Molecular cloning and nucleotide sequence of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 paris of partially overlapping primers which span the entire genome of CSFV strain Shimen were designed and synthesized. Each cDNA fragment of strain Shimen was amplified by RT-PCR method from the anticoagulant blood of strain Shimen infected pig. The PCR fragments were cloned into pGEM-T vector respectively and sequenced. The results show that we have obtained the nucleotide sequence of strain Shimen. The viral RNA consists of 12 297 nucleotides including noncoding regions of 373 and 227 bases at the 5′ and 3′ end, respectively, and a single large open reading frame spanning 11 697 nucleotides in the middle, which encodes an amino acid sequence of 3 989 residues with a calculated molecular weight of 437.6×10³. The precisely sequencing of 5′ and 3′ termini is undertaking. Supported by the National Pandeng Project Huang Qianhua: born in 1968. Graduate student     

Molecular cloning and polymorphism of the human immune deficiency virus type 2

We recently reported the isolation of a novel retrovirus, the human immune deficiency virus type 2 (HIV-2, previously named LAV-2), from patients with acquired immune deficiency syndrome (AIDS) originating from West Africa. This virus is related to HIV-1, the causative agent of the AIDS epidemic now spreading in Central and East Africa, as well as the USA and Europe (see ref. 3 for review) both by its morphology and by its tropism and in vitro cytopathic effect on CD4 (T4) positive cell lines and lymphocytes. But preliminary hybridization experiments indicated that there are substantiated differences between the sequences of the two genomes. Furthermore, the proteins of HIV-1 and HIV-2 have different sizes and their serological cross-reactivity is restricted to the major core protein, as the envelope glycoproteins of HIV-2 are not immunoprecipitated by HIV-1-positive sera. We now report the molecular cloning of the complete 9.5-kilobase (kb) genome of HIV-2, the observation of restriction site polymorphism between different isolates, and a preliminary analysis of the relationship of HIV-2 with other human and simian retroviruses.     

Molecular cloning and nucleotide sequence of the attenuated hog cholera virus Lapinized Chinese strain

The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 239 nt at the 5′ and 3′-noncoding region, respectively. The complete genome sequence contained one large open reading frame which encoded an amino acid sequence of 3 898 residues with a calculated molecular weight of 437×10³. Although there were mostly only small differences between the sequence of the HCLV strain and the published sequences of strains ALD, GPE⁻, Alfort and Brescia, there was one notable insertion of 12 nucleotides, TTTTCTTTTTTC in the 3′ non-coding region of HCLV strain. Supported by the National Pandeng Project, Genbank accession number AF091507 Wang Jiafu: born in 1972, Ph. D.     

Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.     

Molecular cloning of human telomeres in yeast

Telomeres are the DNA sequences found at the ends of linear chromosomes. They define the boundaries of the genetical and physical maps of such chromosomes and so are particularly important for the complete mapping of large genomes that is now being attempted. Telomeres have been intensively studied in the yeast Saccharomyces cerevisiae and in ciliated protozoa: in these organisms the telomeric DNA consists of arrays of tandemly repeated short sequences in which one strand is guanosine-rich and oriented 5' to 3' towards the chromosome end. The conservation of these structural features is reflected in the observation that telomeric DNA from a variety of protozoa will function as telomeres on artificial linear mini-chromosomes in yeast. Tandem arrays of the sequence TTAGGG have been identified at the telomeres of humans and other mammals and also of trypanosomes. This indicates that the structural features of telomeres are conserved between higher and lower eukaryotes and implies that human telomeric DNA could function in yeast. I have used this idea to develop a strategy to isolate a specific human telomere as a molecular clone in yeast and have devised a simple and effective way of cloning other human telomeres and their associated sequences.     

Molecular cloning and expression of brain-derived neurotrophic factor

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.     

Molecular cloning of cDNA for murine interleukin-3

The cDNA sequence for murine interleukin-3, one of the colony stimulating factors that regulate haematopoiesis, codes for a polypeptide of 166 amino acids including a putative signal peptide. The predicted amino acid sequence indicates that formation of mature interleukin-3 involves proteolytic removal of not only the signal peptide but additional amino-terminal amino acids.     

Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides

Molecular cloning and DNA sequencing have yielded considerable structural information about proenkephalin. All previously characterized intermediate peptides of the enkephalin pathways in bovine adrenal medulla have now been aligned into an unambiguous primary structure. Two basic amino acid residues serve as processing signals for release of each of the different components.     

Molecular cloning and expression of human hepatocyte growth factor

Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.     

成都麻羊NSTN基因的克隆测序

根据得克萨斯山羊MSTN基因序列设计引物,并进行PCR扩增,克隆成都麻羊肌肉生成抑制素(MSTN)基因exon1、部分intron1、exon2、部分intron2、exon3及部分intron3,通过DNAman生物软件分析获得MSTN完全编码序列.研究结果,成都麻羊MSTN编码序列含1128个碱基,编码含375个氨基酸的蛋白,编码序列最后三个碱基为终止密码子.exon1全长372bp,翻译第1～124个氨基酸;exon2全长375bp,翻译第125～249个氨基酸;exon3全长381bp,翻译第250～375个氨基酸.成都麻羊MSTN基因exon1变异程度最大,次之为exon2,exon3变异程度最小.     

Molecular cloning and characterization of a cotton phosphoenolpyruvate carboxylase gene

Phosphoenolpyruvate carboxylase （PEPC） plays diverse physiological functions during plant development. In this study, a new phos- phoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton （Gossypium hirsutum cv, zhongrnian 35） by RACE-PCR, The cloned cDNA of GhPEPC2 is 3364 bp in length, and has an open reading frame of 2913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110,6 kD and pI of 5,56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs, Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome, The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cot- ton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers, In addition, the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km （83,6 p.M） and lower Vmax （8,0 p.mol min^-1 mg^-1） compared with the C3 PEPCs previously reported.     

Molecular cloning of the receptor for human antidiuretic hormone.

Antidiuresis, the recovery of water from the lumen of the renal collecting tubule, is regulated by the hypothalamic release of antidiuretic hormone (ADH), which binds to specific receptors on renal collecting tubule cells, stimulates adenylyl cyclase and promotes the cyclic AMP-mediated incorporation of water pores into the luminal surface of these cells. We report here the isolation of the human ADH receptor gene using a genomic expression cloning approach. The gene was used to clone the complementary DNA from a human renal library. The deduced amino-acid sequence of the receptor yields a hydropathy profile characteristic of receptors with seven putative transmembrane regions. This and the comparison with other cloned receptors indicates that the ADH receptor is a member of the superfamily of G-protein-coupled receptors.     

Molecular cloning and characterization of the rat NMDA receptor.

A complementary DNA encoding the rat NMDA receptor has been cloned and characterized. The single protein encoded by the cDNA forms a receptor-channel complex that has electrophysiological and pharmacological properties characteristic of the NMDA receptor. This protein has a significant sequence similarity to the AMPA/kainate receptors and contains four putative transmembrane segments following a large extracellular domain. The NMDA receptor messenger RNA is expressed in neuronal cells throughout the brain regions, particularly in the hippocampus, cerebral cortex and cerebellum.     

草鱼原肌球蛋白基因的克隆及其组织表达

为揭示原肌球蛋白基因在草鱼肌肉中的作用,利用RT-PCR和RACE技术克隆获得了草鱼原肌球蛋白基因c DNA,并对该基因在普通草鱼和脆肉鲩不同组织中的表达情况进行研究分析。结果表明原肌球蛋白基因c DNA全长序列为1 705 bp,包含387 bp的5′UTR序列,1 307 bp的3′UTR序列和855 bp开放阅读框(ORF)。其ORF编码284个氨基酸。系统进化分析表明普通草鱼与斑马鱼、墨西哥脂鲤的原肌球蛋白基因核苷酸同源性分别是93%和87%,氨基酸同源性分别是96%和93%。在聚类上普通草鱼原肌球蛋白基因与其他鲤科鱼类同源性较高,表明亲缘关系最近,与传统分类相一致。Real time-PCR结果表明原肌球蛋白基因在所检测的普通草鱼和脆肉鲩7个组织中均有表达,原肌球蛋白基因在普通草鱼腹肌中表达最高,其次为前肠。原肌球蛋白基因在脆肉鲩腹肌中的表达低于普通草鱼,而脆肉鲩中肌肉、肝脏、肾脏、前肠、后肠中原肌球蛋白基因表达量大于普通草鱼相对应组织,但差异不显著。     

Rapid cloning of high-affinity human monoclonal antibodies against influenza virus

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.     

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.     

Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C

We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.     

Molecular cloning and expression of cDNAs for the human interleukin-2 receptor

We have purified the human T-cell growth factor (interleukin-2) receptor and have cloned, sequenced and expressed cDNAs corresponding to this receptor. We identify one gene, but two interleukin-2 receptor mRNAs which differ in their polyadenylation signals. We have isolated an additional cDNA that may correspond to an alternatively spliced mRNA that lacks a 216 base segment and appears to encode an altered membrane protein which cannot bind interleukin-2.     

Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) was originally characterized as a survival factor for chick ciliary neurons in vitro. More recently, it was shown to promote the survival of a variety of other neuronal cell types and to affect the differentiation of E7 chick sympathetic neurons by inhibiting their proliferation and by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). In cultures of dissociated sympathetic neurons from newborn rats, CNTF induces cholinergic differentiation as shown by increased levels of choline acetyltransferase (ChAT). This increase is paralleled by a reduction of tyrosine hydroxylase (TH) activity. Moreover, CNTF promotes the differentiation of bipotential 02A progenitor cells to type-2-astrocytes in vitro. To help establish which, if any, of these functions CNTF exerts in vivo, it is necessary to determine its primary structure, cellular expression, developmental regulation and localization. The complementary DNA-deduced amino-acid sequence and subsequent expression of cDNA clones covering the entire coding region in HeLa-cells indicate that CNTF is a cytosolic protein. This, together with its regional distribution and its developmental expression, show that CNTF is not a target-derived neurotrophic factor. CNTF thus seems to exhibit neurotrophic and differentiation properties only after becoming available either by cellular lesion or by an unknown release mechanism.     

Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.