Vol.
177,
No.
June
28,
1991
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1252-1257
Pages
STRUCTURE AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF GENES FOR HUMAN LIVER ARYLAMINE N-ACETYLTRANSFERASES Takashi
Department Institute
for
EBISAWA and Takeo DEGUCHI
of Molecular Neurosciences,
Neurobiology, Tokyo Metropolitan 2-6 Musashidai Fuchu-city Tokyo
183
Japan Received May 18, 1991 SUMMARY: Genomic DNA clones coding for polymorphic and (NAT) of human monomorphic arylamine N-acetyltransferases and their liver were isolated from a genomic DNA library, were restriction maps and partial nucleotide sequences determined. Messenger RNA for monomorphic NAT was coded in one exon, while mR.NA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPS of polymorphic NAT gene were observed in coding and 3'flanking region upon digestion with BamHI and KpnI. Q 1991 Academic
Pres*,
Inc.
N-Acetylation polymorphism the toxicity of amine-containing to certain erythematosus
diseases (1,
in the liver has been implicated drugs a;ld in the susceptibility
such as bladder 2).
cancer
The slow acetylator
be inherited via autosomal recessive isolated and identified cDNA clones
We have
further
phenotype
lupus
was shown to
genes (3). Recently, for monomorphic and
polymorphic NATs from human liver (4). cDNAs coding for polymorphic NAT which NAT activity.
and systemic
we have
There are two types of correspond to high and low
demonstrated
a correlation
between
the genotypes of polymorphic NAT and the acetylator phenotypes, and have found indications that there are three types of Abbreviations: NAT, N-acetyltransferase; fragment length polymorphism. 0006-291X/91 Copwight All rights
$1.50 1991 by Academic Press. of reproduction in any form 0
Inc. reserved.
1252
in
RFLP, restriction
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
corresponds polymorphic NAT gene, one of which activity with the other two genes giving rise activity (5). been performed
RFLP analysis in detail.
of
In this report, we have and monomorphic NAT genes. the
nucleotide
sequence
the
structures
of
the
types
of
NAT gene
using
genes,
polymorphic
genes
were
coded in one exon, while polymorphic two exons. We have further analyzed three
three
cloned and analyzed Although there was
between two
the
to to
various
DNA as probes, and have shown that 3'-flanking regions upon digestion
MATERIALS
COMMUNICATIONS
a high a low
NAT NAT
however,
has
not
the polymorphic a high homology
in
and monomorphic
different;
NAT,
monomorphic
NAT is
NAT gene was separated the RFLP in homozygotes fragments
of
cloned
into of genomic
RFLPs are found in coding with BamHI and KpnI.
and
AND METHODS
Cloning of human N-acetyltransferase genes: A human genomic DNA library constructed in EMBL3 was obtained from the Japanese Cancer Research Resources Gene Bank and screened with 32P-labeled cDNAs for the polymorphic and monomorphic NATs recently cloned in this laboratory (4). Phage DNAs were transferred to nitrocellulose filters which were prehybridized in a solution containing 20% formamide, 5 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, S x Denhardt's solution (1 x Denhardt's = 0.02% bovine pH 7.41, serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.1% SDS, and 100 pg/ml denatured salmon sperm DNA at 42 'C for 5 h, and then hybridized in the same solution containing 32P-labeled polymorphic and monomorphic NAT cDNAs (3-4 x 105 cpm/ml of each cDNA) at 42 "C for 16 h. Filters were washed in 2 x SSC containing 0.1% SDS at 42 OC for 30 min, twice. Nucleotide digestion subcloned (6) .
Positive clones were analyzed by sequence analysis: Restriction fragments were with restriction enzymes. into plasmid pUC18 and sequenced by the dideoxy method
Genomic DNA was prepared from Genomic Southern blot analysis: leukocytes of healthy volunteers and Southern blot analysis was Briefly, DNA was digested with 40-50 performed as described (5). units of EcoRI, BamHI, or KpnI at 37 OC for 5 h, separated on a to GeneScreen 0.8% agarose gel at 25 V for 13 h, and transferred Plus filters. The filters were prehybridized in 50% formamide, 1 1% SDS, and 100 gg/ml denatured M NaCl, 10% dextran sulfate, salmon sperm DNA at 42 'C for 2 h and then hybridized in the same solution containing 5 x 105 cpm/ml of 32P-labeled genomic DNA The filters were washed in 2 X SSC fragments at 42 OC for 14 h. the solution twice, containing 1% SDS at 42 'C for 1 h by changing The genomic and exposed to an X-ray film at -80 'C for 24-36 h. using the MultiprimeTM DNA DNA fragments were 32P-labeled United Kingdom) to a specific activity labelling system (Amersham, of 3-10 x lo8 cpm/pg. 1253
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS AND DISCUSSION Screening of the amplified genomic DNA library (2 X lo6 phages) revealed 7 and 5 independent clones for polymorphic and monomorphic
NAT genes,
qenomic
DNA clone
several
stop
was
codons
respectively. also
in
the
Although
obtained,
sequence
putative
it is a pseudogene (data not shown). polymorphic and monomorphic NAT genes restriction cDNAs were
enzymes sequenced
monomorphic NAT was without interruptions. was separated in one exon
coding The
a third analysis
region, longest
were
and EcoRI fragments that (Fig. 1). The sequence
of
revealed
indicating clones of
analyzed
that the
with
hybridized with of cDNA for
detected in one of the EcoRI In contrast, the cDNA of
into two exons: the 5'-noncoding 8 kb upstream from another exon
type
fragments, polymorphic
region was which comprised
coding
and 3'-noncoding
regions.
Since
the
sequences
coding
and 3'-noncoding
regions
of both
the
polymorphic
of
the
NAT located the the
and
Fiqure 1. Sequences and alignment of human polymorphic (headed by P) and monomorphic (headed by M) NAT genes. Nucleotide residues are numbered in the 5' to 3' direction starting with the initiation codon (ATG) as number 1. The initiation codon is shown in bold letters. Sequences are aligned to maximize homology with inserted gaps represented by dashes (-). Asterisks indicate nucleotide residues identical in polymorphic and monomorphic NAT. Capital letters enclosed in boxes indicate exons and small letters denote introns.
Vol.
177,
No.
3, 1991
monomorphic sequences
NATs
were
only
the
of
BIOCHEMICAL
AND
identical
to
noncoding
regions
sequence
liver, did
cDNAs
and surrounding
(41,
the
regions
are
was 69% homology in nucleotide sequence region of monomorphic NAT and the intron
between
monomorphic
NAT, whereas there sequences of 5'-
and polymorphic
NAT cDNAs.
for
the
5'-noncoding
region.
After
NAT gene upstream this
part
was completed, Grant et. al. (7) reported the a genomic DNA clone for the polymorphic NAT of human
which
was
identical
not analyze the We have recently
polymorphic a BamHI
of the
COMMUNICATIONS
seems to indicate that the polymorphic monomorphic NAT gene and utilized the
as an exon
site,
to that
5 '-noncoding shown that
NAT gene;
BamHI site, BamHI site
RESEARCH
coding region of polymorphic homology in the nucleotide
This observation evolved from the of the study sequence of
those
5'-noncoding
shown in Fig. 1. There between the 5'-noncoding upstream of the was no significant
BIOPHYSICAL
gene
reported
exon there
here,
although
of the polymorphic are three types of
1 contains
a 5.5
kb KpnI
they NAT gene.
fragment
with
gene 2 contains a 5.5 kb KpnI fragment without a while gene 3 contains a 5.0 kb KpnI fragment with a (Fig.2).
We further
showed that
gene 1 corresponds
to
lkbps H
cd t”
Probe KE
BE
EB
BEE
E
Ii
e
BEKBE
e
Genomic fragment
El
E2
E3
-iTi--
E4
E5 CH B3
B2
E6 B4
Kl
*
7F-
K2 BEK
E
d-@Le d
Gene 2 E5
BEKBE
&@A;L
Gene 3
Fisure 2. Restriction maps of three genes (E, EcoRI; B, BamRI; K, KpnI).
types of polymorphic NAT The restriction map of NAT gene 1 was deduced from analysis of genomic DNA clones and those of genes 2 and 3 from Southern blot analysis of genomic DNAs. Hatched and open boxes indicate coding and noncoding regions of cDNAs, respectively. Thin arrows(+)) above gene maps show Probe a, Sal1 in vector-BamHI genomic fragments used as probes. fragment; b, EcoRI-BamHI fragment; c, HindIII-BamHI fragment; d,
e,
BamHI-Sal1
BarnHI-BarnHI
fragment;
arrows(*) blot analysis. restriction
show genomic The regions maps were identical
in
vector
fragments detected of gene 2 and gene to that of gene
1255
fragment.
Thick
in genomic Southern 3 in which 1 are omitted.
Vol.
177,
No.
3, 1991
BIOCHEMICAL
Probe
I
Subject No
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
3 II 1 I 2 I 3
23.1kb3 9.4kb+ 66kb+
+23.Ikb 4-J% *
+-El
4.4kb+
.,ji
2.3kb-+ 2.0kb*
Eco RI
4-ES
23.lkb+ 9.4kb+ 6.6kb-*
+B4
4.4kb+ 2.3kb-b
Barn HI
*f
1*
*
+Bl
2.0kb'
*-(c
t +
9.4kb 6.6kb
f
4.4kb
f f
23kb 2.0kh
+23.lkb + 9.4kb f-
6.6kb
+-
4.4kb
+ +
2.3kb 2.0kb
+23.Ikh f 9.4kh + 6.6kh +K3, 44kh
++
Kpn I
Figure 3. Southern blot analysis of genomic DNA. Filters were _hybridized with one of the jzP-labeled genomic fragments (a-e) shown in Fig 2. El-E6, Bl-B5, and Kl-K3 correspond to genomic fragments shown in Fig.2. Wild type k-phage DNA digested with Hind111 was used for size markers, shown on both sides in kilobases.
a high
NAT activity,
while genes 2 and 3 give rise to a low NAT activity (5). To further analyze the structures of the three NAT genes, genomic Southern blot analysis was performed with genomic DNAs from homozygotes of the three NAT genes using 32P-labeled 1256
2.3kh 2.0kb
Vol.
177,
No.
genomic
3, 1991
BIOCHEMICAL
DNA fragments
3 were structure
homozygotes of the
genomic
(probes
a-e
of genes polymorphic
DNA clones
AND
and
BIOPHYSICAL
in
Fig.
RESEARCH
2).
b, or
no RFLP in
from
genomic
subjects.
In
digests.
digests
contrast,
B3 fragment
subject B5, was
Southern
blot
subject consistent
a 5.5
from
RFLP was
observed
in
BamHI
gene 2.
fragment
NAT genes
and 3 '-flanking
in 2.
sites
has
information
taken
together
of
use
and in the acetylator
in
are
subjects
Instead,
the
investigation phenotypes.
with
identification of
Bl-
as by
digested 3). There the
was
three
and KpnI
1 and 3, but a longer
not
in
fragment,
Digestion of genomic DNA by KpnI (K2) in subjects 1 and 2, while (f(3). (5),
This which
generated
the
observation indicated
by mutations
previous of
gene
of
as El-E6,
is that in
report
genotypes
mutations
of
associated
(5)
the
the
No other obvious mutation detected so far. The present
been
and
(Fig.
regions.
restriction prove
DNAs
detected
2,
the NAT gene EcoRI digests
represent partially gene fragments (Fig.
3 showed a 5.0 kb fragment with our previous report
polymorphic
coding
kb
denoted of in
genomic
was
1,
The analysis
analysis
between
2, a homozygote of detected in subject
demonstrated
three
d, and e probably other homologous
EcoRI
Subjects
1, 2, and 3, respectively. NAT gene was deduced from
in Southern blot analysis 2) . The fragments should correspond to regions B5, and Kl-K3 shown in Fig. 2. The faint bands detected probes a, fragments
COMMUNICATIONS
of should
polymorphic
NAT
with
ACKNOWLEDGMENTS This research was supported of the Science and Technology grants from the Ministry of Japan.
in part by Special Agency of Japan, Education, Science,
Coordination Funds and by research and Culture of
REFERENCES 1. 2. 3. 4. 5.
Weber,W.W.(1984) Fed.Proc.43,2332-2337. Evans,D.A.P.(1984) J.Med.Genet.21,243-253. and Hein,D.W.(1985) Pharmacol.Rev.37,25-79. Weber,W.W., Ohsako,S., and Deguchi,T.(1990) J.Biol.Chem.265,4630-4634. Deguchi,T.,Mashimo,M., and Suzuki,T.(1990) J.Biol.Chem.265, 12757-12760. and Coulson,A.R.(1977) Proc.Natl.Acad. 6. Sanger,F.,Nicklen,S., Sci.U.S.A.74,5463-5467. and Meyer,U.A.(1989) Nucleic 7. Grant,D.M.,Blum,M.,Demierre,A., Acids Res.17,3978.
1257
177,
No.
June
28,
1991
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1252-1257
Pages
STRUCTURE AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF GENES FOR HUMAN LIVER ARYLAMINE N-ACETYLTRANSFERASES Takashi
Department Institute
for
EBISAWA and Takeo DEGUCHI
of Molecular Neurosciences,
Neurobiology, Tokyo Metropolitan 2-6 Musashidai Fuchu-city Tokyo
183
Japan Received May 18, 1991 SUMMARY: Genomic DNA clones coding for polymorphic and (NAT) of human monomorphic arylamine N-acetyltransferases and their liver were isolated from a genomic DNA library, were restriction maps and partial nucleotide sequences determined. Messenger RNA for monomorphic NAT was coded in one exon, while mR.NA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPS of polymorphic NAT gene were observed in coding and 3'flanking region upon digestion with BamHI and KpnI. Q 1991 Academic
Pres*,
Inc.
N-Acetylation polymorphism the toxicity of amine-containing to certain erythematosus
diseases (1,
in the liver has been implicated drugs a;ld in the susceptibility
such as bladder 2).
cancer
The slow acetylator
be inherited via autosomal recessive isolated and identified cDNA clones
We have
further
phenotype
lupus
was shown to
genes (3). Recently, for monomorphic and
polymorphic NATs from human liver (4). cDNAs coding for polymorphic NAT which NAT activity.
and systemic
we have
There are two types of correspond to high and low
demonstrated
a correlation
between
the genotypes of polymorphic NAT and the acetylator phenotypes, and have found indications that there are three types of Abbreviations: NAT, N-acetyltransferase; fragment length polymorphism. 0006-291X/91 Copwight All rights
$1.50 1991 by Academic Press. of reproduction in any form 0
Inc. reserved.
1252
in
RFLP, restriction
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
corresponds polymorphic NAT gene, one of which activity with the other two genes giving rise activity (5). been performed
RFLP analysis in detail.
of
In this report, we have and monomorphic NAT genes. the
nucleotide
sequence
the
structures
of
the
types
of
NAT gene
using
genes,
polymorphic
genes
were
coded in one exon, while polymorphic two exons. We have further analyzed three
three
cloned and analyzed Although there was
between two
the
to to
various
DNA as probes, and have shown that 3'-flanking regions upon digestion
MATERIALS
COMMUNICATIONS
a high a low
NAT NAT
however,
has
not
the polymorphic a high homology
in
and monomorphic
different;
NAT,
monomorphic
NAT is
NAT gene was separated the RFLP in homozygotes fragments
of
cloned
into of genomic
RFLPs are found in coding with BamHI and KpnI.
and
AND METHODS
Cloning of human N-acetyltransferase genes: A human genomic DNA library constructed in EMBL3 was obtained from the Japanese Cancer Research Resources Gene Bank and screened with 32P-labeled cDNAs for the polymorphic and monomorphic NATs recently cloned in this laboratory (4). Phage DNAs were transferred to nitrocellulose filters which were prehybridized in a solution containing 20% formamide, 5 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, S x Denhardt's solution (1 x Denhardt's = 0.02% bovine pH 7.41, serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.1% SDS, and 100 pg/ml denatured salmon sperm DNA at 42 'C for 5 h, and then hybridized in the same solution containing 32P-labeled polymorphic and monomorphic NAT cDNAs (3-4 x 105 cpm/ml of each cDNA) at 42 "C for 16 h. Filters were washed in 2 x SSC containing 0.1% SDS at 42 OC for 30 min, twice. Nucleotide digestion subcloned (6) .
Positive clones were analyzed by sequence analysis: Restriction fragments were with restriction enzymes. into plasmid pUC18 and sequenced by the dideoxy method
Genomic DNA was prepared from Genomic Southern blot analysis: leukocytes of healthy volunteers and Southern blot analysis was Briefly, DNA was digested with 40-50 performed as described (5). units of EcoRI, BamHI, or KpnI at 37 OC for 5 h, separated on a to GeneScreen 0.8% agarose gel at 25 V for 13 h, and transferred Plus filters. The filters were prehybridized in 50% formamide, 1 1% SDS, and 100 gg/ml denatured M NaCl, 10% dextran sulfate, salmon sperm DNA at 42 'C for 2 h and then hybridized in the same solution containing 5 x 105 cpm/ml of 32P-labeled genomic DNA The filters were washed in 2 X SSC fragments at 42 OC for 14 h. the solution twice, containing 1% SDS at 42 'C for 1 h by changing The genomic and exposed to an X-ray film at -80 'C for 24-36 h. using the MultiprimeTM DNA DNA fragments were 32P-labeled United Kingdom) to a specific activity labelling system (Amersham, of 3-10 x lo8 cpm/pg. 1253
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS AND DISCUSSION Screening of the amplified genomic DNA library (2 X lo6 phages) revealed 7 and 5 independent clones for polymorphic and monomorphic
NAT genes,
qenomic
DNA clone
several
stop
was
codons
respectively. also
in
the
Although
obtained,
sequence
putative
it is a pseudogene (data not shown). polymorphic and monomorphic NAT genes restriction cDNAs were
enzymes sequenced
monomorphic NAT was without interruptions. was separated in one exon
coding The
a third analysis
region, longest
were
and EcoRI fragments that (Fig. 1). The sequence
of
revealed
indicating clones of
analyzed
that the
with
hybridized with of cDNA for
detected in one of the EcoRI In contrast, the cDNA of
into two exons: the 5'-noncoding 8 kb upstream from another exon
type
fragments, polymorphic
region was which comprised
coding
and 3'-noncoding
regions.
Since
the
sequences
coding
and 3'-noncoding
regions
of both
the
polymorphic
of
the
NAT located the the
and
Fiqure 1. Sequences and alignment of human polymorphic (headed by P) and monomorphic (headed by M) NAT genes. Nucleotide residues are numbered in the 5' to 3' direction starting with the initiation codon (ATG) as number 1. The initiation codon is shown in bold letters. Sequences are aligned to maximize homology with inserted gaps represented by dashes (-). Asterisks indicate nucleotide residues identical in polymorphic and monomorphic NAT. Capital letters enclosed in boxes indicate exons and small letters denote introns.
Vol.
177,
No.
3, 1991
monomorphic sequences
NATs
were
only
the
of
BIOCHEMICAL
AND
identical
to
noncoding
regions
sequence
liver, did
cDNAs
and surrounding
(41,
the
regions
are
was 69% homology in nucleotide sequence region of monomorphic NAT and the intron
between
monomorphic
NAT, whereas there sequences of 5'-
and polymorphic
NAT cDNAs.
for
the
5'-noncoding
region.
After
NAT gene upstream this
part
was completed, Grant et. al. (7) reported the a genomic DNA clone for the polymorphic NAT of human
which
was
identical
not analyze the We have recently
polymorphic a BamHI
of the
COMMUNICATIONS
seems to indicate that the polymorphic monomorphic NAT gene and utilized the
as an exon
site,
to that
5 '-noncoding shown that
NAT gene;
BamHI site, BamHI site
RESEARCH
coding region of polymorphic homology in the nucleotide
This observation evolved from the of the study sequence of
those
5'-noncoding
shown in Fig. 1. There between the 5'-noncoding upstream of the was no significant
BIOPHYSICAL
gene
reported
exon there
here,
although
of the polymorphic are three types of
1 contains
a 5.5
kb KpnI
they NAT gene.
fragment
with
gene 2 contains a 5.5 kb KpnI fragment without a while gene 3 contains a 5.0 kb KpnI fragment with a (Fig.2).
We further
showed that
gene 1 corresponds
to
lkbps H
cd t”
Probe KE
BE
EB
BEE
E
Ii
e
BEKBE
e
Genomic fragment
El
E2
E3
-iTi--
E4
E5 CH B3
B2
E6 B4
Kl
*
7F-
K2 BEK
E
d-@Le d
Gene 2 E5
BEKBE
&@A;L
Gene 3
Fisure 2. Restriction maps of three genes (E, EcoRI; B, BamRI; K, KpnI).
types of polymorphic NAT The restriction map of NAT gene 1 was deduced from analysis of genomic DNA clones and those of genes 2 and 3 from Southern blot analysis of genomic DNAs. Hatched and open boxes indicate coding and noncoding regions of cDNAs, respectively. Thin arrows(+)) above gene maps show Probe a, Sal1 in vector-BamHI genomic fragments used as probes. fragment; b, EcoRI-BamHI fragment; c, HindIII-BamHI fragment; d,
e,
BamHI-Sal1
BarnHI-BarnHI
fragment;
arrows(*) blot analysis. restriction
show genomic The regions maps were identical
in
vector
fragments detected of gene 2 and gene to that of gene
1255
fragment.
Thick
in genomic Southern 3 in which 1 are omitted.
Vol.
177,
No.
3, 1991
BIOCHEMICAL
Probe
I
Subject No
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
3 II 1 I 2 I 3
23.1kb3 9.4kb+ 66kb+
+23.Ikb 4-J% *
+-El
4.4kb+
.,ji
2.3kb-+ 2.0kb*
Eco RI
4-ES
23.lkb+ 9.4kb+ 6.6kb-*
+B4
4.4kb+ 2.3kb-b
Barn HI
*f
1*
*
+Bl
2.0kb'
*-(c
t +
9.4kb 6.6kb
f
4.4kb
f f
23kb 2.0kh
+23.lkb + 9.4kb f-
6.6kb
+-
4.4kb
+ +
2.3kb 2.0kb
+23.Ikh f 9.4kh + 6.6kh +K3, 44kh
++
Kpn I
Figure 3. Southern blot analysis of genomic DNA. Filters were _hybridized with one of the jzP-labeled genomic fragments (a-e) shown in Fig 2. El-E6, Bl-B5, and Kl-K3 correspond to genomic fragments shown in Fig.2. Wild type k-phage DNA digested with Hind111 was used for size markers, shown on both sides in kilobases.
a high
NAT activity,
while genes 2 and 3 give rise to a low NAT activity (5). To further analyze the structures of the three NAT genes, genomic Southern blot analysis was performed with genomic DNAs from homozygotes of the three NAT genes using 32P-labeled 1256
2.3kh 2.0kb
Vol.
177,
No.
genomic
3, 1991
BIOCHEMICAL
DNA fragments
3 were structure
homozygotes of the
genomic
(probes
a-e
of genes polymorphic
DNA clones
AND
and
BIOPHYSICAL
in
Fig.
RESEARCH
2).
b, or
no RFLP in
from
genomic
subjects.
In
digests.
digests
contrast,
B3 fragment
subject B5, was
Southern
blot
subject consistent
a 5.5
from
RFLP was
observed
in
BamHI
gene 2.
fragment
NAT genes
and 3 '-flanking
in 2.
sites
has
information
taken
together
of
use
and in the acetylator
in
are
subjects
Instead,
the
investigation phenotypes.
with
identification of
Bl-
as by
digested 3). There the
was
three
and KpnI
1 and 3, but a longer
not
in
fragment,
Digestion of genomic DNA by KpnI (K2) in subjects 1 and 2, while (f(3). (5),
This which
generated
the
observation indicated
by mutations
previous of
gene
of
as El-E6,
is that in
report
genotypes
mutations
of
associated
(5)
the
the
No other obvious mutation detected so far. The present
been
and
(Fig.
regions.
restriction prove
DNAs
detected
2,
the NAT gene EcoRI digests
represent partially gene fragments (Fig.
3 showed a 5.0 kb fragment with our previous report
polymorphic
coding
kb
denoted of in
genomic
was
1,
The analysis
analysis
between
2, a homozygote of detected in subject
demonstrated
three
d, and e probably other homologous
EcoRI
Subjects
1, 2, and 3, respectively. NAT gene was deduced from
in Southern blot analysis 2) . The fragments should correspond to regions B5, and Kl-K3 shown in Fig. 2. The faint bands detected probes a, fragments
COMMUNICATIONS
of should
polymorphic
NAT
with
ACKNOWLEDGMENTS This research was supported of the Science and Technology grants from the Ministry of Japan.
in part by Special Agency of Japan, Education, Science,
Coordination Funds and by research and Culture of
REFERENCES 1. 2. 3. 4. 5.
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