphate pool analyses .
512
CONTRASTING RESULTS IN HEPATOCYTE UDS ASSAYS USING AUTORADIOGRAPHY 0R LIQUID
SCINTILLATION COUNTING e b r, C .Meli and R .Forster, Dept . Genetic Toxicology,
Life Science Research Roma ox co ogy Centre, 00040 POMEZIA, Rome, Italy
Contrasting findings in rat hepatocyte UDS assays performed using liquid
scintillation counting after nuclear separatlon (LSC) or by autoradiographlc
estimation of radiolabel uptake (ARG) have been observed with several products
received for routine testing . Further experiments were with product X, which gave
reproducible increases in UJS of up to 110% over control values in the LSC method . In
the ARG method, negative results were obtained ; both cytoplasmic and nuclear grain
counts remained similar to control values at all treatment-levels . Possible
explanations for these results were examined : (1) The responses obtained with the
positive control (2-acetylaminofluorene) suggested that the ARG method is the more
sensitive method of detection . (ii) The percentage of cells in S-phase was determined
in the autoradiographic preparations . There was no lncrease in S-phase cells which
could explain the results obtained in the LSC method, but calculations show that the
sensitivity of S-phase scoring may be insufficient to detect increases which will
result in positive responses in LSC . (ii1) The experimental methods follow the same
tine-plan and kinetic differences cannot explain the contrasting results . (iv)
Parallel LSC assays were perfot7ned with or without hydroxyurea treatment ; product X
produced increases in radiolabel uptake 1n the LSC method both in the absence and
presence of HU, indicating that the observed UDS did not result from the swtagenic
properties of HU . At present the basis of the differing results between the LSC and
ARG methods cannot be satisfactorily explained .
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