Fine mapping and functional analysis of the multiple sclerosis risk gene CD6

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2nd SRCR domain with susceptibility to MS (Pmax(T) permutation=161024). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. – CD4+ naı¨ve cells, P = 0.0001; CD8+ naı¨ve cells, P,0.0001; CD4+ and CD8+ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4+ and CD8+ T cells.

Molecular Cloning and Sequence Analysis of Two cDNAs Encoding Metalloproteinase2disintegrin from Trimeresurus jerdonii

　从菜花烙铁头蛇的毒腺中,利用RT2PCR 进行体外扩增,克隆到2 个金属蛋白酶2去整合素基 因,命名为TJM21、TJM22. TJM21 cDNA 全长为1 528 bp ,编码481 个氨基酸;TJM22 cDNA 全长为1 578 bp ,编码484 个氨基酸. TJM21 和TJM22 都属于Ⅱ型蛇毒金属蛋白酶,由信号肽、前肽、金属蛋白酶、 间隔肽和去整合素5 部分组成. Ⅱ型蛇毒金属蛋白酶氨基酸序列的比较及进化分析显示,它可进一 步分为两类,一类包括大多数Ⅱ型蛇毒金属蛋白酶(其中含有TJM21) ,而TJM22 和agkistin 则组成了 另一类. 并且TJM22 和agkistin 的第407 位和第426 位残基都是半胱氨酸,而在其它Ⅱ型金属蛋白酶 的相应位置,407 位是丝氨酸,426 位则缺失. TJM22 和agkistin 均有可与整合素α2 Ⅰ区域特异性结合 的片段QPNRKRHDNAQ(残基276～284) ,这个片段在其它Ⅱ型金属蛋白酶中并没有发现. 因此推 断, TJM22 和agkistin 可能属于一类新型的Ⅱ型蛇毒金属蛋白酶.

Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis.

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

Molecular phylogenetic systematics of twelve species of acipenseriformes based on mtDNA ND4L-ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.

Genomic organization and sequence analysis of the vomeronasal receptor V2R genes in mouse genome

Two multigene superfamilies, named V1R and V2R, encoding seven-transmembrane-domain G-protein coupled receptors (GPCRs) have been identified as pheromone receptors in mammals. Three V2R gene families have been described in mouse and rat. Here we screened

DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

Objective To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. Methods Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. Results Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. Conclusion Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

High penetrance of sequencing errors and interpretative shortcomings in mtDNA sequence analysis of LHON patients

For identifying mutation(s) that are potentially pathogenic it is essential to determine the entire mitochondrial DNA (mtDNA) sequences from patients suffering from a particular mitochondrial disease, such as Leber hereditary optic neuropathy (LHON). Howe

Cloning,Expression and Sequence Analysis of A Luciferase Gene from the Chinese Firefly Pyrocoelia pygidialis

从一种来自中国日行性萤火虫(云南窗萤)发光器官mRNA中克隆、测序并表达了有功能的荧光素酶.云南窗萤荧光素酶的cDNA序列有1647个碱基,编码548个氨基酸残基.从推测得到的氨基酸序列的比对分析得出:云南窗萤的荧光素酶与来自Lampyris noctiluca,L.turkestanicus和Nyctophila cf.caucasica三种萤火虫的荧光素酶有97.8%的序列一致性.从推测得出的氨基酸序列进行系统发育分析,其结果表明:云南窗萤和Lampyris+Nyctophila聚在一起,与同属的发光强夜行性的萤火虫不形成的单系.云南窗萤荧光素酶在大肠杆菌中表达的条带大约70kDa,并且在有荧光素存在时发出黄绿色荧光.对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点.云南窗萤和窗萤属的其他3种萤火虫的荧光素酶卡目比,有13个不同氨基酸位点,位于模拟分子结构的表面.对于这些多肽环、不刚氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异.

Cloning and sequence analysis of Sox genes in a tetraploid cyprinid fish, Tor douronensis

A PCR survey for Sox genes in a young tetraploid fish Tor douronensis (Teleostei: Cyprinidae) was performed to access the evolutionary fates of important functional genes after genome duplication caused by polyploidization event. Totally 13 Sox genes were obtained in Tor douronensis, which represent SoxB, SoxC and SoxE groups. Phylogenetic analysis of Sox genes in Tor douronensis provided evidence for fish-specific genome duplication, and suggested that Sox19 might be a teleost specific Sox gene member. Sequence analysis revealed most of the nucleotide substitutions between duplicated copies of Sox genes caused by tetraploidization event or their orthologues in other species are silent substitutions. It would appear that the sequences are under purifying selective pressure, strongly suggesting that they represent functional genes and supporting selection against all null allele at either of two duplicated loci of Sox4a, Sox9a and Sox9b. Surprising variations of the intron length and similarities of two duplicated copies of Sox9a and Sox9b, suggest that Tor douronensis might be an allotetraploidy.

Molecular phylogeny of three subspecies of common carp Cyprinus carpio, based on sequence analysis of cytochrome b and control region of mtDNA

The complete cytochrome b and the control region of mtDNA (about 2070 bp in total) of 10 strains belonging to three subspecies of the common carp, including three wild subspecies (the Yangtze River wild common carp - Cyprinus carpio haematopterus, Yuanjiang River wild common carp Cyprinus carpio rubrofuscus and Volga River wild common carp - Cyprinus carpio carpio) and seven domestic strains (Xingguo red carp, Russian scattered scaled mirror carp, Qingtian carp, Japanese Koi carp, purse red carp, Big-belly carp, German mirror carp) were sequenced. Phylogenetic analysis indicated that the 10 strains form three distinct clades, corresponding to C. c. haematopterus, C. c. rubrofuscus and C. c. carpio respectively. Purse red carp, an endemic domestic strain in Jiangxi province of China, showed a higher evolution rate in comparison with the other strains of C. c. haematopterus, most probably because of intensive selection and a long history of domestication. Base variation ratios among the three subspecies varied from 0.78% (between C. c. haematopterus and C. c. rubrofuscus) to 1.47%(between C. c. carpio and C. c. rubrofuscus). The topography of the phylogenetic tree and the geographic distribution of three subspecies closely resemble each other. The divergence time between C. c. carpio and the other two subspecies was estimated to be about 0.9 Myr and about 0.5 Myr between C. c. haematopterus and C. c. rubrofuscus. Based on phylogenetic analysis, C. c. rubrofuscus might have diverged from C. c. haematopterus.

Sequence analysis of cytochrome b gene indicates that East Asian group of cyprinid subfamily Leuciscinae (Teleostei : Cyprinidae) evolved independently

The sequences of mitochondrial cytochrome b gene of cyprinid subfamily Leuciscinae are analyzed. Phylogenetic trees generated with methods of neighbor-joining, maximum likelihood and maximum parsimony with Phenacogrammus as an outgroup indicate that Leuciscinae is not a monophyletic group but includes two discrete subgroups. The East Asian group of the subfamily Leuciscinae, including the genera Ctenopharyngodon, Elopichthys, Luciobrama, Mylopharyngodon, Ochetobius, and Squaliobarbus, is close to Aristichthys and Hypophthalmichthys, and they form a monophyletic group which is distant from the leuciscine genera in Europe, Siberia and North America, such as Phoxinus, Leuciscus, Abramis, Rutilus, Chondrostoma, Alburnus, Opsopoedus, Lythrurus, and Pimephales. Our study suggests that the diversified East Asian group of the subfamily Leuciscinae should have an independent origination.

Molecular phylogenetic systematics of twelve species of acipenseriformes based on mtDNA ND4L-ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.

Genomic organization, nucleotide sequence analysis of the core histone genes cluster in Chlamys farreri and molecular evolution assessment of the H2A and H2B

This work represents the nucleotide sequence of the core histone gene cluster from scallop Chlamys farreri. The tandemly repeated unit of 5671 bp containing a copy of the four core histone genes H4, H2B, H2A and H3 was amplified and identified by the techniques of homology cloning and genomic DNA walking. All the histone genes in the cluster had the structures in their 3' flanking region which related to the evolution of histone gene expression patterns throughout the cell cycle, including two different termination signals, the hairpin structure and at least one AATAAA polyadenylation signal. In their 5' region, the transcription initiation sites with a conserved sequence of 5'-PyATTCPu-3' known as the CAP site were present in all genes except to H2B, generally 37-45 bp upstream of the start code. Canonical TATA and CAAT boxes were identified only in certain histone genes. In the case of the promoters of H2B and H2A genes, there was a 5'-GATCC-3' element, which had been found to be essential to start transcription at the appropriate site. After this element, in the promoter of H2B, there was another sequence, 5'-GGATCGAAACGTTC-3', which was similar to the consensus sequence of 5'-GGAATAAACGTATTC-3' corresponding to the H2B-specific promoter element. The presence of enhancer sequences (5'-TGATATATG-3') was identified from the H4 and H3 genes, matching perfectly with the consensus sequence defined for histone genes. There were several slightly more complex repetitive DNA in the intergene regions. The presence of the series of conserved sequences and reiterated sequences was consistent with the view that mollusc histone gene cluster arose by duplicating of an ancestral precursor histone gene, the birth-and-death evolution model with strong purifying selection enabled the histone cluster less variation and more conserved function. Meanwhile, the H2A and the H2B were demonstrated to be potential good marks for phylogenetic analysis. All the results will be contributed to the characterization of repeating histone gene families in molluscs.