A new mechanism for generating TCR diversity: A TCR Jalpha-like gene that inserts partial nucleotide sequences in a D-gene manner

delta-TCR genes of two gammadelta-T cell hybridomas were found to contain an identical 19-nt sequence in their non-germline N-regions. To determine if this sequence represented a third murine TCR Ddelta gene, genomic PCR was performed by using it as a primer together with primers for interspersed repetitive elements (IRE). Sequencing revealed that the 19-nt segment is part of a 61-nt gene with flanking 5' and 3' recombination signaling sequences (RSS). Southern blot analysis confirmed the presence of this 61-nt gene in the genome of several mouse strains. The gene is unusual in that the distal 24 nucleotides of its 3' RSS region are contributed by the 5' portion of a B2 IRE sequence that includes an apparently non-functional RNA splice site within the 3' nonamer sequence. It has sequence similarities with the Ddelta1 gene (81%) at its 5' end and with Jalpha genes (73%) overall. Tyramide-FISH analysis identified the gene to exist within or adjacent to the TCR alpha/delta locus on chromosome 14. Surveys of available TCR sequences reveal possible partial insertions of the 61-nt gene in other delta-TCR and in alpha-TCR gene sequences. Thus, the unique 61-nt gene is Jalpha gene-like in structure but D gene-like in function.     

Evolutionarily conserved organization and sequences of germline diversity and joining regions of the rabbit T-cell receptor beta 2 chain.

We previously reported that there are three different copies of T-cell receptor beta chain constant region (C beta) genes in some rabbits, two of which are present on an approximately 16-kb and one on an approximately 6-kb Eco RI fragment. We also reported that one of the C beta genes on the approximately 16-kb fragment was chimeric, with a 5' cluster of J beta 2 segments and a 3' untranslated region of beta 1 type. Here we report the complete genomic sequences of the D beta 2 and J beta 2 segments associated with the chimeric C beta gene. The rabbit D beta 2 gene segment has very strong similarity to both its human and mouse counterparts. The sequence similarity also extends rather far from the coding region in both 5' and 3' directions. The content and organization of rabbit J beta 2 gene segments is similar to those found in both human and mouse. The rabbit J beta 2 cluster has six functional segments and one pseudogene, as well as a remnant of another pseudogene between J beta 2.2 and J beta 2.3 equivalent to the one found in man in the same location. The J beta 2.5 gene segment of rabbit has lost the splice signal and is a pseudogene unlike its counterparts in man and mouse. Overall analysis of the rabbit D beta 2-J beta 2 region reveals a closer similarity to human than mouse. However, the general organization of the gene segments in the D beta 2-J beta 2 regions of all three species is remarkably conserved over long stretches of DNA sequence.     

Comparative analysis of human and mouse 3' Igh regulatory regions identifies distinctive structural features

Immunoglobulin heavy chain (Igh) locus rearrangements are controlled in part by an approximately 30 b complex 3' regulatory region located 3' of C alpha: this region contains several enhancers. We report here the comparison of the genomic sequences of the 3' regulatory region and further downstream sequences from mouse, rat, human and chimpanzee. Only short segments of homology were detected in the 3' regulatory region, and these were located in the vicinity of the known 3' enhancers. The nearest highly conserved segment is the nearest non-Igh gene, hole, which is located approximately 62 kb downstream of mouse C alpha. Analysis of murine 3' Igh sequences by single nucleotide polymorphism (SNP) and restriction fragment length polymorphism (RFLP) detected a transition region (high to low SNP or RFLP density) approximately 120 kb downstream of mouse C alpha. Although there is only limited sequence identity between rodent and primate 3' Igh regulatory regions, all of these regulatory regions contain a palindrome and locally repetitive elements. Locally repetitive elements in primates comprise blocks of "switch-like" sequences that differ from the families of inverted and tandem repeats that are present in rodents. We propose that together with enhancers, these "conserved" structural features are essential for the activity of the 3' Igh regulatory region in vivo.     

低密度脂蛋白受体相关蛋白5基因的基因组结构

目的 确定2低密度脂蛋白受体相关蛋白5（low density lipoprotein receptor related protein,5 LRP5）的基因组结构。方法 以LRP5基因的cDNA序列为线索，采用计算机杂交方法首先通过对比分析该基因的cDNA序列和基因组序列，初步确定LRP5基因的基因组结构，按分析得到的基因组结构设计引物，扩增并测定外显子序列和外显子内含子接头序列，确定该基因的基因组结构。结果 LRP5基因的基因组DNA全长为131.6kb，含4有23个外显子和22个内含子；在编码序列中检测到3个单核苷酸多态，即位于第2外显子的A459G，位于第10外显子的C2220T和位于第21外显子的G4416C；LRP5基因含有4个已知的短串联重复序列，即D11S1917，D11S4087，D11S1337和D11S4178，它们分别位于该基因的5'端和第1，4，13内含子内。结论 LRP5基因的基因组结构的确定，为分析该基因突变和功能研究奠定了基础。     

cDNA clones coding for the complete murine B chain of complement Clq: nucleotide and derived amino acid sequences

We have isolated cDNA clones covering the complete B chain of the complement subunit Clq from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide; all of the 228 amino acids of the mature protein; and 140 nucleotides of the 3' untranslated region, including a poly A additional signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of Clq. By comparing the nucleotide sequence of the mouse B chain of Clq with the human B chain (Reid, K.B.M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.     

cDNA clones coding for the complete murine B chain of complement C1q: nucleotide and derived amino acid sequences

We have isolated cDNA clones covering the complete B chain of the complement subunit C1q from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide, all of the 228 amino acids of the mature protein and 140 nucleotides of the 3' untranslated region, including a poly A addition signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of C1q. By comparing the nucleotide sequence of the mouse B chain of C1q with the B chain from human (Reid, K. B. M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.     

The genomic organization of the mouse CD94 C-type lectin gene.

The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C-type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa-1b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5' untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV-VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells.     

Structure of nuclear-localized <Emphasis Type="Italic">cox3</Emphasis> genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and in its colorless close relative <Emphasis Type="Italic">Polytomella</Emphasis> sp.

Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.     

Genomic organisation of rainbow trout, Oncorhynchus mykiss TGF-beta

The genomic organisation of Oncorhnchus mykiss TGF-beta has been determined through the generation of contiguous clones by PCR. The O. mykiss TGF-beta gene is approximately 3.4 Kb in length and consists of 7 coding exons with no introns in the 5'-UTR. Whilst this is the same number of exons found in TGF-beta genes of amphibians, birds and mammals, in the O. mykiss gene intron 2 of other vertebrates is absent and an additional intron is present at the 3' end of the molecule, splitting exon 7 of the other known TGF-beta genes into two exons (trout exons 6 and 7). Comparison of exon sizes in the coding region support the suggestion that the Xenopus TGF-beta5 and trout TGF-beta sequences are the forerunners of TGF-beta1. Conservation of exons coding for the mature TGF-beta peptide is relatively high (63-73% identity) but other exons show lower identities (37-58%). Comparison of the TGF-beta intron sequences reveals that in general the O. mykiss introns are considerably shorter than the avian homologs. The impact of the teleost TGF-beta gene organisation on theories of the gene evolution of this cytokine family are discussed.     

Cloning, characterization and genomic structure of the natural killer cell enhancement factor (NKEF)-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Natural killer cell enhancement factor (NKEF) belongs to the antioxidant protein family. In the human, NKEF has the ability to enhance natural killer cell cytotoxic activity in vitro. In the present work, the cDNAs of NKEF from three strains of homozygous clones of rainbow trout were cloned from the splenic cDNA library of one of the strains, OSU142, and then by RT-PCR for the Hot Creek (HC) and Arlee (AR) strains. The HC sequence has 99% sequence identity with both OSU142 and AR. OSU142 and AR have only one nucleotide difference in the cDNA sequence. All three sequences have the same deduced NKEF peptide, which contains 199 amino acids. The 6. 5 kb genomic DNA of OSU142 containing NKEF was sequenced and contains six exons and five introns. Tissue specific expression of NKEF was studied by RT-PCR in eight different tissues of OSU142 and revealed that all tissues expressed NKEF. A southern blot revealed that the gene for NKEF is present in a single copy. The cDNA and amino acid sequences of trout NKEF have high similarity with human, rat, mouse and carp sequences, therefore, indicating that NKEF is a very conserved gene.