T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments.

Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general.     

水稻条叶枯病毒基因组组分3的克隆与序列分析

利用ＲＴ－ＰＣＲ技术,合成并扩增了水稻条叶枯病毒（ＲＳｔＶ）中国云南分离 物基因组组分３的全长ｃＤＮＡ。将ＰＣＲ产物克隆在载体ｐＣＲⅡ上,进行全序列测定。将所得核苷酸序列及其所推导的氨基酸序列与日本分离物Ｔ进行同源性比较,结果表明,在核苷酸水平上,两分离物的５’端非编码区序列相同,ｖＯＲＦ、ｖｃＯＲＦ及基因间非编码区序列的同源性分别为９７．６％、９６．８％及８７．６％,而３’端非编码区同源性为９８     

Restricted usage of T cell receptor V alpha/J alpha gene segments with different nucleotide but identical amino acid sequences in HLA-DR3+ sarcoidosis patients.

BACKGROUND: Sarcoidosis is a granulomatous disease characterized by the accumulation of activated T cells in the lungs. We previously showed that sarcoidosis patients expressing the HLA haplotype DR3(17),DQ2 had increased numbers of lung CD4+ T cells using the T cell receptor (TCR) variable region (V) alpha 2.3 gene segment product. In the present study, the composition of both the TCR alpha- and beta-chains of the expanded CD4+ lung T cells from four DR3(17),DQ2+ sarcoidosis patients was examined. MATERIALS AND METHODS: TCR alpha-chains were analyzed by cDNA cloning and nucleotide sequencing. TCR beta-chains were analyzed for V beta usage by flow cytometry using TCR V-specific monoclonal antibodies or by the polymerase chain reaction (PCR) using V beta- and C beta-specific primers. J beta usage was analyzed by Southern blotting of PCR products and subsequent hybridization with radiolabeled J beta-specific probes. RESULTS: Evidence of biased J alpha gene segment usage by the alpha-chains of V alpha 2.3+ CD4+ lung T cells was found in four out of four patients. Both different alpha-chain nucleotide sequences coding for identical amino acid sequences and a number of identically repeated alpha-chain sequences were identified. In contrast, the TCR beta-chains of FACS-sorted V alpha 2.3+ CD4+ lung T cells were found, with one exception, to have a nonrestricted TCR V beta usage. CONCLUSIONS: The finding of V alpha 2.3+ CD4+ lung T cells with identical TCR alpha-chain amino acid sequences but with different nucleotide sequences strongly suggests that different T cell clones have been selected to interact with a specific sarcoidosis associated antigen(s). The identification of T cells with restricted TCR usage, which may play an important role in the development of sarcoidosis, and the possibility of selectively manipulating these cells should have important implications for the treatment of the disease.     

水稻条叶枯病毒(RStV)基因组组分4的克隆与序列分析

利用RTPCR技术合成并扩增了水稻条叶枯病毒(RStV)中国云南分离物基因组组分4的全长cDNA,将PCR产物克隆在载体pCRII上,并进行全序列测定,所得核苷酸序列及推测的氨基酸序列与日本分离物T进行比较。结果表明,在核苷酸水平,两分离物的vORF、vcORF及基因间非编码区序列的同源性分别为94.9%、94.1%、86.1%,5’端非编码区序列相同,而3’非编码区同源性为96.1%,仅有两个核苷酸不同;在氨基酸水平,vORF及vcORF编码蛋白的同源性分别为99.4%和98.3%。可见,编码区的大小及其氨基酸序列和两末端序列都是很保守的。因此,中国云南分离物Y与日本分离物T可能有很近的亲缘关系。     

Isolation of monotreme T-cell receptor α and β chains

Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the mammalian immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR -chain cDNAs (TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR -chain cDNA (TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.     

A clonotype nomenclature for T cell receptors

T cell receptor (TCR) nucleotide sequences are often generated during analyses of T cell responses to pathogens or autoantigens. The most important region of the TCR is the third complementarity-determining region (CDR3) whose nucleotide sequence is unique to each T cell clone. The CDR3 interacts with the peptide and thus is important for recognizing pathogen or autoantigen epitopes. While conventions exist for identifying the various TCR chains, there is a lack of a concise nomenclature that would identify both the amino acid translation and nucleotide sequence of the CDR3. This deficiency makes the comparison of published TCR genetic and proteomic information difficult. To enhance information sharing among different databases and to facilitate computational assessment of clonotypic T cell repertoires, we propose a clonotype nomenclature. The rules for generating a clonotype identifier are simple and easy to follow, and have a built-in error-checking system. The identifier includes the V and J region, the CDR3 length as well as its human or mouse origin. The framework of this naming system could also be expanded to the B cell receptor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.     

cDNA cloning of luteinizing hormone subunits from brushtail possum and red kangaroo

Luteinizing hormone (LH) plays an important role in the reproductive cycles of all mammals. There is a large amount of both nucleotide and amino acid sequence data available for LH from eutherian mammals, but little is known about the primary structure of LH in marsupials. We have used consensus PCR primers to generate specific probes for screening pituitary cDNA libraries and report the cloning of the cDNAs encoding the α-subunit of LH (also shared by a number of other glycoprotein hormones) and the LH-specific β-subunit, from the common brushtail possum, Trichosurus vulpecula, and the red kangaroo, Macropus rufus. Southern blotting experiments indicated that both genes are probably present as single copies. Comparison of the deduced marsupial protein sequences with homologous sequences from other vertebrates revealed a high degree of conservation, especially for the α-subunit. These sequences represent the first complete primary structures for a marsupial glycoprotein hormone to have been elucidated. Received: 30 January 1998 / Accepted: 10 April 1998     

草鱼瘦素受体基因片段序列的克隆及其组织表达分析

根据斑马鱼、大西洋鲑和人等物种巴知的瘦素受体基因核苷酸保守区序列设计一对简并引物,通过RT-PCR法从草鱼肝胰脏中首次克隆获得草鱼瘦素受体基因的片段序列.该片段序列长713 bp,编码237个氨基酸,氨基酸序列分析表明草鱼瘦素受体基因片段氨基酸序列与其他物种的相似性在35％ -86％之间.通过邻接法(Neighbor Joining,NJ)构建系统进化树显示,鱼类的瘦素受体独立聚成一支,草鱼与金鱼、斑马鱼聚成一支,再与日本青鳉、黑点青鳉、红鳍东方鲀和大西洋鲑聚成一支.通过实时荧光定量PCR分析草鱼瘦素受体基因的组织差异表达,结果表明,草鱼瘦素受体基因在肝胰脏、肌肉、脑、心脏、脾和肠系膜脂肪组织中均有表达,其中在脾脏组织中表达量最多,显著高于其他组织(P＜0.05),其次是心脏、脑、肌肉和肠系膜脂肪组织,在肝胰脏组织中表达量最低,且显著低于其他组织(P＜0.05).     

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.     

The T cell receptor beta locus of the channel catfish,Ictalurus punctatus,reveals unique features

Previously, a series of clonal alloantigen-dependent T cell lines established from the channel catfish revealed distinctly different TCR beta rearrangements. Here, a follow-up study of the junctional diversity of these TCR gene rearrangements focuses on characterization of the genomic organization of the TCRB locus. Surprisingly, a total of 29 JB genes and two substantially different CB genes were identified downstream of a single DB gene. This is in contrast to the situation in mammals, where two clusters of a DB gene, six or seven JB genes, and a CB gene are found in tandem. The catfish CB genes are approximately 36% identical at the amino acid level. All 29 catfish JB gene segments appear functional. Thirteen were used in the 19 cDNAs analyzed, of these eight were used by the 11 catfish clonal alloantigen-dependent T cell lines. As might be expected, CDR3 diversity is enhanced by N-nucleotide additions as well as nucleotide deletions at the V-D and D-J junctions. Taken together, compared with that in mammals, genomic sequencing of the catfish TCR DB-JB-CB region reveals a unique locus containing a greater number of JB genes and two distinct CB genes.     

A variable region gene subfamily encoding T cell receptor beta-chains is selectively conserved among mammals

Studies of murine T cell receptor genes indicate that the VT beta repertoire, in contrast to the large VT alpha, VH, and VK repertoires, is limited to approximately 21 genes. Large differences between the various VT beta sequences allow classification into distinct subfamilies consisting of one or few members. The VT beta sequence of a gene, RTB92, expressed in a rabbit T cell line is most closely related to a VT beta gene expressed in the human cell line Molt-4. Genomic blots with RTB92 VT beta region used as probe indicated conservation of related VT beta gene subfamilies in man and pig, but no related sequences were seen in rat, mouse, or hamster. The relationship among these VT beta genes mirrors similarities and differences in MHC genes of the compared species and suggests coordinate evolution of these functionally related gene families. The constant region of RTB92 was shown to be rabbit CT beta 2 by reference to genomic clones. Comparisons with constant region sequences of human and murine CT beta 1 and CT beta 2 chains reveal no similarities in amino acid or nucleic acid sequence in the coding region characteristic of the respective isotypes. Intraspecies homologies between CT beta 1 and CT beta 2 sequences were much greater than those between CT beta 1 or CT beta 2 from other species. By contrast, comparisons of JT beta and 3' untranslated region sequences showed significant interspecies conservation of sequences in the beta 1 and in the beta 2 regions.     

GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis.     

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.     

Complexity and sequence identification of 24 rat V beta genes

Twenty-four TCR V beta genes were cloned by anchored PCR from the Lewis rat strain and identified by nucleotide and amino acid sequence comparisons to known mouse V beta genes. Rat V beta genes exist in 17 single-member and 3 multimember subfamilies and exhibit 86 to 94 and 72 to 92% nucleotide and amino acid sequence similarities, respectively, to their mouse counterparts. A single rat gene, designated V beta 20, having no previously known mouse counterpart was identified; a closely related gene was subsequently isolated from the C57BL/6 mouse strain. Characterization of the rV beta genes provides the basis for addressing rat T cell tolerance mechanisms and V beta gene usage in immunologically mediated rat disease models.     

Structural analysis of the gene for the beta chain of the antigen-specific receptor of mammalian T-lymphocytes

The antigen specific receptor of T cells (TCR) is composed of alpha and beta chains and is normally present on the T cell surface complexed with the components which make up T3. In the case of beta chain, multiple somatic DNA rearrangements bring together V beta (variable), D beta (diversity) and J beta (joining) gene segments before a mature messenger RNA can be transcribed. So far beta chain genes have been extensively studied in the human and in the mouse system and we have very little information on other mammals. Our aims were to obtain information that may provide a structural basis for understanding developmental as well as evolutionary aspects of the TCR gene system in mammals. In this study we compare the hybridization pattern between a human cDNA probe coding for the beta chain constant region and restricted genomic DNA extracted from lymphocytes deriving from human as well as from rat and lamb. The comparison of the hybridization data represent a first piece of information about the variation of the structure of the TCR beta chain genes in mammals.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.