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.     

Sequence and diversity of rat T-cell receptor α-chain-encoding genes

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L20983-L21005.     

Sequence and diversity of rabbit T-cell receptor gamma chain genes

The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes.The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D38134-D38144 and D42090     

Toll-like receptor 4 genetic diversity among pig populations

The transmembrane glycoprotein encoded by the Toll-like receptor 4 gene ( TLR4 ) acts as the transducing subunit of the lipopolysaccharide receptor complex of mammals, which is a major sensor of infections by Gram-negative bacteria. As variation in TLR4 may alter host immune response to lipopolysaccharide, the association between TLR4 polymorphisms and immune traits of the respiratory and gut systems has important implications for livestock. Here, a sequence dataset from 259 animals belonging to commercial and traditional European pig populations, consisting of 4305 bp of TLR4, including the full transcribed region, a portion of intron 2 and the putative promoter region, was used to explore genetic variation segregating at the TLR4 locus. We identified 34 single nucleotide polymorphisms, 17 in the coding sequence and 17 in the non-coding region. Five non-synonymous mutations clustered within, or in close proximity to, the hypervariable domain of exon 3. In agreement with studies in other mammals, a major exon 3 haplotype segregated at high frequency in the whole sample of 259 pigs, while variants carrying non-synonymous substitutions showed frequencies ranging between 0.6% and 8.7%. Although results on exon 3 provided suggestive evidence for purifying selection occurring at the porcine TLR4 gene, the analysis of both coding and non-coding regions highlighted the fact that demographic factors strongly influence the tests of departure from neutrality. The phylogenetic analysis of TLR4 identified three clusters of variation (ancestral, Asian, European), supporting the evidence of Asian introgression in European main breeds and the well documented history of pig breed domestication previously identified by mtDNA analysis.     

Direct measurement of T-cell receptor repertoire diversity with AmpliCot

Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) beta chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.     

Predominance of one T-cell antigen receptor BV haplotype in African populations

 The human T-cell antigen receptor (TCR) is the counter-receptor for the HLA/peptide complex displayed on the surface of antigen-presenting cells. It confers antigen specificity on T lymphocytes and therefore plays a central role in pathogen recognition and host response. The most frequently used form of the TCR is a heterodimer composed of variable α and β chains. We investigated allele frequencies for four variable-region gene segments of the β chain (2S1, 3S1, 8S3, and 15S1) in 146 Caucasians and 165 Africans. The results reveal significant unexpected differences between the two populations for allele frequencies, phenotypes, genotypes, and haplotypes. Among Caucasians, there are 43 phenotypes, whereas there are 31 among the Africans studied. There are 17 haplotypes in the Caucasian sample but only 10 in Africans. This loss of diversity is largely due to the high frequency of one haplotype in the African sample which represents 65% of the informative chromosomes. At least one copy of this haplotype is present in 90% of informative individuals. As a result, 29% of Africans are homozygous for the common haplotype. Less genetic diversity at TCRBV is unexpected, since Africans usually show greater genetic diversity than other ethnic groups. For example, there are approximately twice as many HLA haplotypes in Africans compared to Caucasians. Homozygosity is also unexpected because it reduces the number of TCR variants available to recognize HLA pathogen-derived peptide complexes. Received: 23 September 1999 / Revised: 2 November 1999     

T-cell repertoire analysis and metrics of diversity and clonality

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Sequence and diversity of variable gene segments coding for rabbit T-cell receptor beta chains

The nucleotide sequences of 11 variable gene segments coding for rabbit T-cell receptor beta (Tcrb-V) chains were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction (CLM-PCR) and by modified anchor PCR without the cloning procedure. The nucleotide sequences in two of these 11 rabbit Tcrb-V gene segments coincided with those in two of the four rabbit Tcrb-V gene segments previously reported; the others have not been described. The percentage similarity of each nucleotide sequence of the 11 rabbit Tcrb-V gene segments was analyzed and the segments were divided into nine families, which were homologous to nine human families (Vb 2, 3, 4, 5, 7, 8, 10, 18, and 22), respectively.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases and have been assigned the accession numbers D17416-D17426.     

Genetic diversity of Toll-like receptor 5 among pig populations

The Toll-like receptor 5 (TLR5) recognizes flagellin of Gram-positive and -negative bacteria and plays an important role in the host defense system. Here, we surveyed single nucleotide polymorphisms (SNPs) in the coding sequence of the porcine TLR5 gene in 83 individuals from five pig breeds, these including Chinese local populations and Western commercial pig breeds. A total of 19 medium polymorphic SNPs (0.25 < PIC < 0.5) were identified, three of which were missense mutations that clustered within the extracellular domain of TLR5. One of the non-synonymous SNPs fell within a 228-amino acid region which has been shown to be important for flagellin recognition. Four SNPs were only found with high frequencies in Oriental pig breeds. The 19 SNPs were found in 30 haplotypes, one of which segregated at high frequency in all samples. Compared with Western pig breeds, Chinese local populations had higher genetic diversity and more haplotypes. Tajima’s test showed no evidence for deviation from neutrality. The data provide useful information for future genetic marker characterization by means of disease association analysis and/or stimulating the mutation carrier with relevant ligands.     

Methods for minimizing the loss of genetic diversity in conserved populations with overlapping generations

Minimization of the average coancestry in a population has been theoretically proven to be the most efficient method to preserve genetic diversity. In the present study, based on a population genetic model, two methods to minimize the average coancestry in populations with overlapping generations were developed. For a given parental coancestry structure, the first method (OG) minimizes the average coancestry in the next generation, and the second method (LT) is designed to minimize the long-term accumulation of coancestry. The efficiencies of the two methods were examined by stochastic simulation. Compared to random choice of parents, the annual effective population sizes under the two proposed methods increased 2–3 folds. The difference among the two methods was small in a population with short generation interval. For populations with long generation intervals, the OG method showed a slightly larger annual effective size in an initial few years. However, in the subsequent years, the LT method gave a 5–15% larger annual effective size than the OG method. From these results, it is suggested that the LT method would be preferred to the OG method in most practical situations.     

Methods of parentage analysis in natural populations

The recent proliferation of hypervariable molecular markers has ushered in a surge of techniques for the analysis of parentage in natural and experimental populations. Consequently, the potential for meaningful studies of paternity and maternity is at an all-time high. However, the details and implementation of the multifarious techniques often differ in subtle ways that can influence the results of parentage analyses. Now is a good time to reflect on the available techniques and to consider their strengths and weaknesses. Here, we review the leading techniques in parentage analysis, with a particular emphasis on those that have been implemented in readily useable software packages. Our survey leads to some important insights with respect to the utility of the different approaches. This review should serve as a useful guide to anyone who wishes to embark on the study of parentage.     

Sequence and diversity of rat T-cell receptor Tcra V8 gene segments

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X80509 (8.2) - X80514 (8.7)     

Structure,diversity, and evolution of the T-cell receptor VB gene repertoire in primates

AB T-cell receptors (TCR) that recognize major histocompatibility complex (MHC)/peptide antigen complexes regulate humoral and cellular arms of the adaptive immune response. Antigen binding sites of MHC and immunoglobulin heavy chain variable regions(Igh-V) are subject to diversity enhancing selection. We sought to establish whether positive Darwinian selection has driven diversity of TCRBV chains in the primate lineage by sequencing rearranged TCR from rhesus monkeys and chimpanzees and comparing them with those of humans. Rates of synonymous (silent) and nonsynonymous (replacement) substitutions indicate selection against amino acid replacements in TCRBV frameworks, and relaxation of these constraints in putative MHC/peptide contact sites. The lack of positive selection for variability in likely ligand contact sites suggests that mechanisms generating somatic diversity in TCR junctional regions have relaxed the pressure for selection of variability in the TCR V region encoded in the germline.     

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

Genetic diversity of bitter taste receptor gene family in Sichuan domestic and Tibetan chicken populations

The sense of bitter taste plays a critical role in animals as it can help them to avoid intake of toxic and harmful substances. Previous research had revealed that chicken has only three bitter taste receptor genes (Tas2r1, Tas2r2 and Tas2r7). To better understand the genetic polymorphisms and importance of bitter taste receptor genes (Tas2rs) in chicken, here, we sequenced Tas2rs of 30 Sichuan domestic chickens and 30 Tibetan chickens. Thirteen single-nucleotide polymorphisms (SNPs) including three nonsynonymous mutations (m.359G >C, m.503C >A and m.583A >G) were detected in Tas2r1 (m. is the abbreviation for mutation); three SNPs were detected in Tas2r2, but none of them were missense mutation; eight SNPs were detected in Tas2r7 including six nonsynonymous substitutions (m.178G >A, m.421A >C, m.787C >T, m.832G >T, m.907A >T and m.943G >A). Tajima’s D neutral test indicates that there is no population expansion in both populations, and the size of the population is relatively stable. All the three networks indicate that red jungle fowls share haplotypes with domestic chickens. In addition, we found that haplotypes H1 and HE1 were positively associated with high-altitude adaptation, whereas haplotypes H4 and HE4 showed a negative correlation with high-altitude adaptation in Tas2rs. Although, chicken has only three Tas2rs, our results showed that both Sichuan domestic chickens and Tibetan chickens have abundant haplotypes in Tas2rs, especially in Tas2r7, which might help chickens to recognize a wide variety of bitter-tasting compounds.