From the Department of Biochemistry, University of
Virginia, Charlottesville, Virginia 22908 and the
§ Department of Chemistry, University of Oklahoma,
Norman, Oklahoma 73019
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ABSTRACT |
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A partial physical map has been constructed of
the human class Mu glutathione S-transferase genes on
chromosome 1p13.3. The glutathione S-transferase genes in
this cluster are spaced about 20 kilobase pairs (kb) apart, and
arranged as
5-GSTM4-GSTM2-GSTM1-GSTM5-3
. This map has been used to localize the end points of the polymorphic GSTM1 deletion. The left repeated region is 5 kb downstream
from the 3
-end of the GSTM2 gene and 5 kb upstream from
the beginning of the GSTM1 gene; the right repeated region
is 5 kb downstream from the 3
-end of the GSTM1 and 10 kb
upstream from the 5
-end of the GSTM5 gene. The
GSTM1-0 deletion produces a novel 7.4-kb HindIII fragment with the loss of 10.3- and 11.4-kb
HindIII fragments. The same novel fragment was seen in 13 unrelated individuals (20 null alleles), suggesting that most
GSTM1-0 deletions involve recombinations between the same
two regions. We have cloned and sequenced the deletion junction that is
produced at the GSTM1-null locus; the 5
- and 3
-flanking
regions are more than 99% identical to each other and to the deletion
junction sequence over 2.3 kb. Because of the high sequence identity
between the left repeat, right repeat, and deletion junction regions,
the crossing over cannot be localized within the 2.3-kb region. The
2.3-kb repeated region contains a reverse class IV Alu repetitive
element near one end of the repeat.
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INTRODUCTION |
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The glutathione S-transferases (EC 2.5.1.18) are a superfamily of catalytic and binding proteins that detoxify chemical carcinogens (1). Based primarily on protein sequence similarity, the soluble glutathione S-transferases have been divided into four protein families: classes Alpha, Mu, Pi, and Theta. Class Alpha and Mu families typically contain four or more members in mammalian genomes. In humans, a large fraction of the population carries polymorphic deletions for the class Mu GSTM1 gene (about 50% of the population is homozygous GSTM1-null; Ref. 2) and the class Theta GSTT1 gene (40% of the population is homozygous GSTT1-null; Ref. 3).
Significant associations between the GSTM1-0 deletion and increased cancer incidence in case/control studies have been reported for lung (4-6), bladder (7, 8), and skin cancer (9-11). Both positive and negative results have been reported for associations between GSTM1-0 and cancer risk. A large study (6) on the effect of the GSTM1 deletion and lung cancer among caucasians and African Americans in southern California suggests that, in these populations at least, the association of the GSTM1-0 deletion and cancer is not strong. London et al. (6) found a significant association between lung cancer and the GSTM1 deletion only in cancer patients with a history of smoking but who had smoked less than 40 pack-years (e.g. 2 packs/day for 20 years) (odds ratio = 1.77, 95% CI1 = 1.11-2.82). After regression to remove age, sex, and smoking history effects, no significant association was found for total lung cancer cases or for cases where the patient had smoked more than 40 pack-years. These authors reviewed the literature on lung cancer and the GSTM1 deletion and argue that while an association may be present, it is not strong for caucasian and African American populations. However, a meta-analysis by McWilliams et al. (12) comes to the opposite conclusion. They examined 12 case/control studies with a total of 1593 cases and 2135 controls and concluded that GSTM1-0 is a moderate risk factor with an odds ratio of 1.41 (95% CI = 1.23-1.61), accounting for about 17% of lung cancer cases (12).
Although there is a substantial literature on epidemiological
associations between the GSTM1-0 deletion and cancer, very
little is known about the structure of the deletion. We do not know the size of the deletion or whether other expressed genes are lost. Likewise, the mechanism of the GSTM1-0 deletion is unknown.
In this paper, we present a physical map of four of the five class Mu
genes in the GSTM cluster on chromosome 1 and show that the GSTM1-0 deletion apparently results from homologous unequal
crossing over between two highly identical regions that flank the
GSTM1 gene, resulting in a 15-kb deletion that contains the
entire GSTM1 gene. The GSTM1 gene and 5- and
3
-flanking regions are excised relatively precisely, leaving the
flanking GSTM4, GSTM2, GSTM5, and
GSTM3 genes intact. There is no change in at least 5 kb 3
to the 5
-flanking GSTM2 gene or in at least 10 kb 5
- to
the 3
-flanking GSTM5 gene. In addition, the same deletion
has occurred in all of the deleted alleles examined. Identification of
the GSTM1-0 recombination region provides a hybridization
probe that can be used to distinguish GSTM1+/
heterozygotes from GSTM1+/+ homozygotes.
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EXPERIMENTAL PROCEDURES |
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Reagents--
[-35S]dATP,
[
-32P]dCTP and [
-32P]ATP were
obtained from ICN Biochemicals. Restriction enzymes were obtained
either from Life Technologies, Inc. or New England Biolabs. T4 DNA
ligase and
-HindIII molecular size markers were obtained
from Life Technologies. T4 polynucleotide kinase and DNA size markers
for pulse field gel electrophoresis (Mid-range PFG Marker II) were
purchased from New England Biolabs. DNA Taq polymerase and
Klenow fragment were obtained from Promega. The Sequenase version 2.0 kit was obtained from Amersham. Zymolase-100T was purchased from ICN
Biochemicals. Zetabind blotting nylon membrane was obtained from Cuno
(Meriden, CT). pEMBL 18+ cloning vector was purchased from Boehringer
Mannheim. Escherichia coli-competent cells (SURE strain) and
reagents for constructing a cosmid library from GSTM-YAC2 (pWE15, T4
ligase, Gigapack II XL packaging extract) were purchased from
Stratagene. T3 and T7 oligonucleotides were also obtained from
Stratagene. Nitrocellulose membranes for colony screening were obtained
from Schleicher & Schuell. COT-1TM repetitive blocking DNA
was obtained from Life Technologies.
Genomic DNA Purification-- Human genomic DNA was isolated from white blood cells as in Refs. 13 and 14. Yeast and cosmid DNAs were prepared using standard methods (13, 14).
Southern Blotting-- Southern blotting was done as described (13) with slight modifications. Restriction enzyme digestions were performed as suggested by the manufacturers. Restriction enzyme-digested DNA samples were electrophoresed on a 0.6% agarose gel. For DNA transfer, the gel was treated with 0.25 M HCl for 10 min and then denatured with 0.5 M NaOH, 1.5 M NaCl for 1 h and neutralized with 0.5 M Tris-HCl (pH 8.0), 1.5 M NaCl for 1 h. DNA was transferred in 10 × SSC overnight to Zetabind (AMF-Cuno) and fixed to the membrane by UV irradiation.
Southern blots to nylon membranes were hybridized with random primer-labeled probe at 65 °C in 0.5 M sodium phosphate buffer and washed at 65 °C in 40 mM sodium phosphate buffer according to Church and Gilbert (15). Hybridization probes containing repeated DNA sequences (probes P1 and P2; Fig. 4) were prehybridized with human COT-1 DNA according to the manufacturer's instructions. A probe/COT-1 DNA mixture was boiled for 5 min, incubated at 65 °C for 1 h, and then added to the hybridization bag. The hybridization was allowed to proceed at 65 °C for 48 h, and the membrane was washed as usual (15).Polymerase Chain Reaction (PCR) Amplification and PCR Cloning-- Conventional PCRs were performed in 100 µl of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, a 200 mM concentration of each dNTP, a 1 mM concentration of each primer, and 3.5 units of Taq polymerase for 35 cycles of 1 min at 94 °C, 2 min at 55 °C, and 3 min at 72 °C. The final cycle was followed by incubation at 72 °C for another 10 min. PCR products to be cloned were treated with Klenow fragment to produce flush ends.
Cosmid Library Construction-- High molecular weight GSTM-YAC2 yeast genomic DNA was isolated as described by Guthrie et al. (16). High molecular weight chromosomal DNA was size-fractionated on a preparative sucrose gradient (5-20% sucrose, 20 mM Tris-HCl, pH 8.0, 20 mM Na2EDTA, pH 8.0, 0.2 M NaCl, and 0.1% Sarkosyl). Fractions were analyzed by electrophoresis on a 0.8% agarose gel; fractions with very little low molecular weight DNA were pooled, dialyzed, and concentrated by CsCl density gradient centrifugation. Fractions from the CsCl gradient were collected, a portion was electrophoresed on a 0.8% agarose gel, and fractions containing only high molecular weight DNA were used for SauIIIA partial digestion. SauIIIA partially digested yeast DNA was size-fractionated on a 5-25% NaCl gradient, and fractions containing DNA enriched in sizes between 35-45 kb were cloned into BamHI-cut pWE15 vector. Packaging and infection was performed using the Gigapack II XL kit as recommended by the supplier.
Cosmid Library Screening--
The resulting GSTM-YAC2 cosmid
library was screened as in Ref. 13. The hybridization probe was the
insert from the PCR-generated clone containing the 3-immediate
flanking sequence of GSTM2. This probe cross-hybridizes with
the 3
-flanking region of GSTM1. Colonies that displayed
positive signals on both replica membranes were picked up from the
master plates and screened a second time at a density of 50 colonies/10-cm membrane. Duplicate replica membranes were made, and
colony hybridization was undertaken as before. Single positive colonies
were grown for cosmid DNA minipreparations (13) through two more cycles
before final storage.
Cosmid Restriction Mapping--
T3 and T7 oligonucleotides
(Stratagene) were end-labeled with T4 polynucleotide kinase as
described (13). Twenty pmol of each oligonucleotide was used in each
labeling reaction. After labeling, the oligonucleotides were purified
by ethanol precipitation once and then dissolved in TE (pH 8.0). Cosmid
mapping was performed according to the procedure provided in the manual
of Stratagene's FLASH Nonradioactive Gene Mapping kit (except that
-32P-end-labeled radioactive T3 and T7 oligonucleotides
were used as probes). The cosmid DNA was prepared using QIAGEN's
Plasmid Mega kit. For obtaining highest quality of DNA, an extra
ethanol precipitation step was employed to reduce the salt
concentration of the DNA preparation. Partial EcoRI and
HindIII digestions of cosmid DNAs were separated on 0.4%
agarose gels.
Cloning of a Deletion Junction Fragment--
A GeneAmp XL PCR
kit (Perkin-Elmer) was used to amplify a 7.4-kb HindIII
junction fragment with the following primers: primer 1, 5-CCTGACCTTCCTTCCTGTTAGTGGT-3
; and primer 2, 5
-GATGTCCCAGTACCCCAGAGTCATG-3
. Primer 1 anneals to the 3
-end of
GSTM2; primer 2 anneals to the 5
-end of GSTM5.
Long range PCR reactions were performed in a 100-µl solution of
1 × buffer (supplied with the PCR kit), a 200 µM
concentration of each dNTP, 40 pmol of each primer, 1.5 mM of Mg(OAc)2, 1 µg of DNA template and 4 units of enzyme.
A "hotstart" protocol with AmpliwaxTM was used to
increase the specificity of the reaction, which was cycled in order for
1 min at 94 °C, 16 cycles of 30 s at 94 °C, 13 min at
64 °C, 12 cycles of 30 s at 94 °C, 13 min at 64 °C with a
cycle extension of 15 s/cycle, and 10 min at 72 °C. PCR products were phenol-extracted, cut with HindIII, and separated on a
low melting agarose gel. The 7.4-kb fragment was recovered and
purified. The 7.4-kb fragment was then ligated to pGEM-7Zf vector and
used to transform SURE-competent cells. Plasmid DNA was purified with a
QIAGEN Plasmid Maxi kit; an extra ethanol precipitation step was used
to reduce the salt concentration for DNA sequencing.
DNA Sequencing and Sequence Assembly-- Subcloned fragments were either sequenced manually using a Taq Cycle Sequencing kit (Amersham) or were sequenced on an ABI PRISM 377 sequencer with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit at the University of Virginia Sequencing Center. Sequence data were further complemented and confirmed at the Human Genome Center at University of Oklahoma. At the University of Oklahoma, cosmid DNAs were isolated free from E. coli host contamination by the cleared lysate, diatomacious earth-based procedure describe earlier (17) and sequenced to a level of 4.5-fold redundancy via the previously described, double-stranded, shotgun-based approach (18) using the ABI PRISM fluorescence-labeled terminators and either forward or reverse universal primers. Sequencing vector regions were removed, and the resulting data were assembled into contiguous fragments initially using the TED and XGAP programs (19) and more recently using the Phred, Phrap, and Consed programs.2 The individual contigs were joined into a final, unique sequence using custom, synthetic primers and Taq DNA polymerase cycle sequencing with fluorescent terminators. Each base was sequenced at least three times. Repeated sequence elements were identified using the RepeatMasker2 program.3
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RESULTS |
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The Structure of the Human Class Mu Glutathione S-Transferase Gene Cluster-- In an earlier paper (14), we reported the identification of three yeast artificial chromosome (YAC) clones that contain human class Mu GST genes. Locus-specific PCR primers were used to show that GSTM-YAC1 contains GSTM1, GSTM3, and GSTM5; GSTM-YAC2 contains all five members of the class Mu family; and GSTM-YAC3 contains GSTM2 and GSTM4. The observation that all five class Mu GST genes are contained within the GSTM-YAC2 clone indicated that all five human class Mu GST genes are located on a single chromosome within the 600-kb insert. Fluorescent in situ hybridization was used to map GSTM-YAC2, and thus the human class Mu GST gene cluster, to chromosome 1p13.3 (14).
As a first step toward mapping the end points of the GSTM1 deletion in humans, we compared the organization of the class Mu glutathione S-transferase genes in GSTM-YAC1, -YAC2, and -YAC3 to their organization in human genomic DNA. Because two of the three YAC clones (GSTM-YAC1 and GSTM-YAC3) contain only a portion of the GSTM2 gene cluster, they can be used to determine the order and orientation of the genes. We first compared the sizes of HindIII and EcoRI restriction fragments containing class Mu glutathione S-transferase genes that cross-hybridized with the GSTM1 cDNA clone GTH411 (22) in GSTM-YAC1, -YAC2, and -YAC3 to restriction fragments in human DNAs. Fig. 1A shows the pattern of hybridization seen with HindIII digests of the YAC and human DNAs; genomic DNA from two individuals, one carrying a non-null GSTM1 allele and one homozygous for the GSTM1-0 deletion are shown. The GTH411 cDNA probe cross-hybridizes to GSTM4, GSTM2, GSTM5, as well as GSTM1, but GTH411 does not cross-hybridize efficiently to GSTM3 under the conditions used (data not shown). The pattern of hybridization in the GSTM-YAC2 lane is identical to the pattern in the GSTM1+/
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A Physical Map of the Class Mu Glutathione S-Transferase
Cluster--
To examine the deletion end points in the intragenic
regions flanking GSTM1, we constructed a cosmid library from
the GSTM-YAC2 clone. The cosmid library contains about 3 × 105 independent colonies with an average insert size of
about 35 kb, equivalent to about 700-fold coverage of a haploid yeast
genome. The library was screened initially with a probe hybridizing
with the 3-sequence immediately flanking both GSTM2 and
GSTM1. Positive clones were confirmed by hybridization of
the GTH411 GSTM1 cDNA clone to HindIII and
EcoRI digests. Two overlapping clones, cGTM1 (38 kb) and
cGTM12 (36 kb), were used for the subsequent mapping of the gene
cluster.
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Localization of the Break Points of the GSTM1 Deletion--
The
physical map of the GSTM gene cluster and cosmid clones
cGTM1 and cGTM12 provided the reagents necessary to map the ends of the
GSTM1 deletion. To localize the break points of the
GSTM1 deletion, the sequences that normally flank
GSTM1 were examined in DNA from a GSTM1/
individual using hybridization probes from sequences flanking the
GSTM1 (Fig. 4). To locate the
5
-boundary of GSTM1 deletion, a 9-kb fragment (P1; Fig.
4E) from cosmid cGTM1 covering the 5
-immediate flanking
sequence of GSTM1 was used as a probe (Fig. 4A).
This fragment contains repetitive DNA sequence elements, so the
hybridization was done in the presence of human COT-1-blocking DNA.
Comparison of the hybridization patterns of the EcoRI
fragments from the GSTM1+/
individual and from the GSTM1
/
individual indicates that the
GSTM1
/
individual lacks 5
-flanking 3.5- and 1.8-kb
EcoRI fragments, although the 1.8-kb fragment is difficult
to see in the reproduction. This result suggests that the 5
-break
point is either within the 1.8-kb fragment or farther upstream.
However, since the 5.2-kb HindIII band containing the 3
-end
of GSTM2 is intact in the GSTM1
/
individual
(Fig. 1A, Fig. 2), the 5
-break point was localized to a
3.4-kb region consisting of a 1.6-kb
HindIII-EcoRI fragment and the 1.8-kb
EcoRI fragment.
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The Sequence of the GSTM1 Deletion Junction Region--
To confirm
the homologous recombination model and to locate the deletion end
points, we sequenced the left and right junction segments predicted by
the restriction mapping to be involved in the homologous recombination.
The sequence of the left junction region (Fig.
6) was determined from subclones of cGTM1
and thus cannot contain sequences from the right recombination region. Sequences for the right recombination region were obtained from subclones of the 11.4-kb HindIII fragment from the
3-flanking region of cGTM12 and thus cannot be contaminated with
sequence from the 5
-flanking region of GSTM1. To locate the
deletion break points, we also cloned and sequenced a portion of 7.4-kb
HindIII deletion junction fragment from the
GSTM1
/
individual shown in Fig. 4.
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DISCUSSION |
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We have determined a physical map of the GSTM1, GSTM2, GSTM4, and GSTM5 class Mu glutathione S-transferase genes on human chromosome 1p13.3 and have identified the left and right junction regions involved in the unequal crossing over event that produces the GSTM1-0 deletion. The human class Mu GST gene cluster contains two almost identical 4.2-kb regions that flank the GSTM1 gene; the GSTM1-0 deletion is caused by a homologous recombination involving the left and right 4.2-kb repeats. However, extensive sequence identity between the left and right repeats conceals the exact break points within a 2.3-kb region (Figs. 6 and 7). Sequences of deletion junctions from additional individuals may narrow the zone. Most GSTM1 deletions appear to be caused by the same homologous recombination, since all 20 null allele chromosomes (3 in Fig. 4 and 17 in Fig. 5) that we examined have the same 7.4-kb HindIII junction fragment.
Gene deletion by homologous unequal crossing over is now well documented (29-31), including a deletion of a member of the cytochrome P450 detoxification gene family (32). Striking features of the GSTM1 deletion are its high frequency in the population and the apparent homogeneity of the recombination region. These features may reflect an ancient acquisition of the deletion, which has been retained at a high frequency in human populations. Alternatively, the 4.2-kb repeat region may be a "hot spot" for unequal crossing over, so that the GSTM1-0 deletion has arisen independently many times. In either case, it is worthwhile to consider the evolutionary history of the sequences involved in the recombination.
Our data suggest that a >8-kb region from the 3-end of
GSTM2 to the 3
-end of the left 4.2-kb repeat is 90-99%
identical to the corresponding region flanking the 3
-end of
GSTM1. This extensive sequence conservation suggests that
the two 4.2-kb repeats probably arose with the original gene
duplication process in the formation of the human class Mu GST gene
cluster, which probably occurred more than 20 million years ago (23).
If the 4.2-kb repeats arose at that time, then their high sequence
identity is not the result of a very recent sequence duplication. The
regions that flank the left and right repeat share about 92% sequence identity, as opposed to >99% identity within the two repeats (Fig. 7). The 8% sequence divergence in the regions flanking the repeats (i.e. 4% divergence of each region from the duplicated
ancestral sequence) suggests that the surrounding region was duplicated about 30 million years ago (4%/0.15%, assuming a divergence drift rate of 0.15% per million years for noncoding regions; Ref. 33). Thus,
the class Mu gene cluster may have been duplicated or rearranged between the divergence of Old World monkeys and apes about 25 million
years ago and New World monkeys and apes about 40 million years
ago.
We searched the GenBankTM DNA and expressed sequence tag
data bases and various protein data bases for possible coding sequences from the 4.2-kb repeated region that might account for its high sequence conservation. In addition to the repetitive elements present
in the 4.2-kb repeat, a search of the GenBankTM (release
102, July 1997) expressed sequence tag data base identified two
overlapping sequences (accession numbers H57626 (490 nucleotides) and
R93679 (497 nucleotides)) that share strong similarity (87% identity
over 497 nucleotides, FASTA E() <1078; Ref. 34) with a
sequence in the 4.2-kb repeat unit. This similarity corresponds to part
of a noncoding alternatively spliced GSTM4 gene exon 9 (35)
and may represent transcripts from this region. We did not detect any
significant matches between these cDNA sequences and any proteins
in the SwissProt (release 34) or OWL (release 29.3) protein data
bases.
If the repeat region does not carry a protein-coding sequence, then the extremely high sequence conservation between the left and the right repeats is the result of gene conversion (36) rather than selective pressure. Gene conversion within the human class Mu glutathione S-transferase gene cluster has been postulated previously (27).
The three Alu elements from the repeat/junction fragments can be used to estimate the age of the class Mu gene cluster and of the most recent gene conversion event. Alu repeats have been divided into six subclasses based on diagnostic substitutions that are shared within each class (37-39). Members of subclasses are descendants of the same source gene. Subclasses have different genetic ages, suggesting that source genes responsible for each subclass were active at different periods during primate evolution. The Alu sequences in the 2.3-kb cross-over region contain characteristic subclass IV-specific nucleotide substitutions at several diagnostic positions (Ref. 37, Fig. 8; referred to as subclass Y in Ref. 40). Among those diagnostic positions, all three Alu elements match the consensus sequence at all but two positions; those two mismatched positions are at highly mutable CpG sites. Subclass IV elements represent about 25% of all of Alu sequences in human genome (37, 38); the majority of these sequences diverge from the subclass IV consensus at 3-4.5% of non-CpG sites. Thus, this subclass was inserted into the genome about 20-30 million years ago (3-4.5%/0.15%/million years; Ref. 37).
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Nineteen nucleotide substitutions (nine non-CpG) and one insertion are shared by the three Alu elements from the left repeat, right repeat, and deletion junction regions with respect to the subclass IV consensus sequence (Fig. 8). Among the three Alu elements, the left and right repeats share 12 differences from the consensus sequence at non-CpG sites, while the junction fragment Alu has 11 differences from the consensus sequence at non-CpG sites. This is consistent with about 35 million years of divergence (12/231/0.15%) from the original insertion events, which implies that the Alu elements in the 2.3-kb repeats were inserted into the genome about 35 million years ago. All but 2 of the 12 non-CpG differences are shared among all three Alu elements, suggesting that a gene conversion event homogenized these Alu sequences as little as 5 million years ago. Multiple gene conversions may have predated the most recent gene conversion event; we cannot determine whether the 10 shared differences were the result of one conversion or accrued from multiple gene conversions. Consistent with the Alu divergence data, the total sequence divergence of less than 1% between the left and right repeats suggests that the last gene conversion in the 4.2-kb region occurred probably no more than about 3 million years ago (1%/2/0.15%).
Given the high frequency of the GSTM1 deletion in human populations and the evidence that gene conversion event(s) have occurred in the 4.2-kb repeat regions, we speculate that this region is a hot spot for homologous recombination. A striking feature of the two 4.2-kb repeats is their near total identity over such a long segment. Homologous recombination frequency has been shown by numerous studies to be related to the length of homology involved. Studies done in mammalian cells using a plasmid-plasmid recombination system (41, 42), as well as recombination between chromosomally inserted plasmids and native chromosome genes (20, 43), have demonstrated that 200 base pairs of uninterrupted identity is required for efficient recombination in mammalian cells, with mismatches reducing recombination efficiency. The recombining regions flanking the GSTM1 gene are considerably longer.
The GSTM1 deletion is very common in human populations, with
about 50% of the individuals having the GSTM1/
genotype, and 45% having the GSTM1+/
genotype, although
the actual numbers vary somewhat in different ethnic groups (2, 21).
Although our study so far has only examined 13 caucasian DNA samples,
we believe the GSTM1 deletions in people from other ethnic
backgrounds are also caused by the same homologous recombination. If
the left and right 4.2-kb repeats are a recombination hot spot, then
the high frequency of the GSTM1 deletion may reflect
multiple independent occurrences of the same homologous recombination.
Alternatively, if the GSTM1-0 deletion is not strongly
selected against, its high frequency may be the result of an ancient
ancestral deletion. Examination of the class Mu GST cluster in other
primates may clarify the evolutionary history of this gene cluster and
the GSTM1 deletion.
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ACKNOWLEDGEMENT |
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We thank Gina Calabrese for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant CN-27D (to W. R. P.) and National Human Genome Research Institute Grant R01 HG00313 (to B.A.R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Box 440 Jordan Hall, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-2818; Fax: 804-924-5069; E-mail: wrp{at}virginia.edu.
1 The abbreviations used are: CI, confidence interval; kb, kilobase pair(s); PCR, polymerase chain reaction; YAC, yeast artificial chromosomes; GST, glutathione S-transferase.
2 B. Ewing, D. Gordon, and P. Green, World Wide Web URL http://www.genome.washington.edu/UWGC/ and personal communication.
3 A. F. A. Smit and P. Green, Internet URL http://ftp.genome.washington.edu/RM/RepeatMasker.html and personal communication.
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REFERENCES |
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