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D I Watkins

Publications and source records attributed to D I Watkins.

At least 73 records · Page 4Linked to original sources
PubMed1997

High-resolution HLA-DRB typing using denaturing gradient gel electrophoresis and direct sequencing.

High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. Here we describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB-specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Importantly, our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing.

Alleles↗
PubMed1997

Further diversification of the HLA-B locus in Central American Amerindians: new B*39 and B*51 alleles in the Kuna of Panama.

Several new HLA-B locus alleles have been discovered in South American Amerindians. By contrast, analysis of the MHC class I alleles of North American native populations has revealed few new HLA-B alleles. This suggests that the HLA-B locus is evolving rapidly in South American populations. Here we describe the HLA-B locus alleles present in individuals from a Central American tribe, the Kuna of Panama. Using a sequence-based typing technique that separates alleles by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we determined the HLA-B alleles from eight Kunas. Two of the HLA-B alleles present in the Kuna have been previously described in other South American Amerindian populations; one allele has been characterized in a Mexican-American. We characterized two new HLA-B alleles in the Kuna, HLA-B*3911 and HLA-B*5110. HLA-B*3911 differed from HLA-B*3905 by only a single nucleotide substitution in exon 3. This substitution resulted in an amino acid replacement of leucine by arginine at residue 156 in the alpha 2 domain. Such a change may affect the repertoire of peptides that are bound by this molecule. HLA-B*5110 differed significantly from other HLA-B*51 alleles in that it is the result of an unusually large intra-locus recombination event of minimally 216 nucleotides. This recombination results in an allele that is part HLA-B*51 and part HLA-B*40. Thus, more dramatic recombination events may also play a role in the rapid evolution of the HLA-B locus in Amerindians.

Alleles↗
PubMed1997

A 25% error rate in serologic typing of HLA-B homozygotes.

The microlymphocytotoxicity technique has been the accepted method for HLA class I typing since the early 1960s. However, it is often difficult to distinguish two related alleles expressed in an individual due to the cross-reactive nature of the alloantibodies used in this technique. This is especially evident at the HLA-B locus, whose more than 180 alleles fall into only 4 major interrelated cross-reactive antigen groups. To estimate the error rate in serologic typing due to the cross-reactive nature of sera, we used polymerase chain reaction with sequence-specific primers (PCR-SSP) amplification to retype 40 individuals who were previously typed as serologic HLA-B locus homozygotes. PCR-SSP revealed that 10 of these 40 individuals (25%) were actually heterozygous at their HLA-B loci. The HLA-B locus alleles of 9 of these 10 discrepant individuals were further analyzed by denaturing gradient gel electrophoresis followed by direct sequencing. The sequence analysis confirmed that all nine individuals were indeed HLA-B locus heterozygotes. This surprisingly high error rate in serologic definition of HLA-B molecules argues for the use of rapid DNA-based techniques in HLA class I typing, even in the setting of solid organ transplantation.

Amino Acid Sequence↗
PubMed1997

A high frequency of Mamu-A*01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing.

SIV infection of rhesus macaques is an excellent model for HIV infection of humans. Unfortunately, it is has been difficult to identify macaques expressing particular MHC class I alleles. Here we describe the use of PCR-SSP for Mamu-A*01 typing of rhesus macaques. The Mamu-A*01 allele was amplified from genomic DNA using Mamu-A*01-specific primers and positive PCR products were directly sequenced. Our technique identified 15 Mamu-A*01-positive animals of 68 tested. We validated our molecular analysis by showing that lymphocytes from 8 Mamu-A*01-positive animals expressed Mamu-A*01 as determined by immunoprecipitation and 1-D IEF. The technical simplicity and accuracy of this typing method should facilitate selection of Mamu-A*01-positive rhesus macaques for AIDS virus pathogenesis and vaccine studies.

Alleles↗
PubMed1996

Identification of the rhesus monkey HLA-G ortholog. Mamu-G is a pseudogene.

HLA-G is a nonclassical MHC class I molecule that is primarily expressed in the placenta. To investigate whether rhesus monkeys possess an HLA-G ortholog, we cloned and sequenced MHC class I cDNAs from the rhesus placenta. We identified two rhesus MHC class I cDNAs with sequence similarity to HLA-G. Each cDNA contained premature stop codons and frameshift mutations, suggesting that it was derived from an MHC class I pseudogene. Gene trees constructed using MHC class I alleles from human and nonhuman primates revealed that the rhesus placental pseudogene alleles clustered with HLA-G orthologs from the human, chimpanzee, and gorilla. These data suggested that this rhesus MHC class I pseudogene is an HLA-G ortholog. This locus was, therefore, designated Mamu (Macaca mulatta)-G. PCR amplification of a portion of Mamu-G from the genomic DNA of five rhesus monkeys resulted in the identification of five additional Mamu-G alleles and revealed the presence of four Mamu-G alleles in one rhesus monkey, suggesting that Mamu-G had been duplicated in this individual. Furthermore, the analysis of 81 MHC class I clones isolated from a rhesus placenta cDNA library did not result in the isolation of Mamu-G cDNAs, nor the isolation of any additional HLA-G homologs, suggesting that Mamu-G was transcribed at negligible levels. Given the similarity of rhesus monkey and human placenta structure and function, these data raise interesting questions regarding the role of HLA-G in pregnancy.

Alternative Splicing↗
PubMed1996

MHC class I-processed pseudogenes in New World primates provide evidence for rapid turnover of MHC class I genes.

The MHC class I genes of the New World primate, the cotton-top tamarin (Saguinus oedipus), are an exception to the high polymorphism and variability displayed by this multigene family. We report the isolation of the first two processed pseudogenes from the MHC region in primates. These two MHC class I-processed pseudogenes (MHC-PS1 and -PS2) were found in several species of New World primates, suggesting a possible explanation for the cotton-top tamarin's limited MHC class I diversity. The pattern of synonymous and nonsynonymous substitutions in PS1 suggests that the gene that gave rise to this processed pseudogene was once subject to selection for variability in the peptide binding region and might, therefore, have been functional. Additionally, PSI is not closely related to the expressed cotton-top tamarin's MHC class I genes, but does show some similarity to So-N1, a tamarin pseudogene from which no transcript has been found. Thus, PS1 may represent a remnant of a once active MHC class I gene that is no longer functional in the cotton-top tamarin. The MHC class I loci in primates, therefore, appear to be evolving by a continual process of duplication and inactivation. This process seems to be exaggerated in New World primates and may in part be responsible for the cotton-top tamarin's limited MHC class I diversity.

Animals↗
PubMed1996

The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates.

Homologues of the human HLA-A and -B MHC class I loci have been found in great apes and Old World primates suggesting that these two loci have existed for at least 30 million years. The C locus, however, shows some sequence similarity to the B locus and has been found only in gorillas, chimpanzees, and humans. To determine the age of the MHC class I C locus and to examine the evolution of the A and B loci we have cloned, sequenced, and in vitro translated 16 MHC class I cDNAs from two unrelated rhesus monkeys (Macaca mulatta) using both cDNA library screening and PCR amplification. Analyses of these sequences suggest that the C locus is not present in the rhesus monkey, indicating that this locus may be of recent origin in gorillas, chimpanzees, and humans. The rhesus monkey's complement of MHC class I genes includes the products of at least one expressed A locus and at least two expressed B loci, indicating that a duplication of the B locus has taken place in the lineage leading to these Old World primates. Comparison of rhesus monkey MHC class I cDNAs to their primate counterparts reveals fundamental differences between MHC class I and class II evolution in primates. Although MHC class II allelic lineages are shared between humans and Old World primates, no such trans-species sharing of allelic lineages is seen at the MHC class I loci.

Animals↗
PubMed1996

Identification of new TAP2 alleles in gorilla: evolution of the locus within hominoids.

Transporters associated with antigen processing molecules (TAP1 and TAP2) mediate the transfer of cytosolic peptides into the lumen of the endoplasmic reticulum for association with newly synthesized class I molecules of the major histocompatibility complex. Previous molecular and functional analyses of rat and human TAP2 homologues indicated major differences in gene diversification patterns and selectivity of peptides transported. Therefore, in this study, we analyzed the alleles of the gorilla TAP2 locus to determine whether the pattern of diversification resembled that in either of those two species. Sequence analysis of the TAP2 cDNAs from gorilla Epstein-Barr virus-transformed B-cell lines revealed four alleles with a genetic distance of less than 1%. The nucleotide substitutions distinguishing the alleles are confined to the 3' half of the coding region and occur individually or within two small clusters of variability. Diversification of the locus appears to have resulted from point substitutions and recombinational events. Evolutionary-rate estimates for the TAP2 gene in gorilla and human closely approximate those observed for other hominoid genes. The amino acid polymorphisms within the gorilla molecules are distinct from those in the human homologues. The absence of ancestral polymorphisms suggests that gorilla and human TAP2 genes have not evolved in a trans-species fashion but rather have diversified since the divergence of the lineages.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The T-cell receptor beta chain-encoding gene repertoire of a New World primate species, the cotton-top tamarin.

The New World primate, the cotton-top tamarin (Saguinus oedipus), expresses major histocompatibility complex (MHC) class I molecules with limited diversity. The uniqueness of the cotton-top tamarin MHC class I loci may contribute to this species' unusual susceptibility to viral infections and high incidence of ulcerative colitis. As a prelude to examining the effect of this limited MHC class I diversity on the tamarin CD8(+) T-cell receptor (TCR) repertoire, we identified expressed tamarin TCR beta chain (TCRB) cDNAs by anchored and inverse polymerase chain reaction. Sequence alignments and phylogenetic comparisons with human and rhesus macaque sequences identified homologues of 21 human variable (V) gene families. Only single variable region genes were identified in each of these tamarin VB families, with the exception of the VB 5, 9, and 13 families which were comprised of two or three distinct members. The multiple genes within these three VB families do not appear to have separate human homologues, but rather aligned equally well to a single human gene from their respective VB families. These genes appear to have arisen, therefore, by duplication of certain VB genes in the tamarin ancestors following their divergence from the lineage leading to Old World primates and hominoids. Homologues of 12 of the 13 human joining (J) region genes were also identified in the tamarin. Comparison of the proportion of nonsynonymous (pN) and synonymous (pS) substitutions occurring per site within tamarin variable region genes demonstrated a reduction in pN in the framework regions compared with pN in the presumed MHC contact regions (CDR1 and CDR2). Taken together, these findings illustrate that the TCR beta chain-encoding genes of the cotton-top tamarin are similar in structure and degree of complexity compared with their Old World primate and human counterparts.

Amino Acid Sequence↗
PubMed1996

HLA-B alleles of the Navajo: no evidence for rapid evolution in the Nadene.

New HLA-B locus alleles have been found in South American Amerindian populations but were largely absent in North American Amerindian tribes also descended from this first Paleo-Indian migration. We have now extended these studies to the Navajo, descendants of the second Nadene migration. No new functional alleles were found at the B locus of this tribe. This limited study supports the notion that while new B locus variants are common in South American Amerindians, it is more difficult to find new B locus alleles in North American native peoples. Whether this dichotomy is due to differences in pathogen environment and/or population structures between North and South America remains a subject of speculation.

Alleles↗
PubMed1995

Chimpanzee MHC class I A locus alleles are related to only one of the six families of human A locus alleles.

There are nearly 50 alleles at the highly polymorphic HLA-A class I locus that fall into six distinct families. To determine the allelic repertoire and the mechanism of generation of diversity of the A locus in primates we have analyzed A locus alleles from 28 apparently unrelated chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). We have, therefore, compared the sequences of 19 HLA-A homologues from chimpanzees and bonobos to 42 HLA-A sequences. HLA-A homologues were well preserved in chimpanzees and bonobos with very few new substitutions present in the A locus alleles of both species of chimpanzee. Surprisingly, all chimpanzees and bonobos expressed A locus alleles related to only one of the six families of human HLA-A alleles. This suggests that the common ancestor of these two species either passed through a genetic bottleneck or that selection has favored the maintenance of the HLA-A1, -A3, -A11 family in chimpanzees.

Alleles↗
PubMed1995

A MHC class I B locus allele-restricted simian immunodeficiency virus envelope CTL epitope in rhesus monkeys.

In light of the importance of virus-specific CTL in the control of the spread of the AIDS virus, it will be important to assess the generation of these effector cell responses in trials of novel vaccine strategies for the prevention of AIDS virus infections. To facilitate such studies in the simian immunodeficiency virus (SIV)/macaque model for AIDS, we have defined a rhesus monkey SIVmac CTL epitope carboxy terminus to both the CD4-binding and V4 regions of the envelope glycoprotein. We also used one-dimensional isoelectric focusing to characterize the MHC class I molecule of the rhesus monkey that binds this 9-amino-acid SIVmac envelope fragment. Cloning and sequencing of the cDNA encoding this rhesus monkey MHC class I molecule demonstrated that it is a newly described HLA-B homologue, Mamu-B*01. The definition of this viral CTL epitope and its restricting MHC class I molecule will facilitate the use of the SIVmac/rhesus monkey model for studies of envelope-based vaccine strategies for the prevention of AIDS.

Alleles↗
PubMed1995

HLA-B alleles of the Cayapa of Ecuador: new B39 and B15 alleles.

Recent data suggest that HLA-B locus alleles can evolve quickly in native South American populations. To investigate further this phenomenon of new HLA-B variants among Amerindians, we studied samples from another South American tribe, the Cayapa from Ecuador. We selected individuals for HLA-B molecular typing based upon their HLA class II typing results. Three new variants of HLA-B39 and one new variant of HLA-B15 were found in the Cayapa: HLA-B*3905, HLA-B*3906, HLA-B*3907, and HLA-B*1522. A total of thirteen new HLA-B alleles have now been found in the four South American tribes studied. Each of these four tribes studied, including the Cayapa, had novel alleles that were not found in any of the other tribes, suggesting that many of these new HLA-B alleles may have evolved since the Paleo-Indians originally populated South America. Each of these 13 new alleles contained predicted amino acid replacements that were located in the peptide binding site. These amino acid replacements may affect the sequence motif of the bound peptides, suggesting that these new alleles have been maintained by selection. New allelic variants have been found for all common HLA-B locus antigenic groups present in South American tribes with the exception of B48. In spite of its high frequency in South American tribes, no evidence for variants of B48 has been found in all the Amerindians studied, suggesting that B48 may have unique characteristics among the B locus alleles.

Alleles↗
PubMed1995

The MHC E locus in macaques is polymorphic and is conserved between macaques and humans.

Although the functions of the molecules encoded by the classical MHC class I loci are well defined, no function has been ascribed to the molecules encoded by the non-classical MHC class I loci. To investigate the evolution and conservation of the non-classical loci, we cloned and sequenced HLA-E homologues in macaques. We isolated four E locus alleles from five rhesus monkeys and two E locus alleles from one cynomolgus monkey, which indicated that the E locus in macaques is polymorphic. We also compared the rate of nucleotide substitution in the second intron of the macaque and human E locus alleles with that of exons two and three. The rate of nucleotide substitution was significantly higher in the introns, which suggested that the E locus has evolved under selective pressure. Additionally, comparison of the rates of synonymous and non-synonymous substitutions in the peptide binding region versus the remainder of the molecule suggested that the codons encoding the amino acids in the peptide binding region had been conserved in macaques and humans over the 36 million years since macaques and humans last shared a common ancestor.

Amino Acid Sequence↗
PubMed1995

The evolution of major histocompatibility class I genes in primates.

MHC class I genes evolve by recombination, largely within loci, and selection (presumably pathogen-driven) maintains these new alleles in the population. Although chimpanzees and humans share an A locus allelic lineage, the B locus molecules of the chimpanzee were less similar to human B locus molecules. The A and B locus molecules in rhesus and cynomolgus monkeys were even less similar to their human counterparts, with little conservation of allelic lineages between macaques and humans. In contrast to the instability of MHC class I alleles and allelic lineages, the MHC class I loci themselves are well conserved during the evolution of Great Ape and Old World primates. Homologues of HLA-A, -B, -E, and -F have been found in macaques. The C locus, however, has only been found in gorillas and chimpanzees, whereas in orangutans and rhesus monkeys it is possible that the A and B loci have been duplicated. Classical New World monkey MHC class I genes are all more similar to the nonclassical HLA-G gene and a nonclassical F-like gene is present in the cotton-top tamarin. Duplication and either subsequent deletion or expansion of MHC class I loci, therefore, appear to be the modus operandi of the evolution of these genes in primates.

Amino Acid Sequence↗
PubMed1994

A uniquely high level of recombination at the HLA-B locus.

Major histocompatibility complex (MHC) loci are some of the most polymorphic genes in the animal kingdom. Recently, it has been suggested that although most of the human MHC loci are relatively stable, the HLA-B locus can undergo rapid changes, especially in isolated populations. To investigate the mechanisms of HLA-B evolution we have compared the sequences of 19 HLA-B homologues from chimpanzees and bonobos to 65 HLA-B sequences. Analysis of the chimpanzee and bonobo HLA-B homologues revealed that despite obvious similarities between chimpanzee and human alleles in exon 2, there was little conservation of exon 3 between humans and the two chimpanzee species. This finding suggests that, unlike all other HLA loci, recombination has characterized the HLA-B locus and its homologues for over 5 million years.

Amino Acid Sequence↗