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Journal of Bacteriology, February 1999, p. 1149-1155, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Spectra of Spontaneous Growth-Dependent and
Adaptive Mutations at ebgR
Barry G.
Hall*
University of Rochester, Rochester, New York
Received 15 July 1998/Accepted 10 December 1998
 |
ABSTRACT |
A comparison of the spectra of spontaneous growth-dependent and
adaptive mutations in ebgR shows that both spectra are
dominated by insertion sequence (IS)-mediated mutations. The difference between growth-dependent mutations (61% IS mediated) and adaptive mutations (80% IS mediated) is highly significant (P < 0.0001). In contrast, the spectra of growth-dependent and adaptive
non-IS-mediated mutations do not differ from each other and therefore
do not provide support for the hypothesis that adaptive and
growth-dependent mutations arise by substantially different mechanisms.
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INTRODUCTION |
Adaptive mutations are spontaneous
mutations that occur in microorganisms during periods of prolonged
stress in nondividing (2) or very slowly dividing
(8) populations. The key feature that distinguishes adaptive
mutations from growth-dependent mutations is that the former are
specific to the selective challenge that is imposed, i.e., the
selective conditions are not generally mutagenic and the only mutations
that are recovered are mutations in the site that is under selection
(10, 12) (but see reference 3 for an
exception to this rule). The phrase "only mutations that are
recovered" was chosen carefully to avoid implying that other mutations do not occur. A variety of experiments have looked very hard
for mutations at sites that are not under selection and have failed to
find such mutations (9, 10), but that does not rule out the
possibility that mutations occur but are lost before they can be
recovered. One possibility that has gained increasing acceptance over
the last few years is that mutations do occur at other sites, but that
the cells that suffer those mutations die (because the mutations confer
no advantage), and thus the mutations are not recovered (6).
Adaptive mutations, which have been shown to occur in both bacteria and
yeast (reviewed in reference 12), are usually
observed by subjecting populations to nonlethal selection for reversion of known mutations in genes for carbon source catabolism or for amino
acid biosynthesis. The first revertants to appear are presumed to be
the result of mutations that were present in the population prior to
plating. Typically, additional revertant colonies continue to appear
for periods ranging from a few days up to a month, and it is those
late-appearing colonies that are said to result from adaptive mutations.
Some of the most important evidence that adaptive mutagenesis is a
different process from growth-dependent mutagenesis comes from showing
that the spectra of adaptive mutations differ significantly from the
spectra of growth-dependent mutations. A series of F'-borne lacZ alleles that could revert only by specific base
substitutions was used to show that the base substitution spectra were
different (7), and both Foster and Trimarchi (4)
and Rosenberg et al. (19) used the F'-borne
lacI33 allele to show that the frameshift-reversion spectra
were different. There are a number of reasons, reviewed in reference
12, to suspect that adaptive reversion of F'-borne alleles may be a special case. Because of concerns about the generality of conclusions based on studies of F'-borne reporter alleles, it is
important to compare the mutagenic spectra of growth-dependent and
adaptive mutations in a chromosomal gene.
Reversion systems are unsatisfactory for determining mutational spectra
because, for any given mutation, there are a very limited number of
sites and kinds of mutations that will produce a revertant phenotype.
Some kinds of mutational events, such as insertion of insertion
sequence (IS) elements, are completely excluded from studies that
employ reversion systems. Because no constraints are placed on the
specific nature of the mutations, it is much more useful to select for
loss of function in the target gene than to select for reversion. The
lacI gene has served as a very powerful system both for
studying spontaneous mutagenesis and for helping to elucidate the
functions of the methyl-directed mismatch repair system (14,
20-22). lacI mutants allow constitutive expression of
lacZY and thus permit growth on phenyl-
-galactoside, which is a substrate for -galactosidase, but is not an inducer of
the lac operon. Aside from a four-base repeat hot spot for frameshifts that causes 70% of spontaneous lacI mutations,
of the remainder about 35% are deletions, 39% are base substitutions, 3.9% result from insertion elements, 18% from single-base
frameshifts, and 3.5% from duplications (14).
Over the long periods of selection required for adaptive mutagenesis
studies, the basal level of lacZ-encoded -galactosidase generates considerable background growth (data not shown); thus the
lacI system is not suitable as a reporter for adaptive
mutagenesis. Adaptive mutations have proven readily detectable in the
Ebg repressor gene ebgR (8), and adaptive
ebgR mutants accumulated continuously over a period of 14 days. The ebgAC-encoded -galactosidase does not generate
significant background growth. Like lacI, ebgR
specifies a repressor that controls expression of an adjacent
-galactosidase gene, and the two genes have 25% homology at the
amino acid level (11). Beyond the issue of adaptive
mutations, a comparison of the spontaneous mutation spectra of these
similar genes could provide insight into the generality of the very
detailed spectra that have been generated for lacI. I have
therefore compared the spectra of forward growth-dependent and adaptive
mutations in ebgR.
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MATERIALS AND METHODS |
Escherichia coli strains.
Strain SJ134
(11) is F
lacZ4680 ebgA51
ebgR+ rpsL. Strain SJ2 is F
lacZ4680 lacY+ ebgR+
ebgA+ rpsL metC.
Transductions.
Transductions were mediated by bacteriophage
P1vir as described by Miller (17).
Media.
Mineral salts (MS) medium consisted of 423 mg of
sodium citrate, 100 mg of MgSO4 · 7H2O,
1 g of (NH4)2SO4, 540 µg of
FeCl3, 1 mg of thiamine, 3 g of
KH2PO4, and 7 g of
K2HPO4 per liter, plus a carbon source.
Lactulose selection medium was MS medium containing 1 g of
lactulose per liter, 0.2 mM IPTG
(isopropyl--D-thiogalactopyranoside) to induce
expression of the lacY-encoded -galactoside permease and
X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside)
(20 mg/liter), a noninducing substrate that produces an intense blue color (to facilitate the detection of colonies). Limiting glycerol medium was MS medium containing 0.01% (vol/vol) glycerol and 0.2 mM
IPTG. X-Gal medium was MS medium containing 0.2% (vol/vol) glycerol,
0.1% (wt/vol) Casamino Acids (Difco), 20 mg of X-Gal/liter, and 0.2 mM IPTG.
L agar consisted of Luria-Bertani-agar (17) plus 1 g of
glucose per liter. Solid media were solidified with Sigma Purified Agar. MacConkey lactulose indicator medium was prepared from MacConkey agar base (Difco) and lactulose according to the manufacturer's instructions and included 0.2 mM IPTG. ebgR colonies of
strain SJ134 are red on MacConkey lactulose plates, while wild-type
colonies are white.
PCR amplification of ebgR.
Amplifications for initial
characterization of mutants used primers 2 and 7 (Fig.
1) and 2.5 µl of crude genomic DNA in a 25-µl reaction that contained 1.5 mM MgCl2, 200 µM
(each) deoxynucleoside triphosphates (dNTP), 0.5 U of Taq
polymerase (Gibco), and 2.5 µl of 10× PCR buffer (Gibco). Crude
genomic DNA was prepared by suspending 600 µl of an overnight
L-broth-grown culture in 50 µl of sterile water, heating the
suspension in a sealed Microfuge tube at 100°C for 15 min,
centrifuging at 4°C for 15 min in a Microfuge, and transferring the
supernate to a fresh tube for storage. PCR products for DNA sequencing
were amplified from genomic DNA prepared by the cetyltrimethylammonium
bromide method (1) using either primers 2 and 7 or, when
sequencing failed to reveal any differences from the canonical
sequence, primers 1 and 8 in a 100-µl reaction that contained 100 ng
of genomic DNA, 1.5 mM MgCl2, 200 µM each dNTP, 2 U of
either AmpliTaq Gold (Perkin-Elmer) or Qiagen Taq
polymerase, and 10 µl of corresponding 10× PCR buffer. PCR products
used for DNA sequencing were purified by the QiaQuick (Qiagen) method.
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FIG. 1.
Oligonucleotide primers used for amplification and
sequencing. Base numbering is according to the published sequence of
the ebg operon (13) (GenBank accession no.
M64441) except for primer 1, which is numbered according to GenBank
accession no. AE000389. The ebgR coding region begins at bp
126 and ends at bp 1109 The locations of the primers are as follows:
primer 1, bp 4501 to 4525; primer 2, bp 73 to 94; primer 3, complement
of bp 219 to 243; primer 4, bp 473 to 497; primer 5, bp 624 to 660;
primer 6, complement of bp 660 to 684; primer 7, complement of bp 1144 to 1168; primer 8, complement of bp 1435 to 1459.
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DNA sequencing.
Purified PCR products were sequenced by
cycle sequencing using the primers shown in Fig. 1 and an ABI kit
according to the manufacturer's instructions. Sequencing products were
separated and analyzed on an ABI model 377 automated DNA sequencer.
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RESULTS |
Kinetics of adaptive mutations in ebgR.
Strain SJ134
carries the ebgA51 allele, which encodes a mutant Ebg
-galactosidase that hydrolyzes lactulose effectively, but that
strain cannot utilize lactulose as a carbon source because lactulose is
not an effective inducer of the ebg operon. Mutations in
ebgR, which permit constitutive expression of the
ebg operon, allow ebgA51 strains to utilize
lactulose effectively.
To monitor the accumulation of
ebgR mutants, on day 0 approximately 10
7 SJ134 cells were spread onto lactulose
selection plates, and
the plates were incubated at 30°C. On days 1 through 4 and on
day 7, cells were washed from two plates, suitably
diluted, and
plated onto L agar to determine the number of viable
cells. Once
ebgR colonies began to appear, they were
immediately eliminated
from a subset of plates, with little disturbance
of surrounding
cells, through the use of a diathermy probe (Hyfrecator
Plus model
7-796; Birtcher Medical Supplies), an electrosurgery device
that
delivers an intense spark which kills the cells in the colony.
Those treated plates were then used to determine the number of
viable
cells. During the first few hours after plating, the populations
grew
from 2.4 × 10
7 to 2.3 × 10
8 cells
at the expense of trace contaminants in the medium. After
day 1, the
population declined, with a death rate of
0.41 day
1.
Figure
2 shows the accumulation of
ebgR mutants over the course of 7 days. Reconstruction
experiments have shown that >98%
of preexisting
ebgR
mutants of strain SJ134 form visible colonies
in 3 days under these
conditions (
11), which is consistent with
the first
appearance of
ebgR colonies on day 3. Based on the
assumption
that colonies appeared 3 days after the
ebgR
mutations occurred,
the adaptive mutatation rate on any given day is
the average number
of mutants that appeared 3 days later per plate
divided by the
average number of viable cells per plate. The average
adaptive
mutation rate to
ebgR was (1.9 ± 0.24) × 10
7 mutations per cell per day.
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FIG. 2.
Accumulation of ebgR mutants on lactulose
selection medium. Data along the ordinate are the average numbers of
mutant colonies per plate based on counting at least 10 plates each
day.
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Isolation of growth-dependent and adaptive ebgR
mutants.
A total of 144 independent 0.2-ml cultures of SJ134 were
grown from inocula of about 2 × 103 cells at 30°C
in limiting glycerol medium to ensure that all cultures were at the
same density (about 108 cells per ml). Next, 127 of the
0.2-ml cultures were spread onto lactulose selection plates, and the
remainder were used to determine that the average number of cells per
culture was 2.4 × 107. The lactulose selection plates
were incubated at 30°C, and the colonies on each plate were counted
and marked on day 3. Those early-arising colonies resulted from
growth-dependent mutations that occurred either during the growth of
the cultures prior to plating or during growth at the expense of trace
contaminants to a final density of 2.7 × 108 cells
per plate. This selection constituted a fluctuation test (16), and the growth-dependent mutation rate to
ebgR was estimated from the distribution of the number of
ebgR colonies per plate on day 3 according to the method of
Stewart et al. (23) as implemented by Stewart's DataFit
program. That program estimates separately the average number of
mutations that occurred prior to plating and the average number that
occurred after plating. In this experiment, there was an average of
1.52 mutations per culture prior to plating and 1.3 mutations per
culture after plating. Dividing the average number of mutations per
culture by the number of cells per culture at the time of plating
yielded the estimated growth-dependent mutation rate to ebgR
of 6.3 × 108 per cell division.
To avoid isolating sibling mutants, one early-arising (day-3) mutant
was isolated from each of 100 lactulose selection plates
by restreaking
onto X-Gal
medium.
The incubation of lactulose selection plates was continued at 30°C,
and late-arising colonies were marked on days 4, 5, and
6. One day-5
and one day-6 colony were isolated from each of 100
lactulose selection
plates and restreaked onto X-Gal medium. Those
were designated as
late-arising
mutants.
All mutants were grown in L broth from single colonies and stored at
80° in 7% dimethyl
sulfoxide.
If the spectra of growth-dependent mutations (early-arising mutants)
and adaptive mutations (late-arising mutants) are to
be compared, it is
important to be confident that the sample of
late-arising mutants does
not include growth-dependent mutations
in which the
ebgR
individual either grew slowly or lagged before
starting to grow.
Bacteriophage P1
vir lysates were prepared from four of the
ebgR mutants, including
two missense mutants, one nonsense
mutant, and one frameshift
mutant. The lysates were used to transduce
strain SJ2 (
ebgR+ ebgA+), and the
transduced cells were spread onto lactulose selection
plates that had
previously been scavenged with about 10
8 strain SJ2 cells
to eliminate trace contaminants that would allow
the transduced
population to grow at the expense of nutrients
other than lactulose.
Strain SJ2 cannot easily mutate to lactulose
utilization because it
makes both the functional ebg repressor,
which does not respond to
lactulose as an inducer, and the wild-type
Ebg enzyme, which cannot
hydrolyze lactulose effectively. Indeed,
SJ2 control cells that were
not transduced with P1
vir produced no colonies on the
lactulose selection plates over the
course of this experiment.
Transduction of an
ebgR allele into
an
ebgR+ cytoplasm mimics the situation when a new
ebgR allele arises
by mutation. An average of 191
ebgR transductants were obtained
from each of the four
donors. During incubation at 30°C, no colonies
appeared on day 1 after the transductions, <1% of the final total
of colonies appeared
on day 2, >98% appeared on day 3, <1% appeared
on day 4, and no
additional colonies appeared after day 4. This
result shows that newly
arisen
ebgR mutations do produce colonies
on the
lactulose-selective plates within 3 days and indicates
that the sample
of late-arising mutants isolated on days 5 and
6 was unlikely to have
included any growth-dependent
mutations.
Initial characterization of mutants.
PCR amplification
products generated by using primers 2 and 7 (Fig. 1) were resolved on
0.7% Tris-borate-EDTA agarose gels. Gels were stained with Syber
Green, and the sizes of the PCR products were determined by comparison
with a 1-kb ladder (Gibco) on the same gel. The wild-type PCR product
of amplification with primers 2 and 7 is 1,096 bp. Mutants whose PCR
products were detectably larger fell into two size classes, those whose
PCR product was about 2 kb and those whose PCR product was about 2.5 kb. The 2-kb product was likely to have arisen from insertion of the
768-bp IS1 insertion element, while the larger PCR product
was likely to have arisen from insertion of other IS elements, all of
which are in the 1.2- to 1.5-kb range.
Distribution of IS-mediated mutations.
Several of the putative
IS1 insertion mutants were sequenced with primers 2 and/or 7 and proved to contain authentic IS1 insertions. Putative
IS1-mediated mutants were amplified using a three-primer cocktail consisting of primer 2 (Fig. 1) and primers corresponding to
bp 685 to 709 and the complement of bp 53 to 77 of IS1.
Because amplification was with primer 2 and one of the two
outward-amplifying IS1 primers the position, but not the
orientation, of the IS1 insertion element could be
determined. Over 95% of the putative IS1 insertions were
authentic, and the remainder proved to have been misidentified on the
basis of anomalously migrating bands in the initial determinations.
Several of the mutants whose PCR products were about 2.5 kb were
sequenced with primers 2 and/or 7 and all proved to contain
IS
30 insertions. The remainder of the putative
IS
30 insertion
mutants were amplified with primer 2 and a
primer corresponding
to bp 1043 to 1067 of IS
30. Those that
failed to produce a PCR
product in that reaction were amplified with
the IS
30 primer and
primer 7. Those reactions allowed the
positions of the remaining
IS
30 insertions to be
determined.
The remainder of the putative insertion element mutants were sequenced
with primer 2 and/or primer 7 to determine the identity
and position of
the
element.
Among the 100 early-arising mutants there were 54 IS
1, 1 IS
4, and 6 IS
30 insertions. Their positions are
shown in Fig.
3.
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FIG. 3.
Distribution of IS elements among early-arising and
late-arising ebgR mutants. Data along the abscissa are
positions in the ebg operon numbered according to reference
13 (GenBank accession no. M64441). Data along the
ordinate are the percentages of total isolates with that insertion
element in the indicated 100-bp interval. Note the difference in scale
of the ordinates of panel A and panel B.
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Among the 200 late-arising mutants, one which consistently produced two
bands upon amplification with primers 2 and 7 and
three which could not
be amplified were not further considered.
Among the remaining 196 late-arising mutants, there were 70 IS
1,
78 IS
30,
1 IS
2, and 8 IS
4 insertions. Over half of the
late-arising
IS
30 insertions were in the 400- to 500-bp
region (Fig.
3); therefore
four of those mutants were sequenced. All
proved to have IS
30 inserted at bp 472. The region centered
at bp 472 matches the
consensus target for IS
30 insertion
(
18) at 20 of the 24 bp,
and that region is the best match
to the consensus found within
ebgR. Thus, it appears that
for late-arising mutants that site
is a hot spot for IS
30 insertion.
Sequence changes in non-IS-mediated mutants.
The remaining
sample of 39 early-arising and 39 late-arising mutants that were not
mediated by insertion elements was not large enough for the comparison
of mutational spectra; thus another 100 early-arising and 200 late-arising mutants were isolated as described above. As before, they
were amplified with primers 2 and 7 to identify those that were caused
by insertion of IS elements, but no attempt was made to identify those
elements or the positions of the insertions. In total, an additional 34 early-arising and 44 late-arising non-IS-mediated mutants were isolated.
Sequence changes in those mutants were determined by sequencing with
the primers shown in Fig.
1. Mutations were identified
by aligning the
sequences to bp 73 to 1168 of the wild-type sequence
(GenBank accession
no.
M64441). Base changes were accepted
as authentic only if they were
found on both strands, but regions
in which the sequence was
unambiguous and did not differ from
the wild-type sequence were not
necessarily completely sequenced
on both
strands.
In a few cases, the sequence changes could not be identified, although
the mutants were clearly phenotypically
ebgR. In those
cases, the region extending from the 234 bp 5' to
ebgR
through
the first 142 bp of
ebgA was amplified with primers
1 and 8 and
sequenced as
above.
Table
1 and Fig.
4 show
the distribution of non-IS-mediated growth-dependent
(early-arising) and adaptive
(late-arising mutations)
in
ebgR. These include two multiple
mutations, a deletion of G
393 and C
397
A, and
AA
1211-1212GC. There were four duplications
ranging
from 2 to 5 bp, one of which was an imperfect duplication
(CCTA instead
of CCTG).
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FIG. 4.
Distribution of non-IS-mediated mutations. The scale
indicates base pair numbers according to reference
13 (GenBank accession no. M64441). Symbols above the
scale indicate growth-dependent mutations, and those below the scale
indicate adaptive mutations. Symbols: x, transition; o, transversion;
f, single base insertion or deletion frameshifts; d, multibase
duplication; , multibase deletion. Positions of symbols indicate
sites of mutations within 100-bp region.
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|
This study adds to previous knowledge of the
ebg operon.
Three insertion mutations, involving IS
1 at bp 57, IS
1 at bp 76,
and IS
4 at bp 75, permitted
identification of a regulatory region
70 bp upstream of the EbgR coding
region.
In three of the mutants, instead of being constitutive for expression
of Ebg, the repressor had become sensitive to lactulose
as an inducer.
Nine such mutants have previously been identified
(
5), and
the mutations responsible have been identified as
resulting in amino
acid substitutions Asp
190Glu, Ala
195Thr,
and Phe
196Cys (
13). The two novel mutants
resulted from Gly
215Val
and Ser
217Phe
replacements. This observation expands the sugar-binding
domain to the
188- to 217-amino-acid region of the
repressor.
One mutation, AA
1211-1212GC, is well outside of the
ebgR coding region and is probably in the
ebgA
operator. The region
from bp 1205 to 1225, half of which overlaps the
previously identified
35 region of the promoter, forms a perfect
palindrome and is
probably the
operator.
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DISCUSSION |
Both the spectra of growth-dependent (early-arising) and adaptive
(late-arising) mutations in ebgR are clearly dominated by IS-mediated mutations. Although domination by IS-mediated mutations is
not the case for spontaneous lacI mutations (14),
where only about 4% are IS mediated, IS element domination of the
mutation spectrum has also been reported for spontaneous mutations in
tonB (15). There is a highly significant
difference between the growth-dependent mutants, of which only 60% are
IS mediated, and the adaptive mutants, of which 80% are IS mediated
(P < 0.0001 by Fisher's exact test of 2 × 2 contingency tables). There is also a highly significant difference
between early-arising and late-arising mutations in terms of the
relative frequencies with which the different IS elements cause those
mutations (P < 0.0001 by the likelihood ratio [G
test] of contingency tables).
The large contribution of IS30 insertions at the hot spot in
late-arising mutants raises the question of whether that hot spot alone
accounts for those highly significant differences. It does not.
Elimination of those mutants with IS30 inserted at the hot
spot still results in a highly significant difference (P = 0.0065) between early and late arising in terms of the relative frequencies with which the different IS elements cause those mutations, and a significant difference (P = 0.04) between
growth-dependent and adaptive mutants in terms of the contributions of
IS elements to the mutational spectrum. It is emphasized that there is
neither a good statistical reason nor a good biological reason to
eliminate those hot spot IS30 mutants from consideration.
Despite these differences, the distribution of IS-mediated mutations
may tell us more about IS biology in starving cells relative to growing
cells than it does about adaptive mutagenesis in general.
Table 2 compares the spectra of
growth-dependent (early-arising) and adaptive (late-arising) mutations.
There is no significant difference between the spectra of
growth-dependent and adaptive non-IS-mediated ebgR mutations
(P = 0.66 by likelihood ratio [G test] of contingency
tables). This result sharply contrasts with the results of earlier
studies that employed F'-borne targets for mutagenesis (4, 7,
19) and suggests that concerns about the generality of
conclusions from F'-borne reporter genes are well justified.
The lacI gene is the most thoroughly studied gene in
E. coli with respect to mutational spectra. Several studies
have used the entire lacI gene, in which 59% of the
mutations occur by frameshifts at one hot spot, as a target
(14). If the spectra reported for lacI reflect
general spontaneous mutation processes, then spontaneous mutations in
other genes should exhibit spectra that do not differ significantly
from the lacI spectra. Table 3
compares the spontaneous spectrum of lacI mutations
(excluding the frameshift hot spot) with the spectrum of
ebgR mutations (growth-dependent and adaptive mutations
combined). The spectra are highly significantly different (P < 0.0001 by the likelihood ratio test). Transitions dominate the
base substitutions in lacI, while transversions dominate in ebgR. Like ebgR, lacI specifies a
repressor that controls expression of an adjacent -galactosidase
gene, and indeed the two genes share 25% homology at the amino acid
level (13). The spectra of spontaneous mutations at
lacI and ebgR differ significantly, and we do not
know which, if either, spectrum is typical, but the difference between
the spectra of these reasonably similar genes suggests that we should
be cautious when generalizing about spontaneous mutational spectra on
the basis of even extremely thorough and detailed studies of one gene.
It is almost certainly not the case that there is a single
mechanism for adaptive mutagenesis, any more than there is a single mechanism for DNA repair. At this time, the weight of the evidence indicates that (i) adaptive mutagenesis of F'-borne genes and chromosomal genes is dominated by different processes, and (ii) growth-dependent and adaptive mutagenesis of F'-borne genes are dominated by different processes. The finding that the spectra of
non-IS-mediated growth-dependent and adaptive mutations do not differ
fails to provide evidence for different processes acting at the
chromosomal locus ebgR. In order to determine whether
replicon location is a major factor, it will be necessary to compare
the spectra of adaptive mutations at the same locus on F' and on the chromosome.
| |
ACKNOWLEDGMENTS |
This study was supported by grant NP-932 from the American Cancer Society.
I am grateful to Jacqueline Toner for expert technical assistance and
to George Kampo of the University of Rochester Core Nucleic Acid
Facility for his expert advice and helpfulness.
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FOOTNOTES |
*
Mailing address: Biology Department, River Campus,
University of Rochester, Rochester, NY 14627. Phone: (716) 275-0721. Fax: (716) 275-2070. E-mail:
drbh{at}uhura.cc.rochester.edu.
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Journal of Bacteriology, February 1999, p. 1149-1155, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
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