Assessment of the nucleotide sequence variability in the bovine T-cell receptor alpha delta joining gene region

The sequence of 2,193 nucleotides from the bovine T-cell receptor alpha/delta joining gene region (TCRADJ) was determined and compared with the corresponding human and murine sequences. The identity was 75.3% for the comparison of the Bos taurus vs. the Homo sapiens sequence and 63.8% for the Bos taurus vs. the Mus musculus sequence. This comparison permitted the identification of the putatively functional elements within the bovine sequence. Direct sequencing of 2,110 nucleotides in nine animals revealed 12 variable sites. Estimates, based on direct sequencing in three Holstein Friesian animals, for the two measures of sequence variability, nucleotide polymorphism (u) and nucleotide diversity (p), were 0.00050 (60.00036) and 0.00077 (60.00056), respectively. The test statistic, Tajima's D, for the comparison of the two measures indicates that the difference between u and p is close to significance (P < 0.05), suggesting the possibility of selective forces acting on the studied genomic region. Allelic variation at 5 of the 12 variable sites was analysed in 359 animals (48 Anatolian Black, 56 Braunvieh, 115 Fleckvieh, 47 Holstein Friesian, 50 Simmental and 43 Pinzgauer) using the oligonucleotide ligation assay (OLA) in combination with the enzyme linked immunoabsorbant assay (ELISA). Nine unambiguous haplotypes could be derived based on animals with a maximum of one heterozygous site. Four to seven haplotypes were present in the different breeds. When taking into account the frequencies of the haplotypes in the different breeds, especially in Anatolian Black, an ancestral cattle population, we could establish the likely phylogenetic relationships of the haplotypes. Such haplotype trees are the basis for cladistic candidate gene analysis. Our study demonstrates that the systematic search of single nucleotide polymorphisms (SNPs) is useful for analysing all aspects of variability of a given genomic region.     

Evolutionary dynamics of the T-cell receptor VB gene family as inferred from the human and mouse genomic sequences

The diversity of T-cell receptors is generated primarily by the variable-region gene families, each of which is composed of a large number of member genes. The entire genomic sequence of the variable region (VB) of the T- cell receptor beta chain from humans and mice has become available. To understand the evolutionary dynamics of the VB gene family, we conducted a phylogenetic analysis of all VB genes from humans and mice, as well as a detailed analysis of internal DNA duplications in the human genomic VB region. The phylogenetic tree obtained shows that human and mouse VB genes intermingle extensively rather than forming two separate clusters and that many gene duplications occurred both before and after the divergence between primates and rodents. Analyzing the genomic maps of transposable elements (e.g., LINEs and SINEs) and relic VB genes in the VB gene region, we present evidence that a 20-kb VB region duplicated tandemly four times in the human lineage during the last 32 Myr, and 6 out of the 15 VB genes in this region have become nonfunctional during this period. Our results show that the VB gene family is subject to evolution by a birth-and-death process rather than to concerted evolution.     

Novel competitive PCR methods for quantitation of T-cell receptor delta (TCRD) gene rearrangements

Since the invention of the polymerase chain reaction (PCR) several quantitative PCR-based approaches have been described. Recently, the real-time PCR method became a standard in quantitative PCR, although high costs of the necessary equipment and reagents make it unaffordable for many laboratories. In this paper we describe two novel competitive PCR techniques, which were used to determine the frequency of T-cell receptor delta gene (TCRD) rearrangements in peripheral blood leukocytes. In the reference gene competitive PCR (rgc-PCR) the rearranged TCRD gene competes with the reference gene (RAG1) for common reagents (dNTPs and Taq polymerase). The intensity ratio of amplification products, TCRD/RAG1, corresponds to the portion of cells containing a rearrangement. A series of reactions was performed, in which RAG1 primers were added to the PCR after different numbers of cycles. On the basis of the number of cycles needed to obtain equal band intensity, the frequency of cells containing a rearrangement was calculated. In the common primer competitive PCR (cpc-PCR), two gene rearrangements, Vdelta1-Jdelta1 and Vdelta2-Jdelta1, compete for the common Jdelta1 primer. The competing genes are amplified from the same genomic DNA template; therefore unlike in the method using the internal competitor, the results are not affected by the quantity or quality of the analysed sample. We showed that the rgc-PCR and cpc-PCR are reliable and give reproducible results. The methods do not require any expensive equipment or reagents, and can be used to determine the frequency of gene rearrangements.     

Evolutionary comparison of murine and human delta T-cell receptor deleting elements

The gene for the T-cell antigen receptor (TCR) delta chain is a gene within a gene, being located in the TCR alpha chain gene in both mice and humans. The human delta locus is flanked by delta deleting elements that undergo preferential rearrangement in the thymus, resulting in deletion of internal delta coding segments. The mouse has conserved analogous elements, m delta Rec and m phi J alpha, which separate delta from alpha and undergo a m delta Rec/m phi J alpha rearrangement in polyclonal thymus. The 5' element, m delta Rec, which is an isolated heptamer-spacer-nonamer (h-s-n), lies within 200 kb of D delta 1, and displays two areas of nearly 80% homology to human delta Rec. The downstream element, m phi J alpha, lies 12.5 kb 3' to C delta, lacks the consensus amino acids for J alpha, and retains 80% homology to human phi J alpha. Cells from murine neonatal thymus show three prominent m delta Rec rearrangements consisting of the m delta Rec/m phi J alpha recombination, a delta Rec/D delta 1/D delta 2/J delta 1 recombination, and two hybrid recombinations. A consequence of the m delta Rec/M phi J alpha rearrangement is a deletion of internal D delta and J delta coding segments that would prevent their incorporation into alpha TCR products. The conservation of noncoding deleting elements flanking the delta TCR in mice and humans is similar to the evolutionarily preserved kappa deleting element of the B-cell lineage and argues for an important role in receptor utilization.     

Nucleotide sequence and expression of the human gene encoding apolipoprotein H (beta 2-glycoprotein I).

Human apolipoprotein H (ApoH), also called beta 2-glycoprotein I, is a 50-kDa serum glycoprotein whose function is not clearly defined. We have cloned and sequenced ApoH cDNAs both from human liver and from a human hepatoma cell line (HepG2). Both cDNA sequences predict a protein 345 amino acids (aa) in length. This sequence includes a 19-aa hydrophobic, N-terminal signal sequence which is not present in the mature protein [Lozier et al., Proc. Natl. Acad. Sci. USA 81 (1984) 3640-3644]. It differs from this previously reported aa sequence at two positions, both of which strengthen the conservation among the four short consensus repeats within the ApoH molecule. COS-1 cells transiently transfected with the ApoH cDNA in a eukaryotic expression vector produced a single species of ApoH mRNA and secreted in the ApoH protein. The level of ApoH mRNA expressed by HepG2 cells is downregulated by incubation with inflammatory mediators, implying that ApoH is a negative acute-phase protein.     

Genomic structure and functional expression of a human alpha(2)/delta calcium channel subunit gene (CACNA2).

CACNA2 encodes the alpha(2)/delta subunit of the human voltage-gated calcium channels and is located in the candidate region of malignant hyperthermia susceptibility type 3 (MHS3). We determined the structural organization of CACNA2 by isolation of overlapping genomic DNA clones from a human phage library. The gene consists of at least 40 exons, 2 of which are alternatively spliced, spanning more than 150 kb of genomic DNA. Exons range from 21 to 159 bp, and introns range from 98 bp to at least more than 20 kb. We constructed a full-length cDNA and cloned it into a mammalian expression vector. Cotransfection of the CACNA2 cDNA with alpha(1A) and beta(4) cDNA into HEK293 cells led to the expression of Q-type calcium currents. The alpha(2)/delta subunit enhanced the current density 18-fold compared to cells transfected with only alpha(1A) and beta(4) cDNA. The sequence analysis provides the basis for comprehensive mutation screening of CACNA2 for putative MHS3 individuals and patients with other channelopathies.     

Structure and partial genomic sequence of the human retinoblastoma susceptibility gene

This report describes the genomic organization of the human retinoblastoma susceptibility locus. This gene spans approximately 200 kb of DNA within human chromosome 13, band q14. The previously determined cDNA sequence comprises 27 exons, ranging in size from 31 bp to 1873 bp, and 26 introns, ranging in size from 80 bp to 70,500 bp. We have mapped the positions of the exons and the positions of the recognition sites for six restriction endonucleases. We also present the sequence of 9.2% of the locus (18,335 bp), including approximately 200 bp of intron sequence immediately flanking each exon. This map of a wild-type allele will form the foundation for future studies of mutant, oncogenic alleles at this locus.     

Fibroblast growth factor receptor 2 (FGFR2): genomic sequence and variations

Fibroblast growth factor receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.     

Sequence and diversity of the rat delta T-cell receptor

The cDNA sequence of the delta T-cell receptor (TCRD) in the adult Lewis rat thymus was determined using the technique of rapid amplification of cDNA ends. Sixteen variable region genes (TCRDV), two diversity regions (TCRDD), two joining regions (TCRDJ), and a single constant region gene (TCRDC) were identified. The sixteen unique TCRDV genes identified represented eight different subfamilies in the rat and were highly conserved (>80% nucleotide identity) to corresponding mouse sequences. Extensive junctional diversity was observed in the rat, with both TCRDD regions (TCRDD1 and TCRDD2) utilized in the majority of cDNA clones identified. The two TCRDJ genes were highly conserved and corresponded to TCRDJ1 and TCRDJ2 in the mouse; the majority of clones utilized TCRDJ1. The TCRDC region in the rat was 91.1% identical to the mouse TCRDC gene and was highly conserved to other species. Although extensive sequence information about mouse gamma-delta T-cell receptor genes is available, current knowledge of rat gamma-delta T-cells is limited. The sequence analysis presented in this study adds to our understanding of gamma-delta T-cells in general, and it may be utilized to study the role of gamma-delta T-cells in immune-mediated disease and transplantation models previously established in the rat.     

Cloning,expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.