Laboratory of Molecular Genetics, National
Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina 27709
 |
INTRODUCTION |
The bacteriophage T4 rnh
gene encodes Rnh, an RNase H that removes the RNA primers of DNA
replication (11). This nuclease, whose structure has been
determined by X-ray crystallography (21), acts as a 5'-to-3'
exonuclease on RNA-DNA and DNA-DNA duplexes and also as a flap
endonuclease (2). An rnh null mutation
(rnh
) reduces total DNA synthesis only slightly, causing
instead an accumulation of short, nascent DNA fragments and reducing
the burst size roughly twofold (10). rnh is
partly complemented by the host rnhA gene, whose impairment
reduces the rnh mutant burst size to about 10% of
normal, while rnh is more strongly complemented by the
5'-exonuclease function encoded by the host polA gene, whose
impairment almost abolishes the rnh mutant burst size
(10). In addition to its role in DNA replication, Rnh acts in recombination repair: rnh increases sensitivity to the
lethal effects of both UV radiation and the topoisomerase inhibitor
4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and this survival defect is epistatic to mutations
in two genes required for T4 recombination repair, uvsW and
uvsX (33).
T4 Rnh is related by sequence and/or structure to a family of flap
endonucleases that includes examples from the archaebacteria Methanococcus jannaschii (13) and
Pyrococcus furiosus (12), a eubacterium, a
mammal, and the Saccharomyces cerevisiae DNA repair protein
RAD27 (reviewed in references 26 and
30). These endonucleases remove the single-stranded
flaps that can arise either as a result of displacement synthesis past
the beginning of an Okazaki fragment or during genetic recombination
and/or repair. However, T4 Rnh flap endonuclease is strongly inhibited by the gene-32 single-stranded-DNA-binding protein whereas
its RNase H activity is not (2). In the T4 multiprotein in
vitro DNA replication system, Rnh preferentially uses its
5'-exonuclease activity rather than its flap endonuclease to remove
primers and adjacent DNA from the 5' end of lagging-strand fragments
(3). A rad27 mutation has a strong mutator
phenotype at microsatellite and minisatellite sequences (14,
16) and a remarkable duplication mutator activity
(31). These duplications comprised 5 to 108 bp, arose at
sequences flanked by repeats of 2 to 12 bp, and were proposed to occur
in a process triggered by the failure to excise DNA flaps bearing
short, separated sequence repeats.
Defects in genes of T4 DNA metabolism often result in a mutator
phenotype (8). Thus, T4 rnh mutations might
display mutator activity, and this activity might include a bias toward
duplications. An rnh null mutation was reported previously
to increase the frequency of T4 acriflavin-resistant (Acr)
mutants (10). While such an increase might result from
mutator activity, the relation between mutant frequency and mutation
rate is often complicated and can be strikingly distorted when a
mutational target (such as the ac gene whose knockout
generates acriflavin resistance) interacts with a gene (such as
rnh) whose product is involved in DNA metabolism. Acriflavin
itself is both a topoisomerase inhibitor (19, 24) and a
powerful mutagen (22). Because of the unknown impact of
these potential interactions and also in order to determine whether a
T4 rnh mutator activity had a duplication bias, we decided
that the increase in Acr mutant frequency in an
rnh
background should be examined in some
detail. The unexpectedly complex results of this investigation are
described here.
| |
MATERIALS AND METHODS |
Strains.
The translated portion of the rnh gene
comprises 915 bp. The rnh(10-777) mutation, obtained
from Ken Kreuzer, is an in-frame deletion of codon-encoding bases 10 through 777 which leaves intact a middle-mode promoter near the end of
rnh and is thus unlikely to affect downstream genes
(33). The rnh(352-847) mutation (renamed from
the original) (10) removes this middle-mode promoter; in
addition, because it disrupts the reading frame, it retains the first
117 rnh codons and then adds VAKFIHIL before encountering an
ochre stop codon. The rI, rII, and rV
mutants are from the Drake collection; rIIUV58 is
hyperrevertible by proflavin (5). The rnh mutants
are in a T4D background, and the r mutants are from the
Drake collection in a T4B background.
The Escherichia coli strains are from the Drake collection.
B cells are su, and tight mutations of all the
major T4 r loci produce the r plaque morphology (large,
sharp-edged plaques) on B cells. B40 suI+ cells
are B cells containing an amber-suppressing allele. BB cells are
su; rII mutants produce an
r+ plaque morphology on BB cells. KB cells are K-12(
)
and restrict the growth of T4 rII mutants.
Media and growth conditions.
Luria-Bertani (LB) medium and
Drake agars (4) were used throughout at 37°C. Stocks were
grown with BB cells.
Scoring rnh alleles.
Individual plaques were
resuspended in 40 µl of water, and the rnh gene in each
was amplified by PCR. A 1,034-bp sequence (including primers) was
amplified from 
99 through 935 where the AUG initiation codon begins
with bp 1. The upstream primer was 5'-TGAAAACACAATAGGAGCCCG-3'
(99 through 79), and the downstream primer was
5'-TTTAGCCATTATTCACCTC-3' (917 through 935). The PCR consisted of 25 cycles of 1 min at 94°C, 1 min at 62°C, 1 min at
72°C, and a final extension time of 10 min at 72°C with Display Taq polymerase (Display System Biotech). PCR products were
separated on a 1.2% agarose gel. The rnh(10-777)
mutation produces a product of 266 bp; the rnh(352-847)
mutation produces a product of 538 bp.
Screening and sequencing Acr mutants.
T4 stocks
were plated on BB cells with acriflavin neutral (Sigma) at 0.2 µg/ml
in the top agar and 0.5 µg/ml in the bottom agar to select
Acr mutants and on unsupplemented agars to score total
phages when an Acr mutant frequency was desired; the plates
were incubated overnight. To obtain Acr mutants of
independent origin, individual acriflavin-sensitive plaques were
isolated and a single Acr mutant was isolated from each;
the rnh+ background was T4B.
The sequence of the ac gene (formerly open reading frame
52.2) (32) comprises 156 bp encoding 51 amino acids plus an
ochre termination codon and extending from T4 genomic coordinate 165493 through 165338 (17). To determine their ac
sequences, each mutant plaque was resuspended in 40 µl of water and
its ac gene was PCR amplified with the upstream primer
5'-TCGAAGAAATGAACCGTATGT-3' (complementary to 165618 through
165638) and the downstream primer 5'-CTACCAATAAAGCAGCAAGGG-3'
(complementary to 165294 through 165314). The PCR consisted of 25 cycles of 1 min at 94°C, 1 min at 50°C, 1 min at 72°C, and a
final extension time of 10 min at 72°C with Display Taq
polymerase. PCR products were purified with the Qiagen purification
kit. Sequencing was performed with an ABI Prizm 377 automatic sequencer
with the above upstream PCR primer and the dRhodamine terminator cycle
sequencing kit (PE Applied Biosystems).
Test for acriflavin mutagenicity.
Working under yellow light
to avoid photodynamic effects, we used rIIUV58 in an
rnh+ or an rnh(352-847)
background to infect concentrated log-phase BB cells at 5 × 108/ml at a multiplicity of infection of 5 in LB broth at
37°C on a rotary shaker. At t = 6 min after
infection, another multiplicity of infection of 5 was applied. At
t = 10 min, acriflavin was added to 0 or 1 µg/ml. At
t = 30 min, the mixture was diluted 80-fold in LB
broth, and incubation was continued. Phage development was terminated
at t = 90 min by adding chloroform.
rIIUV58+ revertant frequencies were determined
by plating phages on KB cells (on which only
rII+ phages grow) and on BB cells (on which all
phages grow).
Screening r mutants.
When T4 is plated on B cells, large,
sharp-edged plaque morphology mutants can be observed. These are called
r mutants for rapid growth, although the number of phage particles per
plaque is reduced. The r phenotype results from loss of lysis
inhibition, and r mutations can arise at any of several
loci. About 70% of spontaneous r mutants contain mutations at the
rII locus; about 25% contain mutations at the rI
locus; and the remainder contain rIII, rV,
unmapped, or leaky or rapidly reverting rII mutations. Although the rII locus is about 10-fold larger than the
rI locus, rII missense mutations are poorly
detected whereas rI missense mutations are better detected,
so that the observed ratio of rII to rI mutants
is only about threefold. A typical median r mutant frequency scored by
plating on B cells is roughly 6 × 104 in a
high-titer stock grown in BB cells but is twofold to threefold lower in
a resuspended plaque. We picked and restreaked all r and ambiguous
plaques to verify their phenotypes.
rII mutants produce the r+ plaque morphology on
BB cells. Thus, when T4 is plated on BB cells, most of the r mutants
contain rI mutations. A typical median r mutant frequency in
a resuspended plaque plated on BB cells is roughly 2 × 105.
Details of plating procedures for detecting r mutants have been
described elsewhere (4). Because of the clonal nature of the
distribution of mutant frequencies among stocks, the best measure of a
mutant frequency is the median rather than the mean; the mean is
excessively sensitive to the small proportion of stocks with high
mutant frequencies, particularly jackpots. Medians from five stocks are
about 95% reproducible to within twofold (25), and those
from seven stocks are about 95% reproducible to within about 1.5-fold.
When stocks contain similar numbers of T4 particles, ratios of mutation
rates are the same as ratios of mutant frequencies. For simplicity,
therefore, we present frequencies rather than rates.
| |
RESULTS |
Mutation to Acr.
We wished first to redetermine
Acr mutant frequencies in the wild-type and
rnh backgrounds and then to characterize the mutations at
the level of DNA sequence. The Acr mutant frequency was
previously estimated to increase 10-fold (from about 4 × 106) in an rnh(352-847) background
compared to an rnh+ background (10).
Using the treatment protocol described in reference
10 but substituting BB cells for B cells, we
observed a wide range (2 × 106 to 20 × 106) of median Acr frequencies in several
experiments in an rnh+ background; in
particular, we were plagued by a variable background of tiny plaques on
the selection plates. Our Acr frequencies were 5 × 106 to 37 × 106 in
rnh(10-777) and 1 × 106 to 50 × 106 in rnh(352-847). Ratios of
Acr mutants in rnh versus
rnh+ were 0.5 to 20 for
rnh(10-777) and 0.5 to 10 for
rnh(352-847).
In order to identify possible changes in the rates of specific kinds of
Acr mutations, we turned to DNA sequencing. We plated
wild-type and (both) rnh strains in the absence of
acriflavin, suspended well-separated plaques (of large to small rather
than pinpoint size), replated these low-titer stocks in the presence of
acriflavin, picked a single Acr mutant from each stock, PCR
enriched the ac DNA, and sequenced. [We analyzed similar
numbers of Acr mutants in rnh(10-777) and
rnh(352-847) backgrounds and discerned no differences
between the two backgrounds in all that follows.] T4-infected cells
take up acridines at an increased rate compared to uninfected cells,
and ac mutations arise in a gene whose inactivation prevents
this uptake (27). The fractions of ac mutants
among Acr mutants were different in the
rnh+ and rnh backgrounds (Table
1). In the wild-type background, 94% of
Acr mutants contained ac mutations, a result
indistinguishable from the 68 of 76 mutants (89%) reported previously
(32). In the rnh background, however, only
57% of Acr mutants contained ac mutations. The
locations and Ac sensitivities of the non-ac mutations were
not investigated and remain unknown, but our primers amplify 125 nucleotides before the start codon and 23 nucleotides after the
termination codon, so that the observed acriflavin resistance is
unlikely to result from a defect in ac function; similar
mutations described previously arose in a small region linked to
ac (32). Our result suggests that roughly half of
the previously described increase in Acr mutants in an
rnh background (10) has nothing to do with
mutation at the ac locus.
The two ac mutational spectra are shown in Fig.
1 (which does not display several large
mutations that are described in the figure legend). None of the
mutations are complex (that is, mixtures of two or more base pair
substitutions, insertions, and/or deletions). These spectra do not
approach saturation, so that many of the smaller differences tend to be
uninformative. However, the spectra are notable for the several sites
and regions sporting multiple mutations in both
rnh+ and rnh backgrounds. In
addition, there is one apparent difference between mutation frequencies
in the two rnh backgrounds at position 108 (three additions
and four deletions in rnh+ versus no mutations
in rnh). A notable observation of multiple differences
with little change in total mutation rate has been described elsewhere
for differing gene-43 backgrounds (32). A much
larger collection of ac mutations would have to be
characterized to determine whether any of the differences in the two
rnh backgrounds are real.
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|
FIG. 1.
Spectra of spontaneous ac mutations in
rnh+ (above sequence) and rnh
(below sequence) genetic backgrounds. Capital letters indicate base
pair substitutions, boldface indicates insertions, and lowercase
letters indicate deletions. The sequence is the ac gene from
ATG (positions 1 through 3) encoding the initiation codon through TAA
(positions 153 to 156) encoding the ochre termination codon; every 10th
base is underlined and numbered beneath when possible. When an
insertion or deletion could have arisen between two or more short
repeated sequences, the mutation is placed in the middle of the
wild-type repeats. Two or more adjacent added or deleted bases
represent a single mutation, and repeated mutations are stacked. There
are no complex mutations containing mixtures of base pair
substitutions, insertions, and/or deletions. Note that several
mutations are not shown: deletions of 32 bases between TTTT at 59 through 62 and at 91 through 94 that occurred three times in
rnh+ and twice in rnh and one
addition of 99 bases that occurred once in rnh (see
text).
|
|
The classes of mutations are summarized in Table
2. In contrast to Table 1, there is no
significant difference between the frequencies of the various kinds of
mutations recovered from the two genetic backgrounds; when comparisons
were made between the 11 categories of observed base pair substitutions
and additions and deletions of base pairs, df = 10, 
2 = 10.5, and P = 0.40. Few mutator mutations produce mutations identical to those arising in the nonmutator background. It therefore seems likely that the previously reported increase in Acr
mutants in an rnh background (10) was due at
least in part to the non-ac mutants that are specifically
selected in that background and perhaps to other less well
characterized factors.
Given the duplication propensity of the yeast rad27 mutator
mentioned earlier, it is notable that the proportion of duplication mutations in the two sets of ac mutations is virtually
identical (10 of 61 in the rnh+ background and 5 of 30 in the rnh background). These duplications are
shorter than most of those observed in the yeast rad27
mutator: the T4 duplications included 12 of 1 bp, 6 of 2 bp, and 1 each of 3, 4, 6, and 10 bp in the rnh+ background and
3 of 1 bp, 2 of 2 bp, and 1 each of 4, 5, and 99 bp in the
rnh background. All of the addition mutations were tandem
duplications. These can be of two general types: those in which no
flanking sequence repeats existed in the wild-type sequence and those
apparently arising from repeats. The latter can be described as RiR
RiRiR where R is a repeated sequence (which can be as short as a single
base pair) and i is an intervening sequence between the repeats. Such
mutations can arise by slipped mispairing (29) and are
characteristic of the yeast rad27 duplication mutations
(31). Of the 22 insertions in the
rnh+ background, 12 have slippage-like structure
and are additions of only 1 or 2 bases. Of the eight insertions in the
rnh background, three have slippage-like structure, three
are additions of only 1 or 2 bases, and one is an addition of 99 bases
where R is CATTT at positions 32 through 36 and 131 through 135. Only
this single mutation of the 30 additions resembles the duplications
arising in the yeast rad27 mutator.
Tests for acriflavin mutagenicity in the screen for Acr
mutants.
Acriflavin is a mixture of
3,6-diamino-10-methylacridinium chloride and 3,6-diaminoacridine
(proflavin), both of which are strong frameshift mutagens when present
during T4 replication (22). Thus, the acriflavin test might
be intrinsically mutagenic. Furthermore, because acridines
preferentially mutate and promote breakage at topoisomerase sites in T4
DNA (19, 24), and because an rnh mutation may
interfere with the repair of such breakage (33), the
acriflavin test may preferentially kill or mutate replicating genomes
carrying an rnh defect.
This possibility was tested by comparing acriflavin-induced reversion
of rIIUV58 in rnh and
rnh+ backgrounds (see Materials and Methods).
rIIUV58 was chosen because its reversion is unusually
strongly promoted by proflavin mutagenesis (5). The results
of three experiments appear in Table 3.
The variation is typical of experiments involving mutagenesis with acridines but the trend is clear: acriflavin (at a concentration twice
that used in our measurements of Acr frequencies, but for a
shorter time) is slightly more mutagenic (2.1-fold on average) in an
rnh background than in an rnh+
background. Although rnh mutations typically reduce burst
sizes, no reduction was seen in these particular experiments. Proflavin treatments also typically reduce burst sizes, and the reduction was slightly greater (85 versus 74%) in the rnh
background. The small difference in relative net mutagenesis
observed with this very sensitive target suggests that the Ac test is
not significantly more mutagenic to rnh than to
rnh+ strains. This surmise is supported by the
lack of any increase in the frequency of base pair additions or
deletions among ac mutants in the rnh
background compared to the rnh+ background
(Table 2).
Absence of an rnh effect on mutation from
r+ to rI.
Most r mutants detected on
E. coli BB cells bear rI mutations. The
rI-encoded protein appears to sense superinfection and to
carry out the first step in establishing lysis inhibition
(23) but is not obviously involved in DNA metabolism. The
97-codon rI locus is large enough to fairly sample average
mutation rates. Therefore, in order to determine whether an
rnh mutation affects rates of spontaneous mutation in a
gene other than ac, we scored frequencies of r mutants on BB
cells. The results appear in Table 4. Two
entries per rnh genotype appear for mutant frequencies: values for sharp-edged plaques displaying the classical r
phenotype and values also including slightly fuzzy-edged
plaques (sr, for "semi-r") which we anticipated would be caused by
leaky missense mutants. To confirm this conjecture, we isolated eight
such sr plaques and sequenced them; four contained rI
missense mutations, one contained a frameshift mutation near the end of
the gene, and three contained no rI mutation. Therefore, the
values in the "r + sr" column were computed by multiplying the
sr contribution by five-eighths. Mutant frequencies such as those in
Table 4 based on seven stocks are usually reproducible to about
1.5-fold (25). The data in Table 4 reveal no
rnh mutator activity on the rI gene.
Epistasis between rnh and rII
mutations.
Before undertaking the experiments detailed in Tables 3
and 4, we sought to determine the frequencies of r plaque morphology mutants in stocks plated on E. coli B cells. On B cells,
roughly 70% of spontaneous r mutants carry typical rII
mutations; about 25% carry rI mutations; and the remainder
carry leaky or rapidly reverting rII mutations,
rIII mutations, or r mutations at other less well
characterized loci. Because the rII locus comprises about
3,135 bp, the frequency of r mutants is usually much higher on B cells
than on BB cells, even though missense mutations are more efficiently
detected in rI than in rII. Thus, an r-mutant screen on B cells was expected to be a simple, sensitive test for
mutator activity in rnh mutants. The results appear in
Table 5. Contrary to our expectations,
frequencies of r mutants are clearly reduced severalfold in a
rnh background. (The data suggest that this effect may
differ in magnitude between the two different rnh
alleles, but this possibility was not further explored.)
Because our results with the ac and rI systems
gave no hint of an antimutator effect, we suspected that the reduced
frequencies of r mutants in Table 5 reflected some special property of
rnh rII double mutants such as synthetic lethality or
epistasis. This suspicion was supported by the results of crosses of
the form rnh × r where r = rII versus rI or rV. If recombinant
rnh r double mutants were lethal or indetectable, then
the frequency of r mutants should fall in the cross progeny compared to
the parental mix. We observed no change in r frequencies in
rnh crosses against rI or rV
(average progeny/parental ratio = 1.02) but a marked decrease in
rnh crosses against rII mutants (average
progeny/parental ratio = 0.48) with both rnh
mutations. In addition, because the rnh allele causes
small plaques, we collected numerous progeny with a small-r
(sharp-edged) phenotype and tested them for the rnh
allele by PCR; we found none.
We next performed crosses between rnh mutants and five
different rII amber mutants with B40
suI+ host cells for the cross and for plating
the progeny (so that all progeny necessarily displayed the
r+ plaque morphology). Ten small-plaque progeny were
isolated from each cross. These were tested for inability to grow
on E. coli su K() cells, a characteristic
of most rII mutations; among the nongrowers, we screened for
the presence of the rnh allele by PCR. Each of the
crosses readily yielded progeny that appeared to bear both mutant
alleles. These progeny were backcrossed against the original
rII parent to confirm the identity of the rII
allele. Thus, rII mutations (including the frameshift
mutation rIIUV58 described previously) display an
r+ plaque morphology in the presence of an rnh
null allele but do not regain the ability to grow on K() cells.
| |
DISCUSSION |
Apparent lack of an rnh effect on mutation
rates.
The evidence as a whole indicates that bacteriophage T4
rnh null mutations do not affect mutation rates to an
appreciable extent. No change was detected with the rI
system, and the increase in mutant frequency reported previously with
the Acr system is unlikely to reflect a corresponding
increase in mutation rate for three reasons. First, we did not find a
reliable increase in the frequency of Acr mutants by the
reference protocol (10), although an alternative protocol
(32) appears to be more robust. (We used a different strain
of B cells, namely, BB or B Berkeley, but we have no reason to suspect
that B and BB would behave differently in rnh phage infections.) Second, while most Acr mutants in an
rnh+ background contain ac mutations,
many in an rnh background do not. Third, there was no
discernible difference in the kinds of ac mutants produced
in the two backgrounds, whereas most mutator mutants display
specificity. Therefore, either the host DNA polymerase I 5'-exonuclease
activity that moderately complements the loss of Rnh or the host RNase
H that weakly complements the loss (10) seems to provide any
fidelity components that might be missing in an rnh mutant.
Note, however, that changes in mutational specificity can occur without
changes in mutation rate (6, 32). The data in Table 2 can
exclude only major changes in mutational specificity. However, they do
appear to exclude any strong slippage-like duplication bias in an
rnh background.
Epistasis between rnh and rII
mutations.
Just as a mutator effect was shown to be an unlikely
explanation for an increased Acr mutant frequency in an
rnh background, an antimutator effect was shown not to be
the cause of a decreased r mutant frequency in an rnh
background. Instead, the severalfold decrease in the median r mutant
frequency in stocks plated on B cells reflected the unanticipated
r+ plaque morphology of rII rnh double
mutants, although such double mutants retain the typical inability of
rII mutants to grow on a lysogen.
The rII locus interacts with several other loci important in
DNA metabolism (20, 23, 28). rII mutations
produce the r+ plaque morphology when accompanied by a
49tsC9 (Holliday resolvase) mutation or by the
gene-32 (single-stranded-DNA-binding protein) mutation
32tsL171. Conversely, rII mutations partly
suppress 32tsL171 and also suppress gene-30 (DNA
ligase) mutations. Thus, rII function is extensively
connected to DNA metabolism, albeit in poorly understood ways. Indeed,
the two rII-encoded proteins not only are associated with
the cell membrane but also cosediment with the huge multiprotein DNA
replication complex (9). The failure of rII
mutations to express the r plaque morphology on BB and K-12 host cells
suggests that the rII locus has only an indirect role in
lysis inhibition (23). However, this pattern of interactions
with DNA metabolism highlights the need for caution when using the
rII system as a mutational reporter gene (7, 8).
The ac mutational spectrum.
Because our spectra
are based on only 91 mutations distributed among many sites and
classes, it is premature to analyze the ac mutational
spectrum in detail. In any case, this is not the purpose of the present
report, and we leave such analysis to our colleague and ac
champion Lynn Ripley (32). Note, however, that the pooled
North Carolina and 43+ New Jersey ac
mutations may differ. The two sets were obtained by somewhat different
selection procedures that may have affected the efficiency of
recovering missense mutations, which comprised 17 of 34 of the North
Carolina base pair substitutions but 27 of 33 of the New Jersey base
pair substitutions. The distribution of mutations among classes may be
different in the two collections (df = 13, 2 = 22.4, P = 0.050), but if they are, much of the
difference is due to five GC (GlyArg) mutations at position 112 that are unique to the New Jersey collection; if those five mutations
are not considered, df = 12, 2 = 15.7, and P = 0.21.
Optimal T4 targets for forward mutation.
The sensitivity of a
mutation test reflects the magnitude of changes produced by physical or
chemical treatments or genetic modifiers. The specificity of a mutation
test reflects the information that it provides about what kinds of
mutations arise and where. Reversion tests tend to maximize sensitivity
and specificity but may provide uncharacteristic responses conditioned
by the local DNA sequence in which the reverting base pairs reside;
some potential responses may escape detection altogether, and little or
no information is generated concerning the effects of local DNA
sequence. In contrast, forward-mutation tests may display reduced
sensitivity because only certain kinds of mutations tend to be
increased experimentally, but such tests may display high sensitivity
with regard to effects of local sequence on mutation. Currently, there
are three useful T4 targets for forward mutation, rII,
ac, and rI, each with its particular advantages
and disadvantages (Table 6).
Until recently, the most frequently used T4 mutation system employed
the powerful rII locus, which can be used to measure both
forward mutation and reversion. A large amount of information is
available about both spontaneous and induced mutation in the rII locus. However, this system has several drawbacks. One
is lack of information about the biochemical roles of the
rII-encoded proteins (reviewed in reference
28 and largely unchanged since then), including
their equivocal role in lysis inhibition (23). Another is
the involvement of rII function in DNA metabolism, as
detailed above. A third is the large size of rII, so that
very large numbers of mutants must be isolated when it is desirable to
identify preferentially mutable sites, and mutations must be mapped
before they can be sequenced efficiently. A fourth is the inefficiency
with which missense mutations are detected.
The ac and rI systems share the advantages of
small size and relatively efficient detection of missense mutations
(1, 23, 32). (We will describe several aspects of the
rI system in more detail in a later article.) Small size has
two advantages, ease of sequencing and ease of detecting differential
mutability from site to site. The ac system is the easiest
for collecting mutants of independent origin but may be difficult to
quantitate, and the Acr screen may perturb DNA metabolism.
The rI system seems to be totally independent of DNA
metabolism (although crucial for lysis inhibition, which leads to more,
apparently quite normal DNA synthesis). The main drawback of the
rI system is the low frequency of spontaneous mutants, which
renders quantitation and mutant collection tedious; however,
this problem disappears under conditions that increase mutant
frequencies by 10-fold or more.
We also investigated the alc system, in which forward
mutations can be selected by plating on a specific host
(18). Unfortunately, the resulting alc plaques
are small under a variety of plating conditions, so that quantitation
is difficult. In addition, alc mutations cause T4 DNA to
contain ordinary cytosine instead of 5-hydroxymethylcytosine, a
difference that perturbs DNA metabolism.
| 1.
| Bebenek, A., H. K. Dressman, and J. W. Drake. Unpublished results.
|
| 2.
|
Bhagwat, M.,
L. J. Hobbs, and N. G. Nossal.
1997.
The 5'-endonuclease activity of bacteriophage T4 RNase H is stimulated by the T4 gene 32 single-stranded DNA-binding protein, but its flap-endonuclease is inhibited.
J. Biol. Chem.
272:28523-28530[Abstract/Free Full Text].
|
| 3.
| Bhagwat, M., and N. G. Nossal. Unpublished
results.
|
| 4.
|
Conkling, M. A., and J. W. Drake.
1984.
Isolation and characterization of conditional alleles of bacteriophage T4 genes uvsX and uvsY.
Genetics
107:505-523[Abstract/Free Full Text].
|
| 5.
|
Drake, J. W.
1963.
Properties of ultraviolet-induced rII mutants of bacteriophage T4.
J. Mol. Biol.
6:268-283.
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| 6.
|
Drake, J. W.
1993.
General antimutators are improbable.
J. Mol. Biol.
229:8-13[Medline].
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| 7.
|
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Abstract
Introduction
