不同地域卵黄萤荧光素酶基因的克隆与序列分析

为了比较不同地域萤火虫荧光素酶基因的进化关系,通过GenBank中已知的荧光素酶基因保守区段设计引物,利用5'-RACE(rapid-amplification of cDNA ends)和3'-RACE技术克隆了来自云南省文山州和西双版纳州的同种卵黄萤荧光素酶基因cDNA和全基因序列.来自不同地域的2种卵黄萤荧光素酶在基因序列上存在3个不同碱基位点,但是它们编码的荧光素酶只存在1个不同的氨基酸.卵黄萤荧光素酶基因全长(从起始密码子到终止密码子)1998 bp,包含7个外显子,6个内含子,其cDNA序列共1976 bp,包含102 bp 5'UTR(untranslated region)、1635 bp的荧光素酶基因开放阅读框和239 bp的3'UTR序列.卵黄萤荧光素酶基因的开放阅读框编码1个544个氨基酸的蛋白质,比同属的其它几种荧光素酶少4个氨基酸.来自2个不同地域的卵黄萤荧光素酶在进化上是比较保守的,它们与北美萤火虫Photinus pyralis荧光素酶在碱基序列上分别有62.9%和63%相似性.

卵黄萤荧光素再生酶基因的克隆与序列分析

目的 克隆和分析荧光素再生酶基因(LRE).方法 通过GeneBank中已知的荧光素再生酶基因保守区段设计引物,利用5'RACE(rapid-amplification of cDNA ends)和3'RACE技术克隆了来自云南省两双版纳州的卵黄萤(Luciola ovalis)荧光素再生酶基因cDNA和全基因序列.通过GeneBank、National Center for Bioteclmology Information和ProDom at the ExPASy Server软件和数据库进行序列分析.结果 卵黄萤荧光素再牛酶的cDNA序列和基因序列存在2个不同碱基位点,但是它们编码的荧光素再生酶是相同的.卵黄萤荧光素再生酶基因全长(从起始密码子到终止密码子)为1131 bp,包含5个外显子4个内含子,其cDNA 序列为1008 bp,包含924bp的荧光素酶基因开放阅读框和84 bp的3'UTR序列.卵黄萤荧光素酶基因的开放阅读框编码1个307个氨基酸的蛋白质.它与北美萤火虫(Photinus pyralis)荧光素再生酶在碱基序列和氨基酸序列上分别有61.8%和53.3%的相似性.结论 成功地克隆了荧光素再生酶的cDNA和基因序列,为其在基因工程中的应用奠定了基础.

Polymorphic Sites at the 3 ' Untranslated Region of the HLA-G Gene Are Associated with Differential hla-g Soluble Levels in the Brazilian and French Population

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Insights on the HLA-G Evolutionary History Provided by a Nearby Alu Insertion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise do polimorfismo da região 3’não traduzida (3’NT) do gene HLA-G de pacientes com esclerose múltipla e neuromielite óptica da região noroeste do estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Papel de la región 3'UTR en la acumulación del RNA mensajero de VEGF-D en células PC12 que expresan constitutivamente el oncogen wnt-1

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

A 1.8-kb insertion in the 3'-UTR of RXFP2 is associated with polledness in sheep

Sheep breeds show a broad spectrum of different horn phenotypes. In most modern production breeds, sheep are polled (absence of horns), whereas horns occur mainly in indigenous breeds. Previous studies mapped the responsible locus to the region of the RXFP2 gene on ovine chromosome 10. A 4-kb region of the 3'-end of RXFP2 was amplified in horned and polled animals from seven Swiss sheep breeds. Sequence analysis identified a 1833-bp genomic insertion located in the 3'-UTR region of RXFP2 present in polled animals only. An efficient PCR-based genotyping method to determine the polled genotype of individual sheep is presented. Comparative sequence analyses revealed evidence that the polled-associated insertion adds a potential antisense RNA sequence of EEF1A1 to the 3'-end of RXFP2 transcripts.

Effect of 5′UTR introns on gene expression in Arabidopsis thaliana

Background The majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5′UTRs and 3′UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plant Arabidopsis thaliana has identified introns residing in both coding and non-coding regions of the genome. Results A bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5′UTRs is similar to that in CDSs but much higher than that in 3′UTRs; (2) the 5′UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5′UTRs are, on average, longer than introns in the CDSs and 3′UTRs; and (4) 5′UTR introns have a different nucleotide composition to that of CDs and 3′UTR introns. Furthermore, we show that the 5′UTR intron of the A. thaliana EFIα-A3 gene affects the gene expression and the size of the 5′UTR intron influences the level of gene expression. Conclusion Introns within the 5′UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3′UTR. In the EFIα-A3 gene, the presence of a long intron in the 5′UTR is sufficient to enhance gene expression in plants in a size dependent manner.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

鳜IL-1β基因的克隆及原核表达

用RACE PCR获得了鳜白介素-1β(IL-1β)的全长cDNA.鳜IL-1β的cDNA全长为1298 nt(核苷酸),其5′非编码区包含UTR 93 nt;3′非编码区包含452 nt;其开放阅读框内包含753 nt,翻译成251个氨基酸.将鳜IL-1β克隆到原核表达载体pET-32a上,在大肠杆菌Rosetta- gami(DE3)内以包涵体形式得以高效表达.

Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappa B and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C. (c) 2008 Published by Elsevier Ltd.

Characterization and expression analysis of TNFR-associated factor 1 (TRAF1) in grass carp Ctenopharyngodon idella

TNF receptor associated factor 1 (TRAF1) plays an important role in regulating the TNF signaling and protecting cells from apoptosis. In the present study, a TRAF1 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA is 2235 bp, including a 250 bp 5' UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3'UTR. The polyadenylation signal (AATAAA, AATAA) and one mRNA instability motif (AUUUA) were found followed by a poly (A) tail in the 3'UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF1 (gcTRAF1). The putative amino acids of gcTRAF1 share 72% identity with the homologue in zebrafish. It is characterized by a zinc finger at the N-terminus and a TRAF domain (contains one TRAF-C and one TRAF-N) at the C-terminus. The identity of the TRAF domain among all the TRAF1 homologues in vertebrates varies from 52% to 58%, while the identities of TRAF-C were almost the same as 70%. The recombinant gcTRAF1 has been constructed successfully and expressed in Escherichia coli by using pET-32a expression vector. The polyclonal antibody for rabbit has been successfully obtained. The expression of gcTRAF1 in different organs was examined by real-time quantitative PCR and Western blotting, respectively. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of TRAF1 homologue molecule found in fish. (c) 2007 Elsevier B.V. All rights reserved.

TRIM5-CypA融合基因与北平顶猴易感HIV-1的机制研究

天然免疫分子TRIM5α（tripartite motif protein 5α）是近年来发现的一种重要的宿主细胞内逆转录病毒限制因子。在灵长类动物细胞中，TRIM5α蛋白可以在病毒进入细胞后、逆转录前的阶段抑制HIV-1、N-MLV和EIAV等逆转录病毒的复制。由于TRIM5α分子的作用，绝大部分旧大陆猴（Old World monkey）都不能感染HIV-1。而在新大陆猴（New World monkey）中，鹰猴是唯一不感染HIV-1的灵长类动物。研究证明，鹰猴细胞中存在的TRIM5-CypA融合蛋白（owl monkey TRIM5-CypA，omTRIMCyp）介导了抗HIV-1的作用，从而使鹰猴不能感染HIV-1。研究证明，平顶猴是旧大陆猴中唯一报道可以感染HIV-1的灵长类动物，但是其感染HIV-1的机制并不清楚。根据现行的灵长类动物分类学，原属平顶猴群体（M. nemestrina group）的三个亚种分为猕猴属的三个不同种：巽他平顶猴（Sunda pig-tailed macaque，M. nemestrina），北平顶猴（Northern pig-tailed macaque，M. leonina）和明打威猴（Mentawai macaque，M. pagensis）。本论文对中国云南境内北平顶猴TRIM5基因座和感染HIV-1的相关性进行了研究。通过PCR和测序对北平顶猴基因组TRIM5基因座进行分析，发现一个CypA假基因的cDNA通过逆转座机制插入至TRIM5基因座的3’-UTR区域，形成了一个不同于鹰猴TRIM5-CypA的新型融合基因npmTRIMCyp（northern pig-tailed macaque TRIM5-CypA）。通过RT-PCR对npmTRIMCyp融合基因的转录本进行分析，我们鉴定出npmTRIMCyp共有3种不同的选择性剪接产物，分别为npmTRIMCypV1-V3。进一步克隆和测序这3种不同选择性剪接体，通过丰度和序列分析证实：npmTRIMCypV2是优势剪接体，可能在该融合基因产物的功能中发挥作用。研究发现北平顶猴npmTRIMCyp融合基因主要转录本中外显子7和8均被剪切掉。外显子7剪接丢失机制源于TRIM5第6内含子内 3’剪接位点的G/T突变。我们克隆了npmTRIMCyp融合基因cDNA的蛋白编码区ORF，并构建了重组表达npmTRIMCyp的载体，转染HeLa和HeLa-T4细胞并获得稳定表达的细胞株。通过感染HIV-1证实，npmTRIMCyp融合蛋白不能够限制HIV-1的感染和复制，这可能是北平顶猴作为旧大陆猴中唯一对HIV-1易感的灵长类动物的重要分子机制之一。通过HIV-1感染灵长类动物PBMCs实验证实，北平顶猴可以感染HIV-1。npmTRIMCyp可以有效地限制HIV-2ROD的复制，但对SIVmac239只有十分微弱的限制活性。通过构建鹰猴omTRIMCyp和北平顶猴npmTRIMCyp的置换剪接体（SWAP-1和SWAP-2），转染融合基因及其置换剪接体的CRFK细胞激光共聚焦实验证明，npmTRIMCyp、SWAP1和SWAP2在细胞内主要存在于胞浆中。稳定表达融合蛋白和置换剪接体的CRFK细胞感染HIV-1-GFP-VSVG分析表明，含omTRIMCyp外显子7的SWAP-1和SWAP-2均具有限制HIV-1活性，但SWAP-1的活性更强一些，这表明TRIM5结构域的外显子7可能在介导对HIV-1的限制活性中发挥了协同辅助作用。免疫共沉淀研究表明，npmTRIMCyp不能识别和结合HIV-1的衣壳蛋白。对北平顶猴中介导识别逆转录病毒区域的基因组部分进行了测序，共鉴定出46个多态性位点，表明在北平顶猴识别逆转录病毒衣壳区域存在较高的多态性。

Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.