Search PubMed⌕ Search

Biomedical subjects

M T Boyce-Jacino

Publications and source records attributed to M T Boyce-Jacino.

15 recordsLinked to original sources
PubMed2003

Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

Alleles↗
PubMed2000

A SNPshot: pharmacogenetics and the future of drug therapy.

Pharmacogenetics holds great promise for the optimization of new drug development and the individualization of clinical therapeutics in the 21st century. In this brief review, we trace the historical roots of pharmacogenetics, discuss its rapidly evolving processes and paradigms, and look towards future applications of pharmacogenetics in enhancing the efficiency of the drug development pipeline and in improving patient care.

Drug Therapy↗
PubMed1999

Immobilization of oligonucleotides onto a glass support via disulfide bonds: A method for preparation of DNA microarrays.

The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 x 10(5) oligonucleotides/microm2 was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm2.

DNA Probes↗
PubMed1999

Solid-phase sequence scanning for drug resistance detection in tuberculosis.

DNA chip arrays hold considerable promise for diagnostic sequencing of polymerase chain reaction (PCR) products. To date, however, arrays have been relatively expensive, complex to use and difficult to interpret, preventing their adaptation to the clinical lab. A moderate density array method has been developed that enables efficient, easy-to-interpret and robust solid-phase PCR product sequencing. Here, the results of Mycobacterium tuberculosis rifampin resistance mutation detection by primer-extension-based sequence scanning of the rpo B gene of M. tuberculosis are presented. Rifampin resistant clinical isolates were identified in as little as 1 h post PCR amplification with visual results detection.

Bacterial Proteins↗
PubMed1997

Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene.

There is a growing and significant demand for reliable, simple and sensitive methods for repeated scanning of a given gene or gene fragment for detection and characterization of mutations. Solid-phase sequencing by single base primer extension of nested GBATM primers on miniaturized DNA arrays can be used to effectively scan targeted sequences for missense, insertion and deletion mutations. This paper describes the use of N-GBA arrays designed to scan the sequence of a 33 base region of exon 8 of the p53 gene (codons 272-282) encompassing a hot spot for mutations associated with the development of cancer. Synthetic DNA templates containing various missense, insertion and deletion mutations, as well as DNA prepared from pancreatic and biliary tumor cells, were genotyped using the exon 8 arrays.

Adenocarcinoma↗
PubMed1994

Frequent HLA class I and DP sequence mismatches in serologically (HLA-A, HLA-B, HLA-DR) and molecularly (HLA-DRB1, HLA-DQA1, HLA-DQB1) HLA-identical unrelated bone marrow transplant pairs.

The rates of graft-versus-host disease (GVHD) and rejection are significantly higher among recipients of unrelated donor marrow (BM) than in recipients of marrow from HLA-identical siblings, even when donors and recipients are mixed lymphocyte culture (MLC) compatible and serologically and Dw identical. It has been hypothesized that phenotypically silent HLA class I and DP sequence mismatches might be associated with these differences, but little is known about their incidence. We have sequenced the HLA-A, HLA-B, HLA-C, HLA-DPA1, and HLA-DPB1 genes expressed by 12 unrelated marrow transplant pairs, 11 of whom were molecularly matched at DRB, DQA1, and DQB1 loci. Nine of these pairs were also HLA-A and HLA-B matched by serology. Six of these nine "HLA-identical" pairs were HLA-A (2 of 6), HLA-B (1 of 6), and HLA-C (6 of 6) mismatched at the sequence level. The mismatched class I alleles of all these pairs had strikingly different sequence motifs in the six specificity pockets of their antigen recognition site, and in five pairs they also had sequence differences at positions implicated in T-cell receptor (TCR) binding. Two of the three pairs who were serologically mismatched for one HLA-A or HLA-B antigen were also sequence mismatched at HLA-C. Finally, 10 of 11 pairs tested expressed different DP sequences. These data indicate that HLA class I, especially HLA-C, and DP sequence mismatches are frequent among unrelated subjects defined as HLA identical by current typing methods. We speculate that these sequence differences may explain, at least in part, the higher incidence of acute GVHD and rejection in unrelated BM transplantation as opposed to transplantation between HLA-identical siblings. Because of their high frequency, the role of HLA-A, HLA-B, HLA-C, and HLA-DP mismatches in transplantation outcome is now amenable to direct study.

Adolescent↗
PubMed1993

HLA class I sequence-based typing.

HLA oligogenotyping has been used successfully to characterize most phenotypically undetectable variants of class II genes. Limitations inherent to the class I system have, however, complicated the application of this and other molecular approaches to HLA class I typing. We have previously shown that HLA class II polymorphism can be analyzed by a SBT approach. Here we present a class I-SBT strategy that provides complete sequence information for the two most polymorphic exons of the HLA-A, -B, and -C alleles. HLA class I SBT is based on direct sequencing of PCR-amplified HLA-A, -B, and -C cDNAs and requires a total of six cDNA -PCR-sequencing reactions (two per locus) and 13 different oligonucleotides. Each combination of oligonucleotides per reaction results in locus-specific sequence ladders and allows identification of both alleles in heterozygotes. Application of HLA-A, HLA-B, and HLA-C SBT to 26 homozygous and 32 serologically heterozygous samples has resulted in the identification of 24 novel class I nucleotide sequences encoding 17 new major histocompatibility complex class I products. An unexpected high degree of heterogeneity was found at the HLA-C locus with 14 novel sequences. Although there was a good correlation between the serologic phenotypes and SBT results, HLA-C SBT of most HLA-C serologically homozygous samples (heterozygous for HLA-A and/or -B) revealed heterozygozity (six of eight). SBT, the first molecular typing approach that has been generalized to both class I and class II genes, may be of special interest in applications demanding high sensitivity and specificity, such as in paternity testing or in the evaluation of the effects of sequence allelism in the outcome of unrelated bone marrow transplantation.

Alleles↗
PubMed1992

HLA class II "typing": direct sequencing of DRB, DQB, and DQA genes.

Routine clinical HLA class II typing is based largely on serological and cellular methods. These methods have many drawbacks that have led to the evaluation of molecular approaches to typing, including restriction fragment length polymorphism studies and oligotyping. We present here an alternative molecular approach, sequence-based typing (SBT), that allows direct determination of the sequences of all HLA class II polymorphic genes, thus providing the most detailed information currently possible in this regard. The data presented here using SBT are based on direct sequencing of polymerase chain reaction (PCR)-amplified DRB, DQB, and DQA cDNAs using a limited number of oligonucleotides. The oligonucleotides are designed to allow simultaneous determination of allelic sequences in any heterozygote as well as characterization of DRB isotypic complexity. Two types of amplification oligonucleotides (nonconserved and/or conserved) are used for DRB typing, which involves a maximum of four simultaneous cDNA/PCR/sequencing reactions. The first of these reactions only uses conserved oligonucleotides and is designed to detect all the different DRB transcripts present in any given heterozygote; the other three reactions use nonconserved oligonucleotides and are designed to ensure the unambiguous interpretation of the most complex DRB heterozygote combinations. Characterization of DQA1 and DQB1 sequences can be performed by using conserved oligonucleotides and only involves one reaction per locus. We have applied SBT to 43 homozygous cell lines and to 38 different heterozygote combinations that had previously been serologically typed. In all cases we were able to determine the allelic composition at DRB1, DRB3/4/5 and/or DQB1, and DQA1 loci of these cell lines and subjects; our results, analyzed by blind protocol, were consistent with the serological phenotypes. SBT can be extended to class I and class III genes and is automatable. We believe that this strategy deserves further evaluation as a possible HLA typing method.

Base Sequence↗
PubMed1992

Novel human endogenous sequences related to human immunodeficiency virus type 1.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.

Amino Acid Sequence↗
PubMed1992

Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus.

We have analyzed the genome of the domestic chicken for the presence of genetic sequences related to the envelope protein-encoding genes of avian sarcoma/leukosis retroviruses to determine the organization, structure, potential functionality, and distribution of such sequences. We have previously identified in the genus Gallus an extensive group of endogenous avian retroviruses termed EAV-0. Southern blot and sequence analysis presented here of EAV-0 elements revealed that the majority of the EAV-0 elements in the domestic chicken genome have large deletions in their env genes. Screening of a line 0 chicken genomic DNA library for potential full-length env gene-containing endogenous elements yielded three provirus clones of a previously unrecognized group of endogenous retroviruses. These three clones, E13, E33, and E51, are more closely related to each other (80% or more sequence identity) than to other avian retroviruses (70% or less sequence identity). The E13 element has a large deletion in env, but the E51 element has full-length and highly divergent SU- and TM-coding domains. Complete sequence analysis of the E51 env gene region revealed a defective SU-coding domain and an intact TM-coding domain. Sequence analysis of the E51, E33, and E13 3' termini revealed highly distinctive long terminal repeats of approximately 360 bp which appear to be the products, in part, of long terminal repeat domain shuffling. Hybridization analysis with E51 and E33 env gene probes indicated that they are members of an extensive group of elements present in all Gallus species, and at least one element, E51, could be shown by polymerase chain reaction amplification and direct sequencing to have integrated prior to Gallus speciation.

Amino Acid Sequence↗
PubMed1991

Alloreactive T cells can distinguish between the same human class II MHC products on different B cell lines.

Certain allele-specific alloreactive T cell clones do not recognize the products expressed by some B cell lines that, according to typing methods other than sequencing, carry the allelic molecules recognized by these clones. In order to characterize the naturally occurring sequence polymorphisms putatively responsible for the differential allorecognition of these class II molecules, we have determined the third and/or second exon nucleotide sequences of HLA-DRB1, -DRB3/4/5, -DQB1, and -DQA1 genes from 35 representative lymphoblastoid cell lines. In some cases, the lack of recognition correlates with the presence of single amino acid substitutions in either the second or third hypervariable region (HVR) of the first domain of these molecules. In other cases, the differentially allorecognized class II molecules have identical second and/or first domain amino acid sequences. These findings indicate that a) class II MHC-alloreactive T cell clones can distinguish between molecules with identical amino acid sequences expressed by B cell lines established from unrelated individuals; b) allorecognition of class II molecules is sensitive to naturally occurring single amino acid substitutions in either the second HVR of class II molecules, which is unavailable to interact with TCR residues, or the third HVR. Our results also suggest that 1) in different B cell lines, identical class II molecules may present different endogenous peptides, which may behave as histocompatibility Ag; 2) the peptide-binding specificity of a class II molecule may be affected by amino acid substitutions in its second HVR (Ag-binding site); and 3) human class II allorecognition may be restricted by epitopes contributed by residues of their third HVR.

Amino Acid Sequence↗
PubMed1991

Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes.

Nucleic acid sequences of the second exons of HLA-DRB1, -DRB3/4/5, -DQB1, and -DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.

Alleles↗
PubMed1990

Phylogenetic distribution of the novel avian endogenous provirus family EAV-0.

A new family of related endogenous proviruses, existing at 50 to 100 copies per haploid genome and distinguishable by remarkably short long terminal repeats, has been described for domestic chickens (Gallus gallus subsp domesticus). In this communication, by using Southern blot analysis and probes derived from both internal viral sequences and locus-specific, cellular flanking sequences, we studied the genetic distribution of this family of moderately repetitive avian endogenous retroviruses within the genomes of four Gallus species. Eight inbred lines of domestic chickens, the evolutionary progenitor to the domestic chicken (red jungle fowl), and two more distantly related species (grey and green jungle fowl) were studied. All Gallus species harbored this class of elements, although the different lines of domestic chickens and different species of jungle fowl bore distinguishable complements of the proviral loci. Jungle fowl appeared to have fewer copies than domestic chickens. For three randomly isolated proviral loci, domestic chickens (G. gallus subsp. domesticus) and red jungle fowl (G. gallus subsp. gallus) showed only a proviral state, whereas the most primitive and divergent of the jungle fowl, the green jungle fowl (G. varius), consistently demonstrated only preintegration states or disparate alleles. The presence of this family in all Gallus species and of related sequences in other genera suggests that a primordial founding integration event occurred prior to the evolutionary separation of Gallus species and possibly related genera. Additionally, at least one proviral locus has been acquired subsequent to speciation, indicating that this family was actively infectious after the primary founding event. This conserved, repetitive proviral family appears to represent the vestigial remnant of an avian retrovirus class related to and evolutionarily more ancient than the Rous-associated virus-0 family of avian endogenous retroviruses.

Animals↗
PubMed1989

Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus.

We have cloned the long terminal repeats and their flanking regions from four different proviruses belonging to a large, highly conserved, novel family of avian endogenous retroviruses. This family, termed the endogenous avian retrovirus (EAV) family, is distinct from the previously characterized avian endogenous and exogenous retroviruses. We have analyzed the sequences of the long terminal repeats and their adjacent noncoding viral sequences, including the gag leader region and the 3' noncoding region, of several different members of the EAV family and have found that the regulatory region of these novel viruses contains several unique features. The LTRs of the EAV proviruses are extremely short (243 bp long) but contain all of the essential regulatory features of longer avian retrovirus LTRs. The gag leader region and the 3' noncoding region of the novel EAVs are only weakly related to those of other avian retroviruses. Northern blot hybridization analysis of RNA from Line-0 chicken embryos reveals several transcripts derived from the EAV proviruses. Primer extension analysis indicates that all transcripts initiated from 5' proviral LTRs are initiated at the predicted +1 position within the EAV LTRs. The relative shortness, sequence divergence from other known LTRs, and the retention of the transcriptional integrity of the EAV LTRs make these LTRs an interesting model system for LTR function and for study of the potential involvement of such highly conserved retroviral elements in development.

Animals↗