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Sequence Comparison of Baboon ABO Histo-Blood Group Alleles: Lesions Found in O Alleles Differ between Human and Baboon
Blood Cells, Molecules, and Diseases (1997) 23(13) July 15: 242–251 Article No. MD970141
D.C. Diamond, et al.
Sequence Comparison of Baboon ABO Histo-Blood Group Alleles: Lesions Found in O Alleles Differ between Human and Baboon Submitted 05/02/97; revised 05/29/97 (communicated by Brian S. Bull, M.D., 06/30/97)
David C. Diamond1,2, Omar R. Fagoaga2, Sandra L. Nehlsen-Cannarella2, Leonard L. Bailey3, Aladar A. Szalay1 ABSTRACT: Histo-blood group O has only rarely been observed in baboon. Recent discovery of such an animal has provided us the opportunity to investigate the molecular genetics of the ABO locus in baboons. The major baboon prototype O allele, observed in two homozygous and several heterozygous animals, is related to the A allele as is the case in humans. Additional apparent prototype O alleles have been observed in heterozygotes, one of which is related to the B allele. The nucleotide changes conferring the O phenotype in the two known human O alleles have not been observed in any baboon allele. This information will aid the identification of baboons useful for the development of xenotransplantation in humans. Keywords: ABO glycotransferase, baboon, glycosyltransferase, histo-blood group, xenotransplantation
INTRODUCTION
attack and can be freely transplanted. Thus, blood group O individuals are considered universal donors of both blood and organs. However, while blood group O is the most common type in humans, it is exceedingly rare in baboons (1,4). This has been an impediment to the development of xenotrans-plantation protocols involving the use of baboon organs (5,6). Previous work on the molecular genetics of the ABO system has established the cDNA sequence and genomic structure of the locus in humans (2, 7, 8), as well as the amino acid residues conferring the different enzymatic activities (9). The coding sequence of the transferase is divided into seven exons. Exon 6 and 7 encode the bulk of the enzyme (13% and 65%, respectively), including its active site. In all examined primate species the residues critical for determining donor
The ABO histo-blood group antigens, the basis of blood type, are found in all anthropoid primates (1). These antigens, which are terminal carbohydrate structures, can be found both in soluble form and on the surface of a variety of tissues, depending on the species examined. Additionally, antibodies to the non-expressed antigen(s) are universally present, agglutinating mismatched blood, and facilitating complement-mediated attack on tissues following transplant. Genetically, the phenotype is controlled by a single glycosyltransferase locus that can be occupied by three fundamental alleles encoding enzymes with either A or B activity or no activity (type O) (for reviews see 2, 3). Cells from blood group O individuals lack both antigens and thus are not subject to
1
Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA 92350; Immunology Center, Department of Pathology, Loma Linda University, Loma Linda, CA 92350; 3 Department of Surgery, Loma Linda University and Medical Center, Loma Linda, CA . Reprint request to: David C. Diamond, Ph.D.,Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA 92350. phone (909)824-4300, ext. 81370, fax (909)478-4177, email: [email protected] 2
Published by Academic Press Established by Springer-Verlag, Inc. in 1975
1079-9796/97 $25.00 Copyright r 1997 by The Blood Cells Foundation, La Jolla, California, USA All rights of reproduction in any form reserved.
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substrate specificity (i.e., A versus B activity) are found at amino acid positions 266 and 268 (numbering according to 2,3), Leu vs. Met and Gly vs. Ala, respectively (10,11). In humans, blood group O arises from either of two mutations in an A-like background; a frameshift in exon 6 leading to premature termination, or a Gly=Arg mutation in exon 7 at position 268 (12-14). As part of our ongoing experimentation in animal models of xenotransplantation (see for example 15), we discovered a blood group O baboon using standard serologic phenotyping methods. To utilize this finding, and to advance their potential as organ donors to humans, we decided to examine the molecular basis of blood group O in baboons. We now report the existence of at least two prototype O alleles in baboon: one, now observed in two O homozygotes and several apparent BO heterozygotes, is derived from an A allele; another, observed in an apparent AO heterozygote, is derived from a B allele. A possible third prototype O allele, A-derived, has also been observed in a pair of BO heterozygotes. None of these share the mutations found in the human O alleles.
were collected from the femoral vein. Buccal mucosa epithelial cells were also collected by swabbing the buccal mucosa and applying the cells to a glass slide. The slides were then dried overnight. Direct typing was performed using the standard immunophenotyping assay with immunofluorescent-stained scrapings of the buccal epithelium (16). Conventional reverse typing is performed using baboon serum (pre-adsorbed with human blood group O erythrocytes) to agglutinate human A and B type erythrocytes.
DNA Isolation Peripheral blood lymphocytes were isolated from whole blood by ficoll gradient centrifugation and used as a source of genomic DNA. DNA isolation was carried out using either the RapidPrepy Macro Genomic DNA Isolation Kit (Pharmacia), initially, or the QIAamp Blood Kit (Qiagen) according to the manufacturers’ directions.
PCR Primers were designed based on the human sequence (7,8,12,17). Three upstream and two downstream primers were used for amplification of genomic DNA: UPex5L, GATGGTCTACCCCCAGCCAAAGGTGCT, covering most of exon 5; UPF, TGGGCCACCGTGTCCACTACTATGTCTT, located toward the 58 end of exon 7; UPH, CCAAGGACGAGGGTGATTTCT, located centrally in exon 7; LPC, TCCGGACCGCCTGGTGGTTCTTG, located near and complementary to the 38 end of exon 7; and Uaftx7, AGCCCTCCCAGAGCCCCTGG, located in the untranslated region on the non-coding strand shortly after the stop codon. UPF-LPC reactions contained each primer at a concentration of 0.5µM, 300-500ng genomic DNA, dNTPs at a concentration of 200µM each, pfu polymerase (2.5 units) (Stratagene), and the supplied buffer, in 100µl. Typical thermocycling conditions were: denaturation at 94°C for 2 min.; 30 cycles at 94°C, 5 sec.,
MATERIALS AND METHODS Animals All of the baboons used in this study were olive baboons (Papio cynocephalus anubis) from the Southwest Biomedical Research Foundation (San Antonio, TX). Animal care followed the guidelines of the standards of the National Society for Medical Research, Principles of Laboratory Animal Care, and the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (Publication no. 86-23, revised, 1985).
Serologic Phenotyping Using ketamine anesthesia (15mg/kg), 10 ml heparinized (100 U/ml) and 5 ml clotted blood 243
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55.9°C, 30 sec., and 72°C for 2.5 min.; followed by 72°C for 7 min. on a GeneAmp PCR System 9600 (Perkin Elmer). A hot start was achieved by adding enzyme while paused at the first annealing step. UPex5L-LPC and UPH-Uaftx7 reactions contained each primer at a concentration of 0.4µM, respectively, 200ng genomic DNA, dNTPs at a concentration of 350µM each, Expand Polymerase - a mixture of Pwo and taq polymerases - (2.6 units)(Boehringer-Mannheim) and supplied buffer 1, in 50µl. For UPex5L-LPC typical thermocycling conditions were: denaturation at 94°C for 2 min.; 30 cycles at 94°C, 5 sec., 56°C, 30 sec., and 68°C for 4 min. plus 20 sec. each cycle for the last 20; followed by 68°C for 7 min., on a GeneAmp PCR System 9600 (Perkin Elmer). UPH-Uaftx7 required a touchdown cycle: denaturation at 94°C for 2 min.; 20 cycles of 94°C, 5 sec., 66°C, 30 sec., reduced one degree each cycle; 68°C, 5 min.; 20 cycles of 94°C, 5 sec., 46°C, 30 sec.; 68°C, 4 min. plus 10 sec. each cycle; followed by 68°C for 7 min., on a GeneAmp PCR System 9600. Hot starts were achieved in the same manner. Success of reactions was judged by agarose gel electrophoresis.
tative animals was sequenced with the complete primer set. Additional animals screened for heterozygosity were generally sequenced with only the primers UPH and UPF initially. DNA from all of the screened animals thus identified as heterozygotes was further amplified and sequenced with UPH and Uaftx7b. Finally, an allele-specific primer, RASAO, ACCGACCCTCCGAAGAACCCCCCCAA, located so that its 38 end paired with N796 of A-like alleles, was used to resolve multiple heterozygosities as appropriate.
RESULTS Phenotyping Forward and reverse typing was performed to establish the ABO blood group of all baboons studied (Table 1). Unlike hominoids (humans and great apes), old world monkeys such as the baboon do not express ABO antigens on erythrocytes (1). Thus, while conventional reverse typing is feasible, forward typing is not. These antigens however, are also expressed on the cells of other tissues, including epithelial cells. Therefore forward typing was performed on scrapings of the buccal epithelium (16). Generally this phenotyping was entirely consistent with subsequent genotyping (below). Figure 1 shows typical staining patterns for group A,B, and O baboons.
DNA Sequencing PCR products were purified either directly from the reaction (UPF-LPC and UPex5L-LPC) or from agarose gel slices (UPH-Uaftx7) using QIAquick spin columns (Qiagen) and then used directly in cycle sequencing using an Applied Biosystems 373A and Amplitaq CS (initially) or FS. Additional primers used for sequencing were: LPex6a1, TTGATGGCAAACACAGTTAACCCAATGG, located near and complementary to the 38 end of exon 6; IB4x6, AGAGGAGGCGGAAACTGAG, located in the intron upstream of exon 6; IB4x7b, TCTGAGCCTTCCAATGTCCGCTG, located in the intron upstream of exon 7; and LPm7, GTCCACGCACACCAGGTAATCCAC, located centrally in and complementary to exon 7. Unlike the other primers, IB4x6 and IB4x7b were both designed from baboon intronic sequences (data not shown). DNA from represen-
Amplification and Sequencing Genomic DNA isolated from PBLs was used as template for the polymerase chain reaction with three primer pairs, UPF-LPC, UPex5L-LPC, and UPH-Uaftx7b. The expected products, a 630 bp central segment of exon 7, an ,2.3 kb segment stretching from exon 5 through most of exon 7, and a 315 bp segment including the 38 end of the coding region of exon 7, respectively, were obtained and used as template in automated fluorescent sequencing. Even using a touchdown cycle, UPH-Uaftx7 generated many extraneous bands and required gel purification prior to sequencing.
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Table 1. Association of DNA Sequence Polymorphisms with ABO Types.
Baboon
N480
N629
N651
N704
N711
N1024
Phenotype
Sequence Type
Genotype
9
G
C
C
C
C
G
A
AA
AA
10
G
C
C
C
C
G/A
A
AA
AA
31
G
C
C
C
C
G
A
AA
AA
7
G
C
C
C
C
A
B
BB
BB
11
A/G
C
C
C
C
G/A
B
BB
BB
12
G
C
C
C
C
G/A
B
BB
BB
13
A/G
C
C
C
C
G/A
B
BB
BB
15
G
C
C
C
C
G/A
B
BB
BB
17
G
C
C
C
C
G/A
B
BB
BB
19
A/G
C
C
C
C
G/A
B
BB
BB
20
G
C
C
C
C
A
B
BB
BB
22
G
C
C
C
C
G/A
B
BB
BB
23
A/G
C
C
C
C
G
B
BB
BB
24
G
C
C
C
C
G/A
B
BB
BB
26
G
C
C
C
C
G
B
BB
BB
27
G
C
C
C
C
A
B
BB
BB
28
G
C
C
C
C
G/A
B
BB
BB
29
G
C
C
C
C
A
B
BB
BB
33
G
C
C
C
C
G/A
AB
AB
AB
30
A/G
C
C
C
C
G/A
A
AB!
AO
3
G
C
C
C
C
A
B
AB
BO
16
G
C
C
C
C
G/A
B
AB
BO
1
A/G
T/C
T/C
G/C
T/C
G/A
A
AA
AO?
2
A/G
T/C
T/C
G/C
T/C
G/A
A
AA
AO?
6
A/G
T/C
T/C
G/C
T/C
G/A
B
AB
BO
8
A/G
T/C
T/C
G/C
T/C
G/A
B
AB
BO
14
A/G
T/C
T/C
G/C
T/C
G
B
AB
BO
21
A/G
T/C
T/C
G/C
T/C
G
B
AB
BO
18
A/G
T/C
T/C
G
T
G
B
AB
BO
5
A
T
T
G
T
G
O
AA
OO
32
A
T
T
G
T
G
O
AA
OO
Effect of polymorphism
Silent
AA210 T5Val C5Ala
Silent
AA235 G5Gly C5Ala
Silent
AA342 G5Ala A5Thr
The nucleotide residue at 6 positions that were polymorphic in baboon is presented for all of the animals used in this study. N629, N704, and N1024 determine which amino acid residue (AA) is found at position 210, 235, and 342, respectively. Phenotype was determined serologically. Sequence Type refers to which nucleotide residues were found in the donor sugar specificity-determining region, specifically N796, N803, and N813. Genotype was inferred from phenotype and sequence type as described in the text. The animals are grouped by putative prototype O allele and subgrouped by phenotype.
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Figure 1. ABO Phenotyping by Immunofluorescent Staining of Buccal Mucosa. The inner panels in each row show immunofluorescence images of buccal mucosa from a group A (baboon 31), group B (baboon 28), and group O (baboon 5) animal stained with anti-A or anti-B antibody and phycoerythrin labeled second antibody. The outer panels show the adjacent fields of view under brightfield illumination. Immunofluorescence that is not cell-associated is due to soluble antigen in saliva.
ber 32) exhibiting these same sequence features (Table 1).
Representative sequences of exons 6 and 7 are presented in figure 2. Three nucleotide differences are consistently found between blood group A and B, T796, G803, and A813 in A become A, C, and G, respectively, in B. The first two differences result in the Leu266=Met and Gly268=Ala amino acid substitutions crucial for conversion between A and B transferase activity. The third difference is silent. DNA sequence from the blood group O baboon (number 5) resembled that of group A baboons at the positions described above. Neither the G261 deletion or the G802=A (Gly268=Arg) mutations found in human O alleles were present. Four nucleotide differences from the baboon A/B consensus were observed. Two, C651=T and C711=T, are silent. The other two, C629=T and C704=G, cause Ala210=Val and Ala235=Gly amino acid changes, respectively. Subsequently, we have identified a second group O baboon (num-
Heterozygotes with O Alleles We realized that, even without identifying the causative mutation for the baboon O phenotype, it would be possible to genotype serologic-group B phenotypes as either BB or BO based on the A-like sequence of the O allele. This screening would also allow us to evaluate the usefulness of the four nucleotide differences seen in the O homozygote as markers for the O allele. To accomplish this task we concluded that all serologic-group B phenotypes that appear to be AB genotypes must be BO heterozygotes. Since we are simultaneously sequencing a mixture of both alleles from single individuals, we cannot definitively resolve multiple heterozygosities. Using
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Figure 2. Comparison of Representative Sequences of the ABO Transferase. The sequences of exon 6, and exon 7 through the termination codon, of representative homozygous baboons of each blood group are aligned with each other and the published sequence for human A transferase (2). The sequence from the group A baboon is explicitly presented, as are differences from it in the other three sequences; identical bases are indicated by a hyphen (-). The boundary between the two exons is indicated by a vertical line (0). Positions where different amino acids are encoded by the different sequences are marked with *’s below the sequences. The three nucleotides used to classify baboon sequences as either A-like or B-like (i.e., sequence type) are marked with W’s, and the four nucleotide positions useful in distinguishing the baboon major O allele are marked with V’s, above the sequences. The positions of the deletion and substitution defining the human major and minor O alleles are marked with D and O, respectively. These sequences can be found in GenBank under the accession numbers AF001425-AF001427 for the baboon A, B, and O alleles, respectively.
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Sequence Variation
Occam’s razor we have chosen to interpret these sequences as resulting from two alleles, each of which most closely resembles one of the alleles found in the homozygotes above. For example, an animal with heterozygosities at N796 and N803 would be interpreted as an AB genotype, not a pair of reciprocal A-B chimeras. By the above criterion, 7 of 22 blood group B phenotype baboons (4 of 14 males and 3 of 8 females) were BO heterozygotes (Table 1). Examining these eleven O allele sequences (from the 7 BO heterozygotes and the two OO homozygotes), we observed that N629 was a T (instead of C) in 9 of the 11 cases, as were N651 and N711. Similarly, N704 was a G (instead of C) in 9 of these 11 cases. All of the B alleles had a C at N629 and N651. However, in baboon 18 N704 and N711 were homozygous indicating that the B allele was similar to the O allele with a G and a T at these positions, respectively. Subsequently these animals were sequenced using an A/O allele-specific primer, RASAO, positioned at the specificity-determining nucleotides. By using this primer to generate sequence data specifically from the A-like allele we were able to confirm that the O-like residues at N480, N629, N651, N704, and N711 were indeed all found in the A-like allele. Due to the similarity of the A and O alleles, and unlike the group B animals above, the phenotype of group A animals is generally not informative in deducing their genotype. Nonetheless, the nucleotide differences observed in the O homozygotes exhibited enough linkage that it was possible to screen for putative AO heterozygotes. By this criterion, 2 of 6 type A baboons (all female) were O heterozygotes (Table 1). All four positions where nucleotide differences were observed in the O homozygotes (N629, N651, N704, and N711 ) were heterozygous in these animals (Table 1). To our surprise, one of the 6 serologic group A animals (baboon 30) gave an apparent genotype of AB based on its sequence type (Table 1). Thus the ‘‘B’’ allele in this animal must not encode an active enzyme, i.e. it is constitutes an additional O allele prototype.
In addition to the group-associated differences discussed above, several other polymorphisms, that were either less rigorously associated with type or represented individual variation, have been observed. N1024 is polymorphic A/G; encoding either ThrACT or AlaGCT. N1024 might always be G in the major O allele (see below). Allele-specific PCR supports this assignment for baboons 6 and 8, but it remains ambiguous for baboons 1 and 2 (data not shown). N480, a silent position that is predominantly G but sometimes A, was invariably A in the major O allele in those animals in which the heterozygosity could be resolved (i.e., baboons 6,8,14,18, and 21; see Table 1). A silent G/T polymorphism was observed at N1056 but no significant pattern was discerned. Finally there were four single instance variations observed: in baboon 1, G1010 was heterozygous G/A resulting in a conservative switch from Arg to Lys; and in baboon 18, A681, G1026 and G1062 are all heterozygous G/A, but the resulting codons are synonymous.
Comparison with human sequences The baboon and human group A sequences from Fig. 2 are 96.5% identical, differing at 29 of the 826 positions (including the baboon polymorphism at N1024 as a difference). Twenty-four of these substitutions are silent, including all 6 found in exon 6. The predicted amino acid sequences thus differ at 5 of 274 residues (all in exon 7), for 98.2% identity. Comparing group B sequences (using human as reported in ref. 2) reveals 31 nucleotide differences, 27 of which are the same as seen in the A to A comparison, and six amino acid differences, including the five positions where the group A sequences differ, although at one of these positions (amino acid 235) a different residue is involved as noted below. Thus comparing the group B sequences, there was 96.2% and 97.8% identity for nucleotides and amino acids, respectively.
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DISCUSSION
Do the three variant amino acids listed above contribute to the lesion in the major O allele? The Val210 and Gly235 residues seen in baboon blood group O are observed in active alleles in a wide array of other primates (10,11); data from other species are not available for amino acid 342. However, Ala210 (the non-O residue) is unique to baboon; (non-O) Ala235 is also seen in macaque (11). It is interesting to note that the particular amino acid residue present at position 235 does influence enzymatic activity in humans, where it is serine for human group B, and glycine for human group A (9,12,18) as it is for baboon group O. However, the occurrence of Gly235 (instead of Ala235) in baboon 18, a BO heterozygote (Table 1), would seem to rule it out as the inactivating mutation. This unusual B allele must be functional, indicating that Gly235 (resulting from C704=G and homozygous in this animal) cannot by itself be the inactivating mutation of the O allele. Similarly, Ala342, which exists in demonstrably functional alleles in baboons 9,14,18,21, 23,26,31, and 33 (Table 1), and was not associated with reduced levels of antigen expression (data not shown), cannot be sufficient to cause the O phenotype. Both might nonetheless be necessary components of the major O allele. To the contrary, Val210 has not been observed in a definitively active allele in baboon (Table 1). The unique occurrence of Ala210, in both active forms of the baboon enzyme, may reflect selection for a residue which is necessary for the retention of enzymatic activity in context of that species’ sequence. Furthermore, the weak activity encoded by human A3 alleles is apparently due in some cases to a F216=I substitution (17). This lends credence to the idea that relatively conservative amino acid substitutions in this region can significantly alter enzymatic activity and that Ala210=Val does account for the lack of enzymatic activity (in those alleles where it occurs). In any event, this possibility is not excluded by our data. The absence of the mutations found in the
In contrast to the human O alleles, the mutations observed in baboon are not immediately compelling candidates as the causative mutations of the O phenotype. Neglecting for the moment the B-like O allele, there are three amino acid substitutions commonly, but not universally, encountered in O alleles, Ala210=Val, Ala235= Gly, and Ala342 (not Thr342). If one assumes that all of these O alleles were derived from the same prototype O allele, the occurrence (in baboons 3 and 16) of two apparent O alleles that do not have these substitutions implies that the causative mutation(s) lie elsewhere. An alternate interpretation that we offer is that the alleles seen in baboons 3 and 16 represent a different prototype and underlying defect than found in the major O allele represented by the O homozygotes. The B-like O allele from baboon 30 clearly must be independently derived from the A-like O allele(s) and thus also constitutes an additional baboon O allele prototype. A B-derived O allele has not been previously observed. In fact, while group O is not uncommon in chimpanzee, which exhibits group A but not B, it has not been found in gorilla, which is monomorphic for group B (1). While we have not been able to identify differences from active alleles for either of these additional prototype O alleles, it has certainly been the case with rare human phenotypes (e.g. A3 and B3), that they are genetically heterogeneous with only some of the alleles having mutations identified by sequencing the same region we have sequenced here (2, 17). The presence of all three of these apparent O alleles in heterozygous animals establishes that the lesion(s) responsible for the lack of enzymatic activity must reside at this locus. Were the lesion to reside at some other locus it would be expected to impact both blood group alleles, but clearly this is not the case. We expect that further sequencing of the upstream regions of the gene or cDNA will clarify the nature of the lesion(s).
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4.
human O alleles from the baboon O alleles, as well as the derivation of the baboon alleles from both an A and a B allele, demonstrates the independent origin of the O phenotype in these species. The appearance of the O phenotype, and in particular its prevalence in humans, combined with the loss of the B phenotype in chimpanzee and the A phenotype in gorilla (1), raises the prospect that this locus is no longer under (strong) selective pressure in the hominoid lineage. However, since our data suggest that O alleles have arisen multiple times in baboon while the phenotype has remained rare, selection may still be operating at this locus in baboon. Nonetheless, group O baboons appear healthy, so it should be possible to breed a population of them to serve as universal histoblood group organ donors in xenotransplantation efforts. The sequence markers we have discovered will enable ABO genotyping for various purposes including the identification of O heterozygous animals, screening of embryos following in vitro fertilization, and differentiation of host and donor derived tissues (e.g. bone marrow) following transplantation.
5.
6.
7.
8.
9.
10.
11.
ACKNOWLEDGMENTS 12.
We thank John Rossi for helpful discussions; Susan Grinde and Cathy Hisey for their expert technical assistance; and the Center for Molecular Biology and Gene Therapy DNA Core Facility, under the direction of Garry Larson, for oligonucleotide synthesis and DNA sequencing. This work was supported by Loma Linda University and the Loma Linda University Medical Center.
13.
14.
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Yamamoto F, Marken J, Tsuji T, White T, Clausen H, Hakomori S. Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuca1=2Gala1=3GalNAc Transferase (histoblood group A transferase) mRNA. J Biol Chem 265:146-1151, 1990. Yamamoto F, McNeill PD, Yamamoto M, et al.
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Sequence Comparison of Baboon ABO Histo-Blood Group Alleles: Lesions Found in O Alleles Differ between Human and Baboon Submitted 05/02/97; revised 05/29/97 (communicated by Brian S. Bull, M.D., 06/30/97)
David C. Diamond1,2, Omar R. Fagoaga2, Sandra L. Nehlsen-Cannarella2, Leonard L. Bailey3, Aladar A. Szalay1 ABSTRACT: Histo-blood group O has only rarely been observed in baboon. Recent discovery of such an animal has provided us the opportunity to investigate the molecular genetics of the ABO locus in baboons. The major baboon prototype O allele, observed in two homozygous and several heterozygous animals, is related to the A allele as is the case in humans. Additional apparent prototype O alleles have been observed in heterozygotes, one of which is related to the B allele. The nucleotide changes conferring the O phenotype in the two known human O alleles have not been observed in any baboon allele. This information will aid the identification of baboons useful for the development of xenotransplantation in humans. Keywords: ABO glycotransferase, baboon, glycosyltransferase, histo-blood group, xenotransplantation
INTRODUCTION
attack and can be freely transplanted. Thus, blood group O individuals are considered universal donors of both blood and organs. However, while blood group O is the most common type in humans, it is exceedingly rare in baboons (1,4). This has been an impediment to the development of xenotrans-plantation protocols involving the use of baboon organs (5,6). Previous work on the molecular genetics of the ABO system has established the cDNA sequence and genomic structure of the locus in humans (2, 7, 8), as well as the amino acid residues conferring the different enzymatic activities (9). The coding sequence of the transferase is divided into seven exons. Exon 6 and 7 encode the bulk of the enzyme (13% and 65%, respectively), including its active site. In all examined primate species the residues critical for determining donor
The ABO histo-blood group antigens, the basis of blood type, are found in all anthropoid primates (1). These antigens, which are terminal carbohydrate structures, can be found both in soluble form and on the surface of a variety of tissues, depending on the species examined. Additionally, antibodies to the non-expressed antigen(s) are universally present, agglutinating mismatched blood, and facilitating complement-mediated attack on tissues following transplant. Genetically, the phenotype is controlled by a single glycosyltransferase locus that can be occupied by three fundamental alleles encoding enzymes with either A or B activity or no activity (type O) (for reviews see 2, 3). Cells from blood group O individuals lack both antigens and thus are not subject to
1
Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA 92350; Immunology Center, Department of Pathology, Loma Linda University, Loma Linda, CA 92350; 3 Department of Surgery, Loma Linda University and Medical Center, Loma Linda, CA . Reprint request to: David C. Diamond, Ph.D.,Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA 92350. phone (909)824-4300, ext. 81370, fax (909)478-4177, email: [email protected] 2
Published by Academic Press Established by Springer-Verlag, Inc. in 1975
1079-9796/97 $25.00 Copyright r 1997 by The Blood Cells Foundation, La Jolla, California, USA All rights of reproduction in any form reserved.
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substrate specificity (i.e., A versus B activity) are found at amino acid positions 266 and 268 (numbering according to 2,3), Leu vs. Met and Gly vs. Ala, respectively (10,11). In humans, blood group O arises from either of two mutations in an A-like background; a frameshift in exon 6 leading to premature termination, or a Gly=Arg mutation in exon 7 at position 268 (12-14). As part of our ongoing experimentation in animal models of xenotransplantation (see for example 15), we discovered a blood group O baboon using standard serologic phenotyping methods. To utilize this finding, and to advance their potential as organ donors to humans, we decided to examine the molecular basis of blood group O in baboons. We now report the existence of at least two prototype O alleles in baboon: one, now observed in two O homozygotes and several apparent BO heterozygotes, is derived from an A allele; another, observed in an apparent AO heterozygote, is derived from a B allele. A possible third prototype O allele, A-derived, has also been observed in a pair of BO heterozygotes. None of these share the mutations found in the human O alleles.
were collected from the femoral vein. Buccal mucosa epithelial cells were also collected by swabbing the buccal mucosa and applying the cells to a glass slide. The slides were then dried overnight. Direct typing was performed using the standard immunophenotyping assay with immunofluorescent-stained scrapings of the buccal epithelium (16). Conventional reverse typing is performed using baboon serum (pre-adsorbed with human blood group O erythrocytes) to agglutinate human A and B type erythrocytes.
DNA Isolation Peripheral blood lymphocytes were isolated from whole blood by ficoll gradient centrifugation and used as a source of genomic DNA. DNA isolation was carried out using either the RapidPrepy Macro Genomic DNA Isolation Kit (Pharmacia), initially, or the QIAamp Blood Kit (Qiagen) according to the manufacturers’ directions.
PCR Primers were designed based on the human sequence (7,8,12,17). Three upstream and two downstream primers were used for amplification of genomic DNA: UPex5L, GATGGTCTACCCCCAGCCAAAGGTGCT, covering most of exon 5; UPF, TGGGCCACCGTGTCCACTACTATGTCTT, located toward the 58 end of exon 7; UPH, CCAAGGACGAGGGTGATTTCT, located centrally in exon 7; LPC, TCCGGACCGCCTGGTGGTTCTTG, located near and complementary to the 38 end of exon 7; and Uaftx7, AGCCCTCCCAGAGCCCCTGG, located in the untranslated region on the non-coding strand shortly after the stop codon. UPF-LPC reactions contained each primer at a concentration of 0.5µM, 300-500ng genomic DNA, dNTPs at a concentration of 200µM each, pfu polymerase (2.5 units) (Stratagene), and the supplied buffer, in 100µl. Typical thermocycling conditions were: denaturation at 94°C for 2 min.; 30 cycles at 94°C, 5 sec.,
MATERIALS AND METHODS Animals All of the baboons used in this study were olive baboons (Papio cynocephalus anubis) from the Southwest Biomedical Research Foundation (San Antonio, TX). Animal care followed the guidelines of the standards of the National Society for Medical Research, Principles of Laboratory Animal Care, and the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (Publication no. 86-23, revised, 1985).
Serologic Phenotyping Using ketamine anesthesia (15mg/kg), 10 ml heparinized (100 U/ml) and 5 ml clotted blood 243
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55.9°C, 30 sec., and 72°C for 2.5 min.; followed by 72°C for 7 min. on a GeneAmp PCR System 9600 (Perkin Elmer). A hot start was achieved by adding enzyme while paused at the first annealing step. UPex5L-LPC and UPH-Uaftx7 reactions contained each primer at a concentration of 0.4µM, respectively, 200ng genomic DNA, dNTPs at a concentration of 350µM each, Expand Polymerase - a mixture of Pwo and taq polymerases - (2.6 units)(Boehringer-Mannheim) and supplied buffer 1, in 50µl. For UPex5L-LPC typical thermocycling conditions were: denaturation at 94°C for 2 min.; 30 cycles at 94°C, 5 sec., 56°C, 30 sec., and 68°C for 4 min. plus 20 sec. each cycle for the last 20; followed by 68°C for 7 min., on a GeneAmp PCR System 9600 (Perkin Elmer). UPH-Uaftx7 required a touchdown cycle: denaturation at 94°C for 2 min.; 20 cycles of 94°C, 5 sec., 66°C, 30 sec., reduced one degree each cycle; 68°C, 5 min.; 20 cycles of 94°C, 5 sec., 46°C, 30 sec.; 68°C, 4 min. plus 10 sec. each cycle; followed by 68°C for 7 min., on a GeneAmp PCR System 9600. Hot starts were achieved in the same manner. Success of reactions was judged by agarose gel electrophoresis.
tative animals was sequenced with the complete primer set. Additional animals screened for heterozygosity were generally sequenced with only the primers UPH and UPF initially. DNA from all of the screened animals thus identified as heterozygotes was further amplified and sequenced with UPH and Uaftx7b. Finally, an allele-specific primer, RASAO, ACCGACCCTCCGAAGAACCCCCCCAA, located so that its 38 end paired with N796 of A-like alleles, was used to resolve multiple heterozygosities as appropriate.
RESULTS Phenotyping Forward and reverse typing was performed to establish the ABO blood group of all baboons studied (Table 1). Unlike hominoids (humans and great apes), old world monkeys such as the baboon do not express ABO antigens on erythrocytes (1). Thus, while conventional reverse typing is feasible, forward typing is not. These antigens however, are also expressed on the cells of other tissues, including epithelial cells. Therefore forward typing was performed on scrapings of the buccal epithelium (16). Generally this phenotyping was entirely consistent with subsequent genotyping (below). Figure 1 shows typical staining patterns for group A,B, and O baboons.
DNA Sequencing PCR products were purified either directly from the reaction (UPF-LPC and UPex5L-LPC) or from agarose gel slices (UPH-Uaftx7) using QIAquick spin columns (Qiagen) and then used directly in cycle sequencing using an Applied Biosystems 373A and Amplitaq CS (initially) or FS. Additional primers used for sequencing were: LPex6a1, TTGATGGCAAACACAGTTAACCCAATGG, located near and complementary to the 38 end of exon 6; IB4x6, AGAGGAGGCGGAAACTGAG, located in the intron upstream of exon 6; IB4x7b, TCTGAGCCTTCCAATGTCCGCTG, located in the intron upstream of exon 7; and LPm7, GTCCACGCACACCAGGTAATCCAC, located centrally in and complementary to exon 7. Unlike the other primers, IB4x6 and IB4x7b were both designed from baboon intronic sequences (data not shown). DNA from represen-
Amplification and Sequencing Genomic DNA isolated from PBLs was used as template for the polymerase chain reaction with three primer pairs, UPF-LPC, UPex5L-LPC, and UPH-Uaftx7b. The expected products, a 630 bp central segment of exon 7, an ,2.3 kb segment stretching from exon 5 through most of exon 7, and a 315 bp segment including the 38 end of the coding region of exon 7, respectively, were obtained and used as template in automated fluorescent sequencing. Even using a touchdown cycle, UPH-Uaftx7 generated many extraneous bands and required gel purification prior to sequencing.
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Table 1. Association of DNA Sequence Polymorphisms with ABO Types.
Baboon
N480
N629
N651
N704
N711
N1024
Phenotype
Sequence Type
Genotype
9
G
C
C
C
C
G
A
AA
AA
10
G
C
C
C
C
G/A
A
AA
AA
31
G
C
C
C
C
G
A
AA
AA
7
G
C
C
C
C
A
B
BB
BB
11
A/G
C
C
C
C
G/A
B
BB
BB
12
G
C
C
C
C
G/A
B
BB
BB
13
A/G
C
C
C
C
G/A
B
BB
BB
15
G
C
C
C
C
G/A
B
BB
BB
17
G
C
C
C
C
G/A
B
BB
BB
19
A/G
C
C
C
C
G/A
B
BB
BB
20
G
C
C
C
C
A
B
BB
BB
22
G
C
C
C
C
G/A
B
BB
BB
23
A/G
C
C
C
C
G
B
BB
BB
24
G
C
C
C
C
G/A
B
BB
BB
26
G
C
C
C
C
G
B
BB
BB
27
G
C
C
C
C
A
B
BB
BB
28
G
C
C
C
C
G/A
B
BB
BB
29
G
C
C
C
C
A
B
BB
BB
33
G
C
C
C
C
G/A
AB
AB
AB
30
A/G
C
C
C
C
G/A
A
AB!
AO
3
G
C
C
C
C
A
B
AB
BO
16
G
C
C
C
C
G/A
B
AB
BO
1
A/G
T/C
T/C
G/C
T/C
G/A
A
AA
AO?
2
A/G
T/C
T/C
G/C
T/C
G/A
A
AA
AO?
6
A/G
T/C
T/C
G/C
T/C
G/A
B
AB
BO
8
A/G
T/C
T/C
G/C
T/C
G/A
B
AB
BO
14
A/G
T/C
T/C
G/C
T/C
G
B
AB
BO
21
A/G
T/C
T/C
G/C
T/C
G
B
AB
BO
18
A/G
T/C
T/C
G
T
G
B
AB
BO
5
A
T
T
G
T
G
O
AA
OO
32
A
T
T
G
T
G
O
AA
OO
Effect of polymorphism
Silent
AA210 T5Val C5Ala
Silent
AA235 G5Gly C5Ala
Silent
AA342 G5Ala A5Thr
The nucleotide residue at 6 positions that were polymorphic in baboon is presented for all of the animals used in this study. N629, N704, and N1024 determine which amino acid residue (AA) is found at position 210, 235, and 342, respectively. Phenotype was determined serologically. Sequence Type refers to which nucleotide residues were found in the donor sugar specificity-determining region, specifically N796, N803, and N813. Genotype was inferred from phenotype and sequence type as described in the text. The animals are grouped by putative prototype O allele and subgrouped by phenotype.
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Figure 1. ABO Phenotyping by Immunofluorescent Staining of Buccal Mucosa. The inner panels in each row show immunofluorescence images of buccal mucosa from a group A (baboon 31), group B (baboon 28), and group O (baboon 5) animal stained with anti-A or anti-B antibody and phycoerythrin labeled second antibody. The outer panels show the adjacent fields of view under brightfield illumination. Immunofluorescence that is not cell-associated is due to soluble antigen in saliva.
ber 32) exhibiting these same sequence features (Table 1).
Representative sequences of exons 6 and 7 are presented in figure 2. Three nucleotide differences are consistently found between blood group A and B, T796, G803, and A813 in A become A, C, and G, respectively, in B. The first two differences result in the Leu266=Met and Gly268=Ala amino acid substitutions crucial for conversion between A and B transferase activity. The third difference is silent. DNA sequence from the blood group O baboon (number 5) resembled that of group A baboons at the positions described above. Neither the G261 deletion or the G802=A (Gly268=Arg) mutations found in human O alleles were present. Four nucleotide differences from the baboon A/B consensus were observed. Two, C651=T and C711=T, are silent. The other two, C629=T and C704=G, cause Ala210=Val and Ala235=Gly amino acid changes, respectively. Subsequently, we have identified a second group O baboon (num-
Heterozygotes with O Alleles We realized that, even without identifying the causative mutation for the baboon O phenotype, it would be possible to genotype serologic-group B phenotypes as either BB or BO based on the A-like sequence of the O allele. This screening would also allow us to evaluate the usefulness of the four nucleotide differences seen in the O homozygote as markers for the O allele. To accomplish this task we concluded that all serologic-group B phenotypes that appear to be AB genotypes must be BO heterozygotes. Since we are simultaneously sequencing a mixture of both alleles from single individuals, we cannot definitively resolve multiple heterozygosities. Using
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Figure 2. Comparison of Representative Sequences of the ABO Transferase. The sequences of exon 6, and exon 7 through the termination codon, of representative homozygous baboons of each blood group are aligned with each other and the published sequence for human A transferase (2). The sequence from the group A baboon is explicitly presented, as are differences from it in the other three sequences; identical bases are indicated by a hyphen (-). The boundary between the two exons is indicated by a vertical line (0). Positions where different amino acids are encoded by the different sequences are marked with *’s below the sequences. The three nucleotides used to classify baboon sequences as either A-like or B-like (i.e., sequence type) are marked with W’s, and the four nucleotide positions useful in distinguishing the baboon major O allele are marked with V’s, above the sequences. The positions of the deletion and substitution defining the human major and minor O alleles are marked with D and O, respectively. These sequences can be found in GenBank under the accession numbers AF001425-AF001427 for the baboon A, B, and O alleles, respectively.
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Sequence Variation
Occam’s razor we have chosen to interpret these sequences as resulting from two alleles, each of which most closely resembles one of the alleles found in the homozygotes above. For example, an animal with heterozygosities at N796 and N803 would be interpreted as an AB genotype, not a pair of reciprocal A-B chimeras. By the above criterion, 7 of 22 blood group B phenotype baboons (4 of 14 males and 3 of 8 females) were BO heterozygotes (Table 1). Examining these eleven O allele sequences (from the 7 BO heterozygotes and the two OO homozygotes), we observed that N629 was a T (instead of C) in 9 of the 11 cases, as were N651 and N711. Similarly, N704 was a G (instead of C) in 9 of these 11 cases. All of the B alleles had a C at N629 and N651. However, in baboon 18 N704 and N711 were homozygous indicating that the B allele was similar to the O allele with a G and a T at these positions, respectively. Subsequently these animals were sequenced using an A/O allele-specific primer, RASAO, positioned at the specificity-determining nucleotides. By using this primer to generate sequence data specifically from the A-like allele we were able to confirm that the O-like residues at N480, N629, N651, N704, and N711 were indeed all found in the A-like allele. Due to the similarity of the A and O alleles, and unlike the group B animals above, the phenotype of group A animals is generally not informative in deducing their genotype. Nonetheless, the nucleotide differences observed in the O homozygotes exhibited enough linkage that it was possible to screen for putative AO heterozygotes. By this criterion, 2 of 6 type A baboons (all female) were O heterozygotes (Table 1). All four positions where nucleotide differences were observed in the O homozygotes (N629, N651, N704, and N711 ) were heterozygous in these animals (Table 1). To our surprise, one of the 6 serologic group A animals (baboon 30) gave an apparent genotype of AB based on its sequence type (Table 1). Thus the ‘‘B’’ allele in this animal must not encode an active enzyme, i.e. it is constitutes an additional O allele prototype.
In addition to the group-associated differences discussed above, several other polymorphisms, that were either less rigorously associated with type or represented individual variation, have been observed. N1024 is polymorphic A/G; encoding either ThrACT or AlaGCT. N1024 might always be G in the major O allele (see below). Allele-specific PCR supports this assignment for baboons 6 and 8, but it remains ambiguous for baboons 1 and 2 (data not shown). N480, a silent position that is predominantly G but sometimes A, was invariably A in the major O allele in those animals in which the heterozygosity could be resolved (i.e., baboons 6,8,14,18, and 21; see Table 1). A silent G/T polymorphism was observed at N1056 but no significant pattern was discerned. Finally there were four single instance variations observed: in baboon 1, G1010 was heterozygous G/A resulting in a conservative switch from Arg to Lys; and in baboon 18, A681, G1026 and G1062 are all heterozygous G/A, but the resulting codons are synonymous.
Comparison with human sequences The baboon and human group A sequences from Fig. 2 are 96.5% identical, differing at 29 of the 826 positions (including the baboon polymorphism at N1024 as a difference). Twenty-four of these substitutions are silent, including all 6 found in exon 6. The predicted amino acid sequences thus differ at 5 of 274 residues (all in exon 7), for 98.2% identity. Comparing group B sequences (using human as reported in ref. 2) reveals 31 nucleotide differences, 27 of which are the same as seen in the A to A comparison, and six amino acid differences, including the five positions where the group A sequences differ, although at one of these positions (amino acid 235) a different residue is involved as noted below. Thus comparing the group B sequences, there was 96.2% and 97.8% identity for nucleotides and amino acids, respectively.
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DISCUSSION
Do the three variant amino acids listed above contribute to the lesion in the major O allele? The Val210 and Gly235 residues seen in baboon blood group O are observed in active alleles in a wide array of other primates (10,11); data from other species are not available for amino acid 342. However, Ala210 (the non-O residue) is unique to baboon; (non-O) Ala235 is also seen in macaque (11). It is interesting to note that the particular amino acid residue present at position 235 does influence enzymatic activity in humans, where it is serine for human group B, and glycine for human group A (9,12,18) as it is for baboon group O. However, the occurrence of Gly235 (instead of Ala235) in baboon 18, a BO heterozygote (Table 1), would seem to rule it out as the inactivating mutation. This unusual B allele must be functional, indicating that Gly235 (resulting from C704=G and homozygous in this animal) cannot by itself be the inactivating mutation of the O allele. Similarly, Ala342, which exists in demonstrably functional alleles in baboons 9,14,18,21, 23,26,31, and 33 (Table 1), and was not associated with reduced levels of antigen expression (data not shown), cannot be sufficient to cause the O phenotype. Both might nonetheless be necessary components of the major O allele. To the contrary, Val210 has not been observed in a definitively active allele in baboon (Table 1). The unique occurrence of Ala210, in both active forms of the baboon enzyme, may reflect selection for a residue which is necessary for the retention of enzymatic activity in context of that species’ sequence. Furthermore, the weak activity encoded by human A3 alleles is apparently due in some cases to a F216=I substitution (17). This lends credence to the idea that relatively conservative amino acid substitutions in this region can significantly alter enzymatic activity and that Ala210=Val does account for the lack of enzymatic activity (in those alleles where it occurs). In any event, this possibility is not excluded by our data. The absence of the mutations found in the
In contrast to the human O alleles, the mutations observed in baboon are not immediately compelling candidates as the causative mutations of the O phenotype. Neglecting for the moment the B-like O allele, there are three amino acid substitutions commonly, but not universally, encountered in O alleles, Ala210=Val, Ala235= Gly, and Ala342 (not Thr342). If one assumes that all of these O alleles were derived from the same prototype O allele, the occurrence (in baboons 3 and 16) of two apparent O alleles that do not have these substitutions implies that the causative mutation(s) lie elsewhere. An alternate interpretation that we offer is that the alleles seen in baboons 3 and 16 represent a different prototype and underlying defect than found in the major O allele represented by the O homozygotes. The B-like O allele from baboon 30 clearly must be independently derived from the A-like O allele(s) and thus also constitutes an additional baboon O allele prototype. A B-derived O allele has not been previously observed. In fact, while group O is not uncommon in chimpanzee, which exhibits group A but not B, it has not been found in gorilla, which is monomorphic for group B (1). While we have not been able to identify differences from active alleles for either of these additional prototype O alleles, it has certainly been the case with rare human phenotypes (e.g. A3 and B3), that they are genetically heterogeneous with only some of the alleles having mutations identified by sequencing the same region we have sequenced here (2, 17). The presence of all three of these apparent O alleles in heterozygous animals establishes that the lesion(s) responsible for the lack of enzymatic activity must reside at this locus. Were the lesion to reside at some other locus it would be expected to impact both blood group alleles, but clearly this is not the case. We expect that further sequencing of the upstream regions of the gene or cDNA will clarify the nature of the lesion(s).
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4.
human O alleles from the baboon O alleles, as well as the derivation of the baboon alleles from both an A and a B allele, demonstrates the independent origin of the O phenotype in these species. The appearance of the O phenotype, and in particular its prevalence in humans, combined with the loss of the B phenotype in chimpanzee and the A phenotype in gorilla (1), raises the prospect that this locus is no longer under (strong) selective pressure in the hominoid lineage. However, since our data suggest that O alleles have arisen multiple times in baboon while the phenotype has remained rare, selection may still be operating at this locus in baboon. Nonetheless, group O baboons appear healthy, so it should be possible to breed a population of them to serve as universal histoblood group organ donors in xenotransplantation efforts. The sequence markers we have discovered will enable ABO genotyping for various purposes including the identification of O heterozygous animals, screening of embryos following in vitro fertilization, and differentiation of host and donor derived tissues (e.g. bone marrow) following transplantation.
5.
6.
7.
8.
9.
10.
11.
ACKNOWLEDGMENTS 12.
We thank John Rossi for helpful discussions; Susan Grinde and Cathy Hisey for their expert technical assistance; and the Center for Molecular Biology and Gene Therapy DNA Core Facility, under the direction of Garry Larson, for oligonucleotide synthesis and DNA sequencing. This work was supported by Loma Linda University and the Loma Linda University Medical Center.
13.
14.
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Yamamoto F, Marken J, Tsuji T, White T, Clausen H, Hakomori S. Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuca1=2Gala1=3GalNAc Transferase (histoblood group A transferase) mRNA. J Biol Chem 265:146-1151, 1990. Yamamoto F, McNeill PD, Yamamoto M, et al.
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