猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析

为了比较猪瘟病毒 (HCV)野毒株、疫苗株及标准株之间E2基因抗原区域的差异 ,采用RT PCR扩增了HCV石门株、兔化弱毒疫苗株、野毒 0 3及 0 7株的囊膜糖蛋白E2 (gp55)全基因的cDNA片段 ,分别克隆于pGEM T载体中并对其进行了核苷酸序列测定及氨基酸序列的推导 ,同时进行了同源性比较及E2结构与功能的分析。所测 4株HCVE2基因的长度均为1 2 73bp,所编码的氨基酸序列均包括部分信号肽序列和完整的跨膜区序列 ,共由 381个氨基酸组成 ;4个毒株E2蛋白N末端的 683位至 690位信号肽序列 (WLLLVTGA)和C末端 1 0 30～1 0 63位跨膜区均为保守序列 ,而且具疏水性 ;N末端抗原功能区中 ,4个E2蛋白与其它所比较序列在位于第 753位至 759位氨基酸处 ,均有一段保守序列RYLASLH ,无一氨基酸发生变异 ,为亲水性 ,在整个E2蛋白抗原谱中抗原性峰值为最高 ,推测对抗原性产生起重要作用 ;4个E2蛋白的氨基酸序列中均含有 1 5个半胱氨酸 (Cys)残基 ,其数量及位置与国外五株HCV(Brescia ,C ,Alfort.ALD和GPE)完全一致。表明…     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.     

Sequence of a cDNA encoding turtle high mobility group 1 protein

In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.     

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.     

Ankyrin repeat: a unique motif mediating protein-protein interactions

Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.     

紫杆柽柳与其它物种脂质转运蛋白基因编码蛋白的同源性比较

根据从柽柳cDNA文库克隆获得的脂质转运蛋白(LTP)的部分序列,用RACE技术克隆出其全长cDNA序列.基因的5'非翻译区96bp,3'非翻译区222bp,开放阅读框285bp,编码94个氨基酸,预计蛋白的分子量为9.9 kD,等电点为8.02.此基因有8个位置保守的Cys残基及26个氨基酸的信号肽,为典型的植物脂质转运蛋白基因.其基因序列数据库(GenBank)登录号为AY574218(基因)和AAS79106(蛋白).     

Characterization of the mouse Cblc/Cbl3 gene

The mouse Cblc/Cbl3 gene was cloned and characterized. It comprises 12 exons and encodes a putative protein of 496 amino acid residues which shares an overall 67% identity with its human ortholog; it also shares 70% of amino acid identity with mouse CBL over their conserved SH2 and Ring finger domains. Mouse Cblc mRNA is expressed in embryo and adult tissues and has a rather ubiquitous distribution.     

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.     

心脏特异表达基因ASBIO的克隆及表达

从人类心脏文库中成功克隆了一个新的含有锚蛋白重复序列及SOCS盒结构域蛋白（ Ankyrin repeat and SOCS box protein, ASB）的家族成员ASBIO。该基因定位于染色体7q36．1,开放阅读框包含1329个核苷酸,由7个锚蛋白重复序列和1个SOCS盒组成,编码452个氨基酸,分子量大约是49．5kD。物种间的进化型分析比较表明该基因是非常保守的,人的ASB10基因和小鼠的Asb10基因有84．8％的同源性。半定量RT-PCR实验结果证明Asb10基因在成年小鼠的心脏和肌肉组织中高表达,提示其可能与心脏和肌肉组织的发育有关。亚细胞定位显示ASB10蛋白在细胞质和细胞核均有表达,为进一步研究奠定了基础。     

鄱阳湖泥鳅黑色素聚集激素1基因及其蛋白结构特征分析

为研究黑色素聚集激素（Melanin-concentrating hormone,MCH）在泥鳅（Misgurnus anguillicaudatus）皮肤黑色素沉着过程中所起的作用,研究采用RACE-PCR技术首次克隆出了鄱阳湖泥鳅前原黑色素聚集激素1（Prepro-melanin-concentrating hormone 1,Pmch1）基因cDNA全长序列,将其命名为MaPmch1。运用生物信息学软件对MaPmch1基因及其蛋白的理化性质和结构特征进行了分析,构建了系统进化树。其cDNA全长为570 bp,存在一个长度为110 bp的CpG岛,位于263-372 bp;该基因开放阅读框共375 bp,编码含124个氨基酸的蛋白质,共有7个潜在的磷酸化位点;PMCH1蛋白经水解产生17个氨基酸的环形神经肽黑色素聚集激素1（MCH1）。PMCH1和MCH1均为亲水性蛋白,二级结构以无规则卷曲为主;MCH1超二级结构具有一个反平行β-折叠片层,通过二硫键连接形成环形神经肽。同源性分析表明鄱阳湖泥鳅MCH1（MaMCH1）氨基酸序列与其他硬骨鱼类的高度一致。系统进化树显示,包括泥鳅在内的鲤形目PMCH1聚为一支,且物种间PMCH1的亲缘关系与传统的分类地位相吻合。在脊椎动物中,MCH存在高度保守的序列RCM*GRVYRPCW（*代表随机氨基酸）,证明该基因在进化上是极为保守的。     

The ankyrin repeat as molecular architecture for protein recognition

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.     

一个可能与T细胞发育相关的新的C2H2型锌指蛋白基因的克隆(英)

一些在组织和细胞分化中起重要作用的蛋白质包含锌指结构域.为了克隆分离和研究与造血细胞分化和发育成熟相关的蛋白基因,利用编码C2H2型锌指蛋白结构域中部分保守氨基酸序列设计简并引物,以骨髓cDNA为模板,进行PCR扩增,得到若干新的锌指蛋白基因EST.用其中一条为探针筛选人骨髓cDNA文库,获得了一个新的锌指蛋白基因全长cDNA,GenBank收录号为AF246126,长3 888 bp,包括一个完整阅读框,编码686个氨基酸,包括17个典型的和2个非典型的C2H2模体,命名为HZF2. RNA印迹、人多组织mRNA斑点杂交分析结果显示, 其在T淋巴细胞发育和定居的器官组织胸腺、淋巴结中有较高表达,在脾脏、胎肝有中度表达,在B淋巴细胞发育的骨髓中表达很低,在外周血几个淋巴细胞系中仅有极微量的表达,提示HZF2可能对于T淋巴细胞发育和增殖有重要功能.该基因也在脑组织的若干部位、胎盘及肾上腺有较高表达,在多种其他组织细胞有微量表达,说明其可能对维持这些组织细胞的生理功能也起一定作用.将编码HZF2读框的DNA顺序克隆到pEGFP-N1载体中,转染3T3细胞,证明表达的HZF2-GFP融合蛋白定位于细胞核,这与根据HZF2蛋白结构推测其可能作为DNA结合蛋白行使调节基因转录的功能是一致的.     

COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins.

Cloning and sequence analysis of cartilage oligomeric matrix protein (COMP) cDNA, representing a cartilage pentameric protein, revealed a protein of 755 amino acid residues with a calculated molecular mass of 82,700 Da. Expression of the cDNA in COS cells showed that COMP is a homopolymer composed of five identical disulfide-linked subunits. COMP is homologous to the carboxyl-terminal half of thrombospondin, and the homologies include 89% and 54% of the residues in COMP and thrombospondin, respectively. The similarities are most pronounced in the carboxyl-terminal domains and in the calcium binding type 3 repeat domains in which about 60% of the amino acid residues are identical. In the type 2/epidermal growth factor repeat domains the two proteins contain 41% identical residues. The sequence of the amino-terminal 84-amino acid residues is unique for COMP. Comparison of the amino acid sequences in the type 2 and type 3 repeat domains of COMP and the thrombospondins shows that COMP is the product of a unique gene and not the result of an alternatively spliced thrombospondin gene.     

The amino acid sequence of the sex steroid-binding protein of rabbit serum

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.     

Molecular cloning of the gene for 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2

The gene encoding a 2,6-beta-D-fructan 6-levanbiohydrolase (LF2ase) (EC 3.2.1.64) that converts levan into levanbiose was cloned from the genomic DNA of Streptomyces exfoliatus F3-2. The gene encoded a signal peptide of 37 amino acids and a mature protein of 482 amino acids with a total length of 1560 bp and was successfully expressed in Escherichia coli. The similarities of primary structure were observed with levanases from Clostridium acetobutylicum, Bacillus subtilis, B. stearothermophilus (51.0-54.3%) and with LF2ase from Microbacterium levaniformans (53.9%). The enzyme from S. exfoliatus F3-2 shared the conserved six domains and the completely conserved five amino acid residues with family 32 glycosyl hydrolases, which include levanase, inulinase, and invertase. These observations led to the conclusion that the enzyme belongs to family 32 glycosyl hydrolases.     

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome. Accession numbers AF015784 and AF015785 at the GenBank.