From The Burnham Institute, La Jolla Cancer Research
Center, La Jolla, California 92037, the § Department of
Legal Medicine, Faculty of Medicine, Toyama Medical & Pharmaceutical
University, Toyama 930-01, Japan, and the ¶ Laboratory of
Evolutionary Genetics, National Institute of Genetics,
Mishima 411, Japan
Received for publication, November 30, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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We have cloned murine genomic and complementary
DNA that is equivalent to the human ABO gene. The murine gene consists
of at least six coding exons and spans at least 11 kilobase
pairs. Exon-intron boundaries are similar to those of the human
gene. Unlike human A and B genes that encode two distinct
glycosyltransferases with different donor nucleotide-sugar
specificities, the murine gene is a cis-AB gene that
encodes an enzyme with both A and B transferase activities, and this
cis-AB gene prevails in the mouse population. Cloning of
the murine AB gene may be helpful in establishing a mouse model system
to assess the functionality of the ABO genes in the future.
Histo-blood group A/B antigens are clinically important antigens
in blood transfusion and organ transplantation. These antigens are
oligosaccharide antigens whose immunodominant structures are defined as
GalNAc A/B antigens are not restricted to humans but are widely present in
nature (16). We therefore investigated the presence/absence of
homologous sequence(s) in the genomes of other species of organisms (17). Hybridization of zoo blots, using the radiolabeled human A
transferase cDNA probe, showed weak signals in chicken genomic DNA
but strong signals, comparable with the signal detected in human DNA,
in genomic DNA from mice and other mammals. No signals were detected in
genomic DNA from lower species of organisms in the evolutionary tree.
We next determined the partial nucleotide sequences of the primate ABO
genes (17). The glycosyltransferases responsible for A or B phenotypes
in primates were shown to conserve amino acid substitutions
corresponding to codons 266 and 268 in humans. A similar study was also
reported by others (18). Through comparative sequence analyses of the
ABO genes from humans and apes, we and others proposed a convergent
hypothesis of evolution that ABO genes arose from independent mutations
after the speciation of humans and apes (19, 20). No apparent
disadvantages are recognized among any of the phenotypes involving the
ABO polymorphism. Hemolytic disease of newborns may be a natural
selection against specific combinations of blood groups between the
mother and fetus. However, serious incompatibility cases are rare with
ABO, since the natural antibodies against A and B antigens are mostly
IgM and do not cross the placenta. Although some anti-A, B antibodies are IgG and capable of crossing the placenta, A/B antigens are not well
developed in fetuses. Therefore, little damage is done. There should be
some reason for the existence of ABO polymorphism in the population. It
has been speculated that the possible role of the ABO system is to
provide resistance against infection (21). Actually, Leb
(Fuc Materials
Mouse genomic DNA library (ML1044j), which was constructed by
replacing the internal BamHI-BamHI stuffer
fragment of the Methods
Isolation of Genomic DNA Clones and Construction of Restriction
Endonuclease Cleavage Maps--
Under low stringency conditions, ~1
million plaques from the mouse genomic DNA library were screened using
a human A transferase cDNA probe by the plaque hybridization method
(25). Radiolabeled probe was prepared by the random hexamer primer
method, using a Prime-It II kit and [ Subcloning, Nested Deletion, and DNA Sequencing--
DNA from
MABO 5'-RACE cDNA Cloning--
RNA from the CMT-93 rectal
carcinoma cell line (ATCC 223-CCL), established from a C57BL strain of
mouse, was prepared and used for the 5'-RACE experiments (29). We
followed the Marathon cDNA amplification protocol provided by the
manufacturer. Briefly, the first strand of cDNA was synthesized
from RNA using Moloney murine leukemia virus reverse transcriptase and
Marathon cDNA synthesis primer. After the second strand was
synthesized with RNase H, E. coli DNA polymerase I, and
E. coli DNA ligase, the Marathon cDNA adaptor was
ligated. Nested PCR was performed, first with AP1 adaptor primer and
MY-1 primer and then with AP2 and MY-2 primers. AP1 and AP2 primers
were provided in the kit. The nucleotide sequences of MY-1 and MY-2
primers were complementary to the sequences in the coding region of the
murine ABO genes. Their sequences were as follows:
5'-TTAGTTTCTGATTGCCTGATGGTCCTTGGGCAC and 5'-TCATGCCACACAGGCTCAATGCCGT
for MY-1 and 2, respectively. PCR products were electrophoresed through
a 3% agarose gel, and the DNA was gel-purified using the GeneClean
kit. DNA fragments were then ligated with pT-Adv vector from the
AdvanTage PCR cloning kit by the T-A cloning method. Nucleotide
sequences of the inserts were determined.
Preparation of Eukaryotic Expression Constructs--
The
BamHI-XhoI fragment containing the last coding
exon of the murine ABO gene was first subcloned from a murine ABO
genomic clone, MABO DNA Transfection and Enzymatic Assays--
Plasmid DNA was
prepared by the SDS-alkaline method (25). The HeLa cell line derived
from a human adenocarcinoma of uterus was used as a recipient of
transient DNA transfection analyses. The HeLa cells express H antigens
on their cell surfaces and have been successfully used in similar
transfection experiments of A and B transferase expression constructs
(5, 30, 31). Following the manufacturer's protocol, we used
LipofectAMINE for transfection. Seventy-two hours after transfection,
the cells were washed and harvested. Cell pellets were then lysed in
buffer (0.1 M NaCl, 25 mM sodium cacodylate, 10 mM MnCl2, and 0.1% Triton X-100). A/B
transferase activity was determined by measuring the transfer of
carbon-14 from [14C]UDP-GalNAc or
[14C]UDP-galactose to the acceptor substrate
2'-fucosyllactose, as described previously (31). After incubation, the
reaction products were separated from unincorporated nucleotide-sugars
by AG1-X8 anion exchange column chromatography. The incorporation of
radioactivity was determined using a scintillation counter.
PCR Amplification and Nucleotide Sequence Determination of the
ABO Gene Fragments from Several Strains of Mouse Species and
Subspecies--
Murine submaxillary glands were used for the
expression analyses of A/B transferases. A and B transferase activities
were measured by the incorporation of carbon-14 from
[14C]UDP-GalNAc or [14C]UDP-galactose to
2'-fucosyllactose. Although the same reaction conditions were used, the
reaction product was separated from the precursor substrate by paper
chromatography rather than column chromatography. Genomic DNA was
prepared by the proteinase K-SDS method and used to amplify a DNA
fragment derived from the murine ABO gene. The names and nucleotide
sequences of the primers used were as follows: SN-16,
5'-GAGACTGCAGAACAACACTT; SN-17, 5'-CAATGCCGTTGGCCTTGTC. The
PCR-amplified DNA fragments were purified through chromatography using
S-300 MicroSpin columns and subjected to direct DNA sequencing reactions with the BigDye cycle sequencing kit. After the sequencing reaction, DNA was purified and then analyzed using an ABI Prism 377 automatic DNA sequencer.
Detection of ABH Antigen Expression--
Expression of the
ABH antigens in murine submaxillary glands was examined immunologically
using extracts spotted on a nitrocellulose membrane. Murine anti-A and
anti-B monoclonal antibody mixtures were biotinylated using EZ-Link
Sulfo-NHS-LC-Biotin, following the protocol provided by the
manufacturer. After biotinylation, the unincorporated biotin was
removed using Microcon 30 centrifugal filter devices. The submaxillary
glands from C57BL and ICR strains of mice were homogenized in buffer
containing 20 mM Tris-HCl (pH 7.5), 0.15 M
NaCl, and 1% Triton X-100. After centrifugation, the supernatant was
diluted with buffer containing 25 mM Tris-HCl (pH 7.5) and
0.1% SDS. The extract was then spotted onto a Duralose-UV membrane. As
controls, the extracts similarly obtained from human colon
adenocarcinoma SW48 cells (AB phenotype) and from group A and O porcine
submaxillary glands were also spotted on the membrane. After drying for
15 min, the membrane was treated with 0.3% hydrogen peroxide and 0.3%
fetal calf serum in phosphate-buffered saline for 5 min to block
endogenous peroxidase activity. After washing, the membrane was
incubated overnight in phosphate-buffered saline containing 4% bovine
serum albumin at 4 °C. The membrane was then cut into four pieces,
which were individually incubated with either biotinylated
murine anti-A monoclonal antibody mixture, biotinylated murine anti-B
monoclonal antibody mixture, biotinylated Ulex europaeus agglutinin I, or bovine serum albumin (negative control) for 1 h
at room temperature. The filters were washed separately and then
incubated collectively with the Elite ABC reagents for 15 min. After washing with phosphate-buffered saline, the membranes were
treated with 4-chloro-1-naphthol substrate for color development.
Mouse Genome Contains the Human ABO Gene Equivalent--
Certain
mammalian cells exhibit Organization of the Murine Gene Is Similar to the Human
Counterpart--
We cloned the genomic DNA sequence encompassing most
of the murine ABO structural gene. By screening 1 million phage plaques from a murine genomic DNA library, we obtained a total of nine independent clones that hybridized with the human A transferase cDNA probe. A preliminary mapping showed that two phage clones named MABO The Nucleotide and Deduced Amino Acid Sequences of the Mouse ABO
Gene Equivalent--
The nucleotide and deduced amino acid sequences
in the coding region of the murine cDNA were aligned with those of
human A1-1 (A101) allele (accession number AF134412 in
GenBankTM) by combining the Clustal method (40) and the
J. Hein method (41) using the MegAlign software. Results are shown
in Fig. 1. Especially high homology was observed in the coding sequence in the last two coding exons. The percentages of identical nucleotide and amino acid residues in the last two coding exons were 78% (642/822) and 81% (222/273) between the two species, respectively. The
amino acid sequence of the murine gene was also aligned with the amino
acid sequences of human A and B transferases, mouse Murine Gene Encodes an Enzyme with both A and B Transferase
Activities--
We examined whether the isolated mouse ABO gene
sequence could encode a functional glycosyltransferase. We first
constructed a human-mouse chimeric construct in an eukaryotic
expression vector pSG-5. A DNA fragment containing coding sequence in
the last coding exon of the mouse genomic sequence was linked
downstream of the human cDNA sequence of exons 1-6 in the human B
transferase expression construct, pBBBB (30). When DNA from the chimera
construct was transiently transfected into HeLa cells, the appearance
of both A and B transferase activity was observed (Table
I). Because there may be inactivating
mutations in the upstream sequence, as observed in human O alleles, we
constructed a mouse cDNA expression construct. The upstream
sequence of the AflII site in the last coding exon of the
mouse gene in the chimeric construct was replaced by the cDNA
sequence obtained from the 5'-RACE experiment. This construction
produced a mouse cDNA expression construct containing the entire
coding sequence. Results from the enzymatic analysis of the transfected
HeLa cells are also shown in Table I. Both A and B transferase
activities were detected in the cell extract. Based on these results,
we concluded that the murine gene is an AB gene that encodes a protein
capable of utilizing both UDP-GalNAc and UDP-galactose donor substrates
to synthesize A and B antigens.
AB Gene Is Prevailing in Mice--
We have determined the partial
nucleotide sequences of the coding region in the last coding exon of
the murine ABO gene using genomic DNA from several species and
subspecies of mice. The results are summarized in Table
II. Mouse strains analyzed were Mus
musculus domesticus (B10 and png2 strains), M. musculus
molossinus (MSF/Msf strain), M. musculus musculus
(BLG/Msf strain), M. musculus breviostris (BFM/2Msf and
NJL/Msf strains), M. musculus castaneus (CAST/Ei and HMI/Msf
strains), M. musculus subspecies (SWN/Msf strain), and
M. spicilegus (ZBN strain). Except for M. spicilegus, all others were subspecies of M. musculus.
The results identified several nucleotide substitutions, some of which
resulted in amino acid substitutions. However, none were found at the
four positions that would distinguish between the human A and B
transferases. Furthermore, both A and B transferase activities were
also detected in the submaxillary gland extracts from those strains of
mice that exhibited amino acid substitutions. Therefore, there are no
mutations that change the donor nucleotide-sugar specificity in the
sequenced and unsequenced regions of the gene in at least those five
strains of mice examined.
A and B Antigens Are Expressed in Mice--
We have shown that the
mouse equivalent of the human ABO gene encodes a protein capable of
transferring both GalNAc and galactose through in vitro
enzymatic assays using extracts from HeLa cells transfected with the
eukaryotic expression constructs of the murine gene. We have also shown
the presence of A and B transferase activity in the murine submaxillary
glands by use of in vitro assays of tissue extracts.
However, the expression of the protein does not necessarily prove the
expression of A and B antigens, since the enzymatic reactions require
the appropriate substrates and reaction conditions. Therefore, we next
analyzed the expression of A and B antigens. Two laboratory strains of
M. musculus domesticus mice were analyzed. We initially
examined the agglutination of murine red blood cells using murine
monoclonal antibody mixtures against A and B antigens under the regular
agglutination conditions used for the ABO blood group typing of human
red blood cells. No agglutination was observed (data not shown).
Because we observed A and B transferase activity in the submaxillary
gland extract, we next performed the inhibition study, using boiled
extracts of the murine submaxillary glands. No inhibition of the
reference human red blood cell agglutination was observed, although the
treatment of murine monoclonal antibody mixtures with the control group
A porcine submaxillary gland extract resulted in some inhibition (data
not shown). These results suggested that A and B antigens are not
expressed in abundance if at all. We therefore examined the expression
of these antigens using the more sensitive immunological method of
nitrocellulose spotting. As shown in Fig.
3, both A and B antigens were detected in
the murine submaxillary glands. Apparently, higher expression was observed with A antigens than with B antigens. This may be attributed to the decreased availability of UDP-galactose substrate, resulting from the competition between B transferase and
We have cloned murine genomic DNA containing most of the coding
sequence that was equivalent to the human ABO gene. The sequence encoding the first few amino acid residues and the promoter region sequence farther upstream remain to be cloned. In humans, ABH antigens
are widely expressed on a variety of cell surface molecules in many
tissues, depending on the ABO genotypes of the individuals. These
include glycoproteins and glycolipids on mucous cells, nerve cells, red
cells, epidermis, and vascular endothelium. ABH antigen expression
seems to be more highly restricted in lower mammals (42, 43). To
understand the differential expression mechanism of A/B antigens
between humans and mice, cloning of the murine gene promoter region
will be necessary.
Functional analysis of the murine gene has shown that the cloned murine
gene is really an AB gene and encodes an enzyme with both A and B
transferase activity. In our prior studies of the human ABO gene, we
determined the molecular causes of two interesting phenomena named
cis-AB (8) and B(A) (7). Human cis-AB alleles encode a protein with strong A and weak B transferase activity, whereas
B(A) alleles encode a protein with strong B and weak A transferase
activity. A mutation was found in these alleles at each of the four
amino acid substitutions that distinguish human A transferase (AAAA)
from B transferase (BBBB). The cis-AB alleles were
represented as AAAB, whereas the B(A) alleles were represented as BABB
in those four substitution positions. By employing DNA transfection
assays of A-B transferase chimeras and in vitro mutagenized expression constructs, we showed that these amino acid substitutions might cause changes in specificity and activity of the enzyme (30, 31).
As shown in Fig. 2B, the amino acid residues at codons 245 and 247 of the murine gene, which corresponded to human codons 266 and
268 (the third and fourth positions of amino acid substitutions between
A and B transferases), were glycine and alanine. The alanine residue is
identical to that of human B transferase, but the glycine residue is
much smaller than the methionine residue of human B transferase.
Therefore, the mouse enzyme is expected to possess a larger space for
donor nucleotide-sugar substrate recognition/binding. Since
N-acetyl-D-galactosamine is more bulky than
galactose, the increase in space may allow for the accommodation of not
only the galactose portion of UDP-galactose but also the GalNAc portion
of UDP-GalNAc, which may account for the dual transferase activity of
the murine enzyme. Dog Forssman glycolipid synthetase contains glycine
and alanine, the same amino acid residues as those of the murine ABO
gene equivalent, at codons 261 and 263. However, the codon 264 in the
Forssman glycolipid synthetase is valine rather than phenylalanine in
the murine AB gene. This difference in size (valine is smaller than
phenylalanine) may possibly render dog Forssman glycolipid synthetase
able to catalyze the transfer of only GalNAc residues and not galactose residues.
To understand the meaning and role of ABO polymorphism during
evolution, the cloned murine AB gene fragments will be useful in the
production of knockout (group O) mice at the ABO locus. Production of
group A and B mice may also be possible by knocking in the genes after
modifying the specificity of the enzymes so that only A or B
transferase activity is retained. Additional manipulation(s) may be a
prerequisite to enhance the expression of A and B antigens. These may
include the knockout of the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
3 (Fuc
1
2) Gal- and Gal
1
3 (Fuc
1
2) Gal- for A and B antigen, respectively. Functional alleles at the ABO
locus encode enzymes that catalyze the final step of synthesis. A
alleles encode for A transferase, which transfers the GalNAc residues from the UDP-GalNAc nucleotide-sugar to the galactose residue
of the acceptor H substrates defined by Fuc
1
2 Gal-. B alleles
encode for B transferase that transfers the galactose residue from
UDP-galactose to the same H substrates. O alleles are nonfunctional,
null alleles. During the past decade, we have been studying the
molecular genetic basis of the histo-blood group ABO system (1). From a
human gastric carcinoma cell line cDNA library, we were able to
clone human A transferase cDNA (2) based on the partial amino acid
sequence of the soluble form of A transferase purified from human lung
(3). Using cross-hybridization with A transferase cDNA probes, we
then cloned B transferase cDNA and nonfunctional O allelic cDNA
from cDNA libraries made with RNA from colon adenocarcinoma cell
lines that exhibited different ABO phenotypes (4). Possible
allele-specific mutations were identified. Four amino acid
substitutions were discovered between A and B transferases. O alleles
were more homologous to A alleles than to B alleles. A single base
deletion was found near the N terminus of the coding sequence in most
of the O alleles, which caused the codon frame to shift. This resulted
in a truncated protein without glycosyltransferase activity. In
addition to the three major alleles (A1, B, and O), we also
identified mutations that modified the enzymatic activity by
determination of the partial nucleotide sequences of subgroup alleles
(A2, A3, Ax, and B3)
(5-7). We also elucidated the molecular mechanisms of two phenomena named cis-AB and B(A) (7, 8). Although the incidence was low, another type of O allele was discovered that lacked the single base deletion but contained an amino acid substitution at the residue
crucial for nucleotide-sugar recognition/binding (9). Although no
functional analyses have been performed to disprove polymorphism,
others have reported additional alterations (10-15). The nucleotide
and deduced amino acid sequences of a variety of ABO alleles are posted
on the Blood Group Antigen Gene Mutation Database developed by
Blumenfeld and colleagues (available on the World Wide Web).
1
2 Gal
1
3 (Fuc
1
4) GlcNAc-), an ABO-related
structure, was demonstrated to be the receptor for a Gram-negative
bacillus, Helicobacter pylori, a causative agent for
gastritis, peptic ulcer, and possibly gastric cancer (22). A and B
transferases modify the Leb structure into ALeb
and BLeb structures, which H. pylori does not
bind to in vitro. This may explain the earlier observation
that group O individuals have a higher incidence of stomach ulcer than
individuals in any other group (21). Antibodies against the
1
3
Gal epitope (Gal
1
3 Gal-) were demonstrated experimentally to
block the interspecies infection of certain retroviruses (23). From
this result, anti-A and anti-B antibodies have been suspected to play a
role in inhibiting the epidemics of certain infections. Some type of
selection based on the advantage/disadvantage of having
antigens/antibodies may have been operating at the ABO locus to secure
the survival of species from extinction during evolution. The report
that an anti-A monoclonal antibody neutralized human immunodeficiency
virus particles produced by lymphocytes from group A individuals but
not from group B or O individuals (24) may support this hypothesis. To experimentally assess the functionality of the ABO genes, establishing an animal model is critical. As an initial step, we cloned and characterized the murine ABO gene equivalent.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EMBL3 SP6/T7 vector with MboI-partially
cleaved genomic DNA fragments of the BALB/c strain of mouse, was
purchased from CLONTECH (Palo Alto, CA). A Marathon
cDNA Amplification Kit and an AdvanTage PCR1 cloning kit were also
from CLONTECH. A GeneClean kit was purchased from
Bio101 (La Jolla, CA), and pT7T3
18 plasmid vector,
-S-dGTP,
-S-dCTP, and S-300 MicroSpin columns were from Amersham Pharmacia Biotech. LipofectAMINE was purchased from Life Technologies, Inc. [14C]UDP-GalNAc, [14C]UDP-galactose, and
[
-32P]dCTP were from PerkinElmer Life Sciences, and
2'-fucosyllactose was from Oxford Glycosystems (Rosedale, NY) and from
Calbiochem. Murine anti-A and anti-B monoclonal antibody mixtures were
purchased from Ortho Diagnostic Systems (Raritan, NJ). Biotinylated
Ulex europaeus agglutinin I, Vectastain Elite ABC
kit, and 4-chloro-1-naphthol substrate kit were from Vector
Laboratories (Burlingame, CA), and EZ-Link Sulfo-NHS-LC-Biotin was
purchased from Pierce. Bluescript SKM13+ vector, Prime-It II kit,
Duralose-UV membranes, and frozen competent XL1-blue strain of
Escherichia coli bacteria were purchased from Stratagene (La
Jolla, CA). The ULTRAhyb hybridization buffer was obtained from Ambion
(Austin, TX). Restriction endonucleases and nucleic acid-modifying
enzymes were from Life Technologies, New England Biolabs (Beverly, MA),
or Roche Molecular Biochemicals. dRhodamine dye terminator cycle
sequencing ready reaction kits and BigDye cycle sequencing kits were
purchased from PerkinElmer Life Sciences.
-32P]dCTP (26).
After four rounds of screening, individual clones were isolated. Phage
DNA was prepared, cleaved with restriction endonucleases,
gel-electrophoresed, and Southern transferred. Hybridization was then
performed to construct restriction enzyme cleavage maps.
16 phage clone was cleaved with HindIII and
SalI and subcloned into the Bluescript SKM13+ vector. Nested
deletion constructs were prepared by the ExoIII-mung bean
nuclease method (27). Where no unique 3'-overhang restriction sites
were available, the thioderivative fill-in reactions were performed
with Klenow enzyme using
-S-dGTP and
-S-dCTP before
ExoIII treatment. After transformation of E. coli
XL1-blue strain, plasmid DNA was prepared from individual clones and
analyzed for insert size. The nucleotide sequences were determined by
Sanger's dideoxy chain termination method using the dRhodamine dye
terminator cycle sequencing ready reaction kit (28). Sequences were
aligned using Lasergene SeqMan II sequencing project management software.
11, into the pT7T3
18 plasmid vector. The
BamHI site was located in the intron preceding the last
coding exon of the murine ABO gene. The XhoI site was in the
EMBL3 SP6/T7 vector next to the BamHI site used to
accommodate genomic DNA. This construct was then digested with
SstI and SnaBI. The SstI site was
within the pT7T3
18 plasmid vector, and the SnaBI site was
located downstream of the stop codon of the mouse ABO gene coding
sequence. The SstI-SnaBI fragment containing the
coding sequence in the last coding exon of the mouse ABO gene was then
isolated. The human B transferase expression construct with intron,
pBBBB (30), was cleaved with BamHI, blunt-ended by the
Klenow filling-in reaction, and then digested with SstI. The
SstI site was in the intron preceding coding exon 7 of the
human ABO gene; BamHI was in the eukaryotic expression
vector (originally pSG-5). The SstI-blunt (BamHI)
vector fragment containing the human B transferase cDNA sequence of
exons 1-6 was then ligated to the mouse
SstI-SnaBI fragment to produce the human-mouse
chimeric gene (pHuman-mouse chimera). A murine cDNA eukaryotic
expression construct was then constructed by replacing the
EcoRI-AflII fragment from the chimeric construct
with the EcoRI-AflII fragment from a 5'-RACE
clone in the pT-Adv vector. The EcoRI site of the clone was
in the plasmid and located 5' upstream of the cDNA end. The
AflII was in the last coding exon. The EcoRI site
in the pSG-5 vector was located downstream of the SV40 early promoter
and upstream of the human cDNA sequence. The resultant construct
(pMouse) contained the entire coding sequence of the mouse ABO gene cDNA.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
3 Gal epitopes. The cDNA encoding
1,3-galactosyltransferase that synthesizes this epitope was
cloned from cow (32), mouse (33), and pig (34, 35). Humans do not
exhibit this epitope but possess the antibody against the epitope in
sera (36). Human sequence corresponding to this gene was shown to be a
pseudogene due to frameshifts and nonsense mutations (32, 37). A/B
transferases utilize the galactose substrate with fucose, whereas
1,3-galactosyltransferase utilizes the substrate without fucose. ABO
genes and
1,3-galactosyltransferase genes share significant homology
at both the nucleotide and deduced amino acid sequence levels (30).
Cloned canine cDNA encoding Forssman glycolipid synthetase
(UDP-GalNAc:globoside
1,3-N-acetyl-D-galactosaminyltransferase) also exhibited sequence homology (38). Therefore, these genes are
believed to have derived from the same ancestral gene and constitute
the ABO gene family. Southern hybridization experiments of murine
genomic DNA showed different banding patterns when murine
1,3-galactosyltransferase cDNA probe and human A transferase cDNA probe were used (17, 33). Accordingly, the ABO gene equivalent was assumed to exist in the mouse genome (17). Results from our cloning
experiments of the murine ABO gene described here concluded that mice
actually do possess an ABO gene equivalent.
11 and MABO
16 contained the entire coding sequence in
the last coding exon. Since the MABO
16 clone contained the sequence
farther upstream, this clone was used for the nucleotide sequence
determination. The MABO
11 clone containing the farther downstream
sequence was used to construct a human-mouse ABO gene chimeric
expression construct as well as a murine gene expression construct. We
sequenced the entire insert in the MABO
16 clone (~11.2 kilobase
pairs) with more than 99.9% accuracy. Almost all of the coding
sequence was contained in the sequenced region. The exon-intron
boundaries were determined and are shown in Fig. 1. Fig. 1, A and B,
represents two probable splicing patterns, although other possibilities
still exist because the sequence encoding the first few amino acid
residues has not yet been identified. There are six coding exons in
Fig. 1A and seven in Fig. 1B. Approximately 4.0 kilobase pairs and 70 base pairs upstream of the splicing acceptor site of coding exon 2 (cEXON 2) in Fig. 1A, there
was a CTCAGAG sequence and a TGAATCTCAG sequence, respectively. These sequences may be portions of the coding sequence, since they are found
upstream in the cDNA preceding the sequence in cEXON 2. Fig.
1B depicts the case where GAATCTCAG of the latter TGAATCTCAG sequence represents the sequence in the preceding exon. In that case,
the acceptor site of cEXON 3 needs to shift 2 nucleotides upstream,
which would break up the GT-AG rule of splice junctions. We determined
the entire nucleotide sequence contained in the MABO
16 clone, which
included ~5.0 kilobase pairs of sequence upstream of the splicing
acceptor site of cEXON2 in Fig. 1A. No sequence
corresponding to the 5'-untranslated region was found. Therefore, the
promoter region of the murine ABO genes must reside farther upstream.
The sequence corresponding to human coding exons 3 and 4 was found in
one exon in the mouse gene. However, the number of amino acid residues
(19 amino acids) was much smaller than that of human exons 3 and 4 combined (35 amino acids). Further studies are needed, since there may
be an alternative splicing that would divide this small exon into two
smaller exons with an intron in between. The entire insert sequence in
the MABO
16 clone and the entire cDNA sequence have been
deposited in the DNA Data Bank of Japan (DDBJ) (accession numbers
AB041038 and AB041039).
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Fig. 1.
Comparison of the exon-intron boundaries and
the nucleotide and deduced amino acid sequences of the coding region of
the murine AB gene with those of the human A1-1 (A101)
allele. The nucleotide sequence of the mouse gene in the MABO 16
phage clone and the sequence of the 5'-RACE-amplified cDNA were
used to determine the exon-intron boundaries. Almost all of the coding
sequence was contained in the cloned genomic DNA fragment. Since the
sequence encoding the first few amino acid residues is still missing,
the earlier exon-intron boundaries have not yet been completely
determined. In Fig. 1, A and B, two possible
splicing patterns are presented. There are six coding exons in
A and seven in B. In A, ~70 base
pairs upstream of the splicing acceptor site in coding exon 2 (cEXON
2), there was a GAATCTCAG sequence. If the acceptor site is shifted
upstream by 2 nucleotides, that sequence may represent the preceding
exon sequence as shown in B. This shift, however, breaks up
the GT-AG rule of splice junctions. In addition, no 5'-untranslated
region sequence is found upstream. Therefore, A may
represent what most likely occurs. XXXXXX, the
nucleotide sequence is unknown. Unlike the human sequence, the sequence
in human exons 3 and 4 was contained in a single exon in the mouse
gene. The numbers shown in parentheses next to
the nucleotide numbers of the coding sequence indicate the nucleotide
residue numbers of the MABO
16 phage clone insert deposited to DDBJ
(accession number AB041038). The nucleotide sequence of the murine AB
gene cDNA has also been deposited (accession number AB041039). Also
shown is the comparison of the nucleotide and deduced amino acid
sequences in the coding region of the murine gene with the human
A1-1 (A101) gene sequences (accession number AF134412 in
GenBankTM). A combined method incorporating the Clustal
method (40) and the J. Hein method (41) was used with the MegAlign
program. High homology was observed in the coding sequence in the last
two coding exons. The percentages of the identical nucleotide and amino
acid residues were 78 and 81% between the two sequences,
respectively.
1,3-galactosyltransferase, and canine Forssman glycolipid
synthetase. Results are shown in Fig.
2A. The percentages of the
identical amino acid residues of the coding sequences in the last two
coding exons are 47% (127 of 272) between the mouse ABO and
1,3-galactosyltransferase genes and 49% (132 of 272) between the
mouse ABO gene and the dog Forssman synthetase gene. Fig. 2B
highlights the amino acid sequences of the region important for the
recognition/binding of nucleotide-sugars. The phylogenetic tree is
shown in Fig. 2C. The cloned mouse gene was evolutionarily
mapped closest to the human ABO gene. It was also mapped closer to the
canine Forssman gene than the murine
1,3-galactosyltransferase
gene.
View larger version (62K):
[in a new window]
Fig. 2.
Comparisons of the amino acid sequences of
the coding regions among murine AB gene, human A1-1 (A101)
and B (B101) genes, murine 1,3-galactosyltransferase gene, and
canine Forssman synthetase gene and an evolutionary tree of the ABO and
related genes. A, the amino acid sequence of the mouse
AB transferase was compared with those of the human A and B
transferases, mouse
1,3-galactosyltransferase (mouse GalT), and
canine Forssman glycolipid synthetase (dog Forssman). The mouse AB
transferase was 47 and 49% identical to the mouse
1,3-galactosyltransferase gene and the dog Forssman synthetase gene
in the last two coding exons, respectively. B, the coding
sequences around the amino acid residues important for the
nucleotide-sugar recognition/binding are compared. The sequences from
the human O03 allele that contains a missense mutation around this area
(9) and the cis-AB01 allele with dual transferase activity
(8) are also included. C, a phylogenetic tree of the ABO
gene family is shown. The following sequences in GenBankTM
were used for the computation of evolutionary distance (46) and
neighbor-joining tree (39): marmoset GalT (S71333), bovine GalT
(J04989), pig GalT (L36152), mouse GalT (M85153), and dog Forssman
synthetase (U66140). Numbers on interior branches denote
bootstrap probabilities in percent. Branch lengths are proportional to
the numbers of amino acid substitutions, and a scale is
given at the bottom of the tree. The dotted line
denotes the probable root node of this tree. This node also corresponds
to a gene duplication event producing both the
1,3-galactosyltransferase gene and ABO-related gene. The latter
lineage further experienced another gene duplication, resulting in
creation of both Forssman glycolipid synthetase gene and ABO gene. The
murine AB gene was mapped closest to the human ABO genes. It also
mapped closer to the canine Forssman gene than the murine
1,3-galactosyltransferase gene.
In vitro enzymatic assays of extracts from HeLa cells transfected with
eukaryotic expression constructs
Nucleotide and amino acid substitutions in mouse species and subspecies
1,3-galactosyltransferase for the same donor nucleotide-sugar. The
low expression of A and B antigens may be similarly explained by the
competition for the same acceptor substrate between
1,3-galactosyltransferase and
1,2-fucosyltransferase. Since
1
3-Gal epitope produced by the
1,3-galactosyltransferase is
abundantly expressed in murine tissues, it is likely that the
1,3-galactosyltransferase has higher affinity for the acceptor
substrates than the
1,2-fucosyltransferase that synthesizes H
antigens.
View larger version (50K):
[in a new window]
Fig. 3.
Immunological detection of A and B antigens
in murine submaxillary glands. The expression of A and B antigens
was analyzed by the immunostaining of the extracts spotted onto a
nitrocellulose membrane. The extracts were derived from the following
sources: 1) human colon adenocarcinoma SW48 cells (AB phenotype); 2)
group A porcine submaxillary gland; 3) group O porcine submaxillary
gland; 4) submaxillary gland from a C57BL strain of mouse; and 5)
submaxillary gland from an ICR strain of mouse. BSA, bovine
serum albumin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-galactosyltransferase gene (44) or
the introduction of the
1,2-fucosyltransferase gene under strong
promoter (45) to either abolish or lessen the expression of the
1,3-galactosyltransferase. These mice with different ABO phenotypes
in the same genetic background may clarify the functionality of the ABO
system in the future.
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ACKNOWLEDGEMENTS |
---|
We thank Emily N. Wang for editing the manuscript and Dr. Sandra S. Matsumoto for critically reviewing the manuscript. We are also indebted to Dr. Toshihiko Shiroishi (National Institute of Genetics, Mishima, Japan) for supplying various strains of mice.
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FOOTNOTES |
---|
* This study was supported in part by funds from The Burnham Institute, the Department of Defense Breast Cancer Research Program (DAMD 17-98-1-8168), and from Kyowa Hakko Kogyo Co. Ltd.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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AB041038 and AB041039.
To whom correspondence should be addressed: The Burnham
Institute, La Jolla Cancer Research Center, 10901 N. Torrey Pines Rd.,
La Jolla, CA 92037. Tel.: 858-646-3116; Fax: 858-646-3173; E-mail:
fyamamoto@burnham.org.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010805200
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ABBREVIATIONS |
---|
The abbreviations used are:
PCR, polymerase
chain reaction;
-S-dGTP, 2'-deoxyguanosine-5'-O-(1-thio-triphosphate);
-S-dCTP, 2'-deoxycytidine-5'-O-(1-thio-triphosphate);
RACE, rapid amplification
of cDNA ends.
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