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Molecular Biology and Genetics |
The Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regardless of their precise genetic function, measurements of microsatellite mutation rates in normal somatic cells are required to fully exploit these sequences for clinical cancer diagnoses. The high degree of polymorphism of microsatellite sequences in the human population is suggestive of a relatively high rate of germ-line variation (12) , but only limited information is available for mutation rates in somatic cells. To date, only GT/CA dinucleotide repeats have been examined quantitatively for genetic stability in normal (13) or nontumorigenic (14 , 15) mammalian cell experimental systems. Among dinucleotide repeat sequences, the TC/AG and GT/CA motifs are the most abundant, constituting 0.2 and 0.5% of the human genome, respectively (16 , 17) . Moreover, the two types of dinucleotides are dispersed throughout the genome in a similar manner (18 , 19) . The relative abundance of TC/AG microsatellites in the human genome and their ability to form DNA structures distinct from GT/CA microsatellites (20) prompted us to perform a comparison of the somatic mutation rates of TC/AG and GT/CA dinucleotides in nontumorigenic human cells. Our strategy quantitated the mutability of the microsatellite sequences relative to coding sequences within the same genetic target, the HSV-tk gene. This bimodal target was incorporated into an oriP shuttle vector that replicates episomally in EBV-transformed lymphoblastoid cells. Episomal vectors avoid potential complications because of random vector integration near endogenous microsatellite sequences. All mutations within the HSV-tk coding region that inactivate the HSV-TK protein, including those deletions or expansions within the dinucleotide motifs that generate alternative reading frames, are selected in bacteria, allowing for characterization of mutants by direct DNA sequence analyses. Our results show that, in the nontumorigenic lymphoblastoid cell line studied, the mutation frequencies and rates at repetitive dinucleotide sequences up to 20 units in length are similar to those of the unique sequences at the HSV-tk locus. However, TC/AG sequences were more mutable than GT/CA sequences of similar allele lengths. Intriguingly, we observed that the simple presence of the microsatellite sequences altered the mutability of the downstream HSV-tk coding sequence in these episomally replicated vectors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
LCL721, an EBV-transformed human lymphoblastoid cell line established
from a clinically normal female donor (21)
, was cultured
as described (22)
. Routine tests for Mycoplasma
contamination were performed, and cultures were negative throughout
this study. Balb 291, a murine fibroblast cell line in which the
hygromycin phosphoryltransferase gene has been integrated into the
genome (23)
, was cultured in DMEM supplemented with 10%
fetal bovine serum, 50 µg/ml gentamicin, and 150 µg/ml hygromycin.
Construction of STR Shuttle Vectors.
Vectors containing various STR sequences in the 5' coding region of the
HSV-tk gene were constructed by oligonucleotide
site-directed mutagenesis (24)
of plasmid pGTK4. pGTK4 is
identical to the pGTK2 plasmid (25)
except for a G
T
base substitution at position 195 of the HSV-tk gene, which
eliminates a potential internal start site for translation of the
HSV-TK protein. The site-directed oligonucleotides were designed to
insert a [GT/CA]9 or a
[TC/AG]9 in-frame between bases 111 and 112 of
the HSV-tk gene (Fig. 1)
. The sequence of the HSV-tk sense strand only will be
referred to from this point. These oligonucleotides also introduced a
silent CA substitution at position 117, generating an
AvaI site at position 113 and allowing for detection of
positive clones. A phagemid with an out-of-frame
[GT]10 insert was also constructed as above.
The HSV-tk gene of each construct was subcloned into the
pND123 shuttle vector (26)
. The resultant pJY shuttle
vectors are depicted in Fig. 1A
. Two positive clones
possessing the [GT]9 (pJY2 and pJY2A) and
[TC]9 (pJY4 and pJY4A) inserts were chosen to
represent two independent DNA preparations of each STR vector. Vector
pJY3, containing the [GT]10 insert, served as
the positive control. Vector pJY1, constructed by subcloning the
HSV-tk gene of pGTK4 into pND123, contained no STR insert
and served as the negative control. Vectors pJY2.1
([GT]12 insert) and pJY2.2
([GT]15 insert) were derived from pJY2 as
spontaneous STR frameshifts in Escherichia coli by
sequential selection, first in the presence of 40
µM FUdR, a pyrimidine analogue that selects for
nonfunctional HSV-tk genes, and second in the presence of 1
µg/ml trimethoprim, a folate analogue that selects for
TK+ activity (27)
. Vectors pJY4.2
([TC]15 insert) and pJY4.3
([TC]18 insert) were derived from pJY4 in a
similar manner. All vectors were propagated in recA13,
upp, tdk E. coli strain FT334 (28)
. The
sequence between HSV-tk positions 83 and 267 of each pJY
vector was confirmed by DNA sequence analysis prior to use in the
mutagenesis assay.
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pJY vector DNA was isolated from 2–3 x 108 lymphoblastoid cells by an alkaline extraction method (30) and incubation of the cell lysate with Proteinase K (100 µg/ml) for 2 h at 37°C. The nucleic acid pellet containing RNA, pJY vector DNA, EBV DNA, and mitochondrial DNA was resuspended in 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) and digested with 25–35 µg RNase A and 150 units XbaI at 37°C for 2 h. XbaI cleaves both EBV and mitochondrial DNA. After ethanol precipitation, the lower molecular weight RNA was removed by centrifugation through a Microcon-100 ultrafiltration device (Amicon, Inc., Beverly, MA) to a final volume of 25–50 µl.
HSV-tk Mutational Analyses of pJY Vectors.
To score for preexisting mutations created by the propagation and
selection of our vectors in E. coli, background
HSV-tk mutant frequencies were determined for each pJY
construct as described previously (25)
. To determine
HSV-tk mutant frequencies of pJY vector DNA isolated from
lymphoblastoid cells, 1.5–2 µl of purified DNA was used to transform
FT334 by electroporation and plated on VBA selective media. Plating by
this method uses the presence of FUdR to select for bacteria with a
plasmid-derived HSV-TK- phenotype. Detectable
mutations include deletions or expansions of any number of repeat units
within the STR motifs that are not a multiple of three, as well as base
substitutions, frameshifts, deletions, and rearrangements within the
1350-bp HSV-tk gene and promoter sequence
(26, 27, 28)
. The HSV-tk mutant frequency is
defined as the number of FUdR-resistant and chloramphenicol-resistant
colonies divided by the total number of chloramphenicol-resistant
colonies. For LCL721 clones, the mutation rate was calculated as the
mutant frequency divided by the number of generations undergone by that
clone at the time of pJY vector isolation. Differences in mutation
rates among clones carrying different vectors were analyzed
statistically using the nonparametric Mann-Whitney test. Both
background pJY vector DNA and pJY vector DNA obtained after replication
in lymphoblastoid cells were used to isolate independent mutants for
mutational spectra as described previously (25)
.
FUdRR mutants with large rearrangements (greater
than 
30 bp in size) of the HSV-tk gene were detected by
digestion of the mutant pJY vector DNA with BglII and
AvaI restriction endonucleases (Fig. 1A)
. DNA
sequence analyses between HSV-tk positions 83 and 267 were
performed on all remaining mutants (Fig. 1B)
. Differences in
proportions of specific types of mutations between different vectors or
between different hosts were analyzed statistically using Fisher’s
exact test (two-tailed).
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RESULTS |
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. These
STR inserts are flanked 5' by an extra GT/CA unit and 3' by two extra
TC/AG units. The total allele length and sequence of the
HSV-tk sense strand for each vector will be reported from
this point. These vectors, along with a negative control vector
containing no inserted repeats, were propagated in E. coli.
Background HSV-tk mutant frequencies representing mutations
arising spontaneously in E. coli are presented in Table 1
. DNA from the same vector preparations was also introduced into LCL721
human lymphoblastoid cells, and plasmid-bearing populations were
expanded for 10 doublings. Shuttle vector DNA was isolated, and
HSV-tk mutant frequencies were determined by selection in
bacteria. These data are reported as the observed human frequencies in
Table 1
. The observed E. coli and human mutant frequencies
were adjusted for large (>30 bp) HSV-tk deletions or
insertions, as determined by AvaI and BglII
restriction enzyme analyses of independent mutant DNA (Fig. 1A)
. Replication of the shuttle vectors in human cells
produced many mutants with large rearrangements of the
HSV-tk gene (Table 1)
, a phenomenon that has been documented
previously (22)
. The frequencies of this type of
mutational event were similar for each vector tested (16–30 x 10-5).
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1 x 10-4
in E. coli, an 18-fold elevation above that of the negative
control vector (Table 1)
. As the allele length of the TC microsatellite
increased to 17 and 20 dinucleotide units, the corrected mutant
frequencies increased 72- and 180-fold, respectively, above the vector
containing no inserted repeat in E. coli. The
HSV-tk mutant frequency of the out-of-frame
[GT]11 positive control vector was 0.55 in
E. coli, indicating that this insertion abolishes HSV-TK
activity in cells harboring these plasmids.
In human cells, the negative control vector showed a 3-fold increased
corrected HSV-tk mutant frequency over the E.
coli background (Table 1)
. The two independently derived
[TC]11 vectors replicated in human cells showed
corrected mutant frequencies that were 2–5-fold elevated above
E. coli background. Corrected mutant frequencies of the
[TC]17 and the [TC]20
vectors isolated from human cells increased only 2-fold above
background (Table 1)
. Replication of the [GT]10
vectors in human cells resulted in a 2-fold or less increase in the
corrected mutant frequencies, relative to E. coli (Table 1)
.
Overall, for all vectors examined, the HSV-tk mutant
frequencies determined after replication in human cells were slightly
elevated above background.
STR Specificity Analyses of TC/AG and GT/CA Vectors in
Lymphoblastoid Populations.
Mutational specificities for independent preparations of both the
[TC]11 and [GT]10
vectors were generated by DNA sequence analyses (Fig. 1B)
and are presented in Table 2
. This rigorous approach permitted us to categorize mutants into either
those with alterations in the STR motif or those with normal
microsatellite allele lengths and alterations in the coding region of
the HSV-tk target gene. DNA sequence analyses also allowed
us to unequivocally determine the specificity of changes within the STR
motifs. For the [TC]11 vectors in E.
coli, a partiality for deletions of one dinucleotide unit at the
STR locus was detected (Table 2)
. We observed a significant difference
in proportions of STR expansions relative to deletions between the two
independent preparations of the [TC]11 vectors
(pJY4 and pJY4A) in E. coli (P = 0.04, Fisher’s exact test), indicating the stochastic nature of these
microsatellites during propagation in E. coli. However, we
observed no significant differences in the STR specificities of these
combined [TC]11 vectors when comparing mutants
obtained from E. coli with those from human cells.
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. After replication in
human cells, only four mutational events from each of the
[GT]10 vectors occurred within the STR locus
(Table 2)
; therefore, the STR specificities for these individual
vectors cannot be assessed statistically. However, combining the STR
mutational data for both [GT]10 vectors reveals
that there are no significant differences between human and E.
coli cells when comparing deletions relative to expansions.
STR versus Coding Region Analyses of TC/AG and GT/CA
Vectors in Lymphoblastoid Populations.
The proportion of mutants with changes in either the STR or coding
region (Table 2)
was multiplied by the corrected frequencies (Table 1)
to determine region mutation frequencies for each vector. In E.
coli, mutation frequencies at dinucleotide STR sequences of 10–11
units were 10-fold greater than frequencies of mutation in the
HSV-tk coding sequences (Table 3)
. As the TC allele length increased, the STR:coding ratios increased to
30-fold in E. coli. In contrast, after replication in
human cells, such large biases for mutations occurring at the STR locus
were not observed (Table 3)
.
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. The STR mutation frequency of the vector containing the
longer [TC]20 allele was 1.7 x 10-3, only 2-fold above background. In contrast,
the mutation frequencies measured at the HSV-tk coding
sequence were increased 10–20-fold after replication of all [TC]
vectors in human cells, relative to background (Table 3)
. The combined
mutational specificities of both [TC]11 vectors
(Table 2)
showed a significant difference between E. coli
cells and human cells when comparing the proportions of mutations
within the microsatellite locus relative to the coding locus
(P = 0.0004, Fisher’s exact test).
Specifically, there were proportionally fewer mutational events within
the STR region in human cells (68%) than in E. coli cells
(93%) for the [TC]11 vectors. These
differences likely are attributable to the increased HSV-tk
coding region mutation frequency observed for the
microsatellite-containing vectors (8.6–33 x 10-5), relative to the control vector
(1.8 x 10-5) after replication
in human cells, a bias not observed in E. coli (Table 3)
.
For the [GT]10 vectors, no increased mutation
frequencies at the STR motif, relative to background, were measured
after replication of these vectors in human cells for 10 population
doublings (Table 3)
. Again, it is intriguing to note that the coding
region mutation frequencies of these vectors isolated from human cells
were 10–20-fold greater than the E. coli coding region
mutation frequencies. Combining the mutational specificities of both
[GT]10 vectors (Table 2)
revealed that the
proportion of mutations occurring at the STR locus relative to the
coding locus was significantly different between E. coli and
human cells (P < 0.0001, Fisher’s exact
test). As was the case for the [TC]11 vectors,
there were proportionally fewer mutations occurring at the STR locus in
human cells (24%) than in E. coli cells (90%), and the
coding region mutation frequencies were elevated above the control only
after replication of the microsatellite vector in human cells (Table 3)
.
We also compared differences between [TC] and [GT] vectors of similar allele lengths. After replication in human cells, there was an extremely significant difference in the proportion of mutations occurring at the STR region relative to the coding region between the [GT]10 and [TC]11 vectors (P = 0.0002, Fisher’s exact test). More mutations occurred at the [TC]11 STR locus (68%) than at the [GT]10 STR locus (24%) in human cells, a difference not observed in E. coli.
Analyses of the Stability of TC/AG and GT/CA Microsatellites in
Clonal Lymphoblastoid Cell Populations.
To quantitate mutation rates at the episomal vector loci,
plasmid-bearing lymphoblastoid cells were cloned as single cells and
expanded for a total of 28–35 generations. The median mutation rate
(mutation frequency/cell generation) of clones carrying the
[TC]17 vector (13 x 10-6) was significantly different from that of
the pND123 parent vector (P = 0.01,
Mann-Whitney test; Fig. 2
). However, the median mutation rates of the clones carrying [GT]
vectors did not vary >2-fold from that of those carrying the parent
vector.
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DISCUSSION |
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. This result is
consistent with the increased frequency of microsatellite changes with
increasing allele length observed for GT/CA loci in yeast
(31)
. Analyses of TC/AG vector-containing human cell
populations revealed that the frequencies of mutations specifically at
the TC/AG loci were 2–4-fold greater than those of the E.
coli background (Table 3)
. Interestingly, the HSV-tk
coding region mutation frequencies for all STR-containing vectors
replicated in human cells were elevated 10–20-fold above those of the
E. coli background (Table 3)
. Thus, in this episomal vector
system, the simple presence of the microsatellite motifs increased the
mutation frequency at downstream unique coding sequences in human
cells, an effect not observed in E. coli.
Our studies demonstrate a differential mutability of TC/AG and GT/CA
microsatellites in human cells. Perhaps the most compelling evidence
suggesting that the TC motifs are less stable than the GT motifs is
shown by combining data from both [TC]11 and
both [GT]10 vectors (Table 2)
and comparing the
proportions of mutations in the STR versus coding regions.
Such a comparison reveals an extremely significant
(P = 0.0002, Fisher’s exact test) difference
between these vectors after replication in human cells, with
proportionally more mutations occurring in the STR motif for the
[TC]11 vectors (68%) than for the
[GT]10 vectors (24%). This difference was not
observed between the TC/AG and GT/CA loci during propagation in
E. coli. Moreover, clonal analyses of mutation rates
revealed significantly different mutation rates for cells carrying the
[TC]17 vector but not the
[GT]16 vector, relative to the control cells
(Fig. 2)
. Because both microsatellite sequences were inserted at the
same position in the HSV-tk gene, the sequence context
surrounding these STR loci is similar (Fig. 1)
, and therefore the
observed instability differences are most likely attributable to the
composition of the microsatellites.
The favored mechanism to explain alterations in microsatellite allele size is slipped strand mispairing between repeat units during replicative or repair DNA synthesis (32, 33, 34) . The observed differential mutagenesis between TC/AG and GT/CA sequences may result from different probabilities of either utilization of misaligned intermediates by DNA polymerases or escape of the misaligned intermediate from DNA repair proteins (34) . Although TC/AG and GT/CA motifs are of identical G·C content, poly TC/AG repeats can form triplex DNA, whereas poly GT/CA repeats can form Z-DNA (20) . Although [TC/AG]12 motifs in plasmid DNA can form triplexes in E. coli (35) , whether these secondary DNA structures are formed within our episomal vector in human cells remains to be tested. The TC/AG motifs also are asymmetric in that one DNA strand is composed of polypurine residues and the other of polypyrimidine residues. In contrast, GT/CA motifs are symmetric in that both strands are composed of alternating purine-pyrimidine residues. Any of these structural differences in DNA may increase the probability of misaligned intermediates forming in poly TC/AG tracts, relative to poly GT tracts, during DNA synthesis (36 , 37) . Alternatively, mutational intermediates formed in TC/AG microsatellites may be repaired less efficiently than those in GT/CA sequences. Although mismatch repair proteins have been shown to recognize and remove loops in GT sequences (31 , 38, 39, 40) , the ability of TC loops to serve as substrates has not been examined. However, mismatch repair studies in E. coli indicate that misaligned TC/AG and GT/CA intermediates are repaired similarly.4 Finally, formation of triplex DNA has been shown to cause an increased level of mutagenesis that is dependent upon a functional nucleotide excision repair pathway (41) .
Other model systems have examined the mutation rate of GT/CA sequences
in eukaryotic cells. In a wild-type yeast strain, an overall mutation
rate of 1.6 x 10-5 events/cell
division was reported for a vector containing a
[GT]16.5 repeat (38
, 39) , a value
similar to the median mutation rate of the
[GT]16 vector in our system (5.2 x 10-6 mutations/cell generation; Fig. 2
).
Also in yeast, 73% of mutants that were obtained from replication of a
vector containing a [GT]7.5 sequence had no
alterations within the STR locus (31)
. We obtained similar
results for the [GT]10 vector replicated in
human cells in which 76% of mutants had no changes in STR allele
lengths (Table 2)
. Mutagenesis at GT/CA sequences has been analyzed
previously in normal human fibroblasts (13)
. In that
system, the mutation rate at a [GT]17 sequence
in an integrated vector was <3.1 to 45 x 10-8 mutants/cell/generation. Because this
system detected only one of three STR frameshifts, an upper limit was
estimated to be as high as 2.7 x 10-6 mutants/cell/generation. In our analyses,
we measured HSV-tk mutation rates ranging from 2.8 to
12 x 10-6 mutations/cell
generation for a [GT]16 episomal vector (Fig. 2)
. However, as demonstrated by our DNA sequence analyses of
lymphoblastoid cell populations bearing the
[GT]10 vectors, a large proportion of
inactivating mutations are HSV-tk rearrangements and point
mutations and thus do not involve STR allele length changes. In fact,
detailed sequence analyses of lymphoblastoid cell clones carrying the
[TC]11 vector revealed that the mutation rate
specifically at the STR locus ranged from only 0.59 to 5.2 x 10-6 mutations/cell generation, whereas
the rate of all HSV-tk mutations ranged from 1.3 to
80 x 10-6. Despite the
differences in mutational detection, cell type, and vector construction
between this and the previous study, our results corroborate the view
that the inherent mutation rate of STR sequences <20 units in length
is low (
10-6) in nontumorigenic human cells.
Furthermore, for the [TC]11 clones in our
system, the median mutation rate at the STR locus was 2 x 10-6 mutations/cell generation, quite
similar to the median mutation rate at the HSV-tk coding
locus (0.47 x 10-6).
In conclusion, we describe a facile system to measure quantitative
mutation frequencies at microsatellites of varying DNA sequence. Our
system has been designed to compare mutagenesis at various
microsatellite loci to that at the coding region within the same target
gene. Our mutational target has been incorporated into an EBV-derived
oriP shuttle vector that is replicated episomally in EBV-transformed or
EBNA-1- expressing human cell lines (42)
, thus eliminating
variability attributable to random vector integration into the genome
and allowing direct comparison of multiple cell lines. We have deduced
somatic mutation frequencies and specificities at the two most abundant
dinucleotide microsatellite loci. The utility of any microsatellite
marker for molecular analyses of genetic alterations in tumor cells is
ultimately dependent upon its inherent mutation rate in normal cells,
for this determines the degree of marker polymorphism in the human
population as well as the baseline frequency of alterations in allele
length. The intrinsic low rates of somatic mutation at microsatellites
that we and others have measured can be used to estimate the
probability of false-positive results in clinical samples that contain
a vast excess of normal cell DNA over tumor DNA, thus allowing more
precise determinations of test specificity (2)
.
Additionally, our system can be used to clarify issues related to the
distinction of low frequency MSI tumors (those that show MSI at one of
five microsatellite loci examined) versus
microsatellite-stable tumors (those that show MSI at zero of five loci)
at various types of microsatellite motifs (4
, 43)
.
The demonstration that TC/AG loci are equally or slightly more mutable
than GT/CA loci can be extrapolated to mean that TC/AG markers will be
as informative as GT/CA markers in genomic studies. On the basis of the
relative abundance of these two dinucleotide sequences (16
, 17)
, the inclusion of TC/AG loci in human genomics may increase
the number of available markers by 40%.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the W. W. Smith Charitable Trust
Foundation and NIH Grant CA 73649. 
2 To whom requests for reprints should be
addressed, at The Jake Gittlen Cancer Research Institute, Milton S.
Hershey Medical Center, P. O. Box 850, Hershey, PA 17033.
3 The abbreviations used are: STR, short tandem
repeat; MSI, microsatellite instability; HSV-tk, herpes
simplex virus thymidine kinase; FUdR, 5-fluoro-2'-deoxyuridine.
4 K. Eckert and G. Yan. Mutational analyses of
dinucleotide and tetranucleotide microsatellites in E. coli:
influence of sequence on expansion mutagenesis, submitted for
publication.
Received 6/25/99. Accepted 2/ 3/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ray-induced loss of expression of HLA and glyoxalase I allele in lymphoblastoid cells. Proc. Natl. Acad. Sci. USA, 77: 4251-4255, 1980.This article has been cited by other articles:
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  S. N. Shah and K. A. Eckert Human Postmeiotic Segregation 2 Exhibits Biased Repair at Tetranucleotide Microsatellite Sequences Cancer Res., February 1, 2009; 69(3): 1143 - 1149. [Abstract] [Full Text] [PDF]
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 A. L. Seyfert, M. E. A. Cristescu, L. Frisse, S. Schaack, W. K. Thomas, and M. Lynch The Rate and Spectrum of Microsatellite Mutation in Caenorhabditis elegans and Daphnia pulex Genetics, April 1, 2008; 178(4): 2113 - 2121. [Abstract] [Full Text] [PDF]
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 Y. D. Kelkar, S. Tyekucheva, F. Chiaromonte, and K. D. Makova The genome-wide determinants of human and chimpanzee microsatellite evolution Genome Res., January 1, 2008; 18(1): 30 - 38. [Abstract] [Full Text] [PDF]
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O. Paun and E. Horandl Evolution of Hypervariable Microsatellites in Apomictic Polyploid Lineages of Ranunculus carpaticola: Directional Bias at Dinucleotide Loci Genetics, September 1, 2006; 174(1): 387 - 398. [Abstract] [Full Text] [PDF]
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V. V. Symonds and A. M. Lloyd An Analysis of Microsatellite Loci in Arabidopsis thaliana: Mutational Dynamics and Application Genetics, November 1, 2003; 165(3): 1475 - 1488. [Abstract] [Full Text] [PDF]
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 K. A. Eckert and G. Yan Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis Nucleic Acids Res., July 15, 2000; 28(14): 2831 - 2838. [Abstract] [Full Text] [PDF]
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) or by
limiting dilution (
) and expanded for 28–35 generations. Shuttle
vector DNA was isolated from each clone, and the HSV-tk
mutation frequency was determined. The mutation rate was calculated as
the mutation frequency of that clone divided by the number of
generations the clone had undergone at the time of DNA isolation. The
negative control vector used in this experiment was the original parent
vector, pND123. N, number of independent clones.