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 of a new transforming gene from a chemically transformed human cell line

Molecular cloning of the transforming gene from a chemically transformed human osteosarcoma-derived cell line enables the gene to be mapped to chromosome 7 (7p11.4-7qter) and by this criterion and by direct hybridization to be shown to be unrelated to known oncogenes.     

藏黄牛褪黑激素合成酶AA-NAT基因cDNA克隆及序列的比较分析

哺乳动物季节性繁殖主要受褪黑激素调控, 而褪黑激素的分泌受其2个合成酶HIOMT和AA-NAT调控. 分布于青藏高原的藏黄牛和牦牛是典型的季节性繁殖动物, 普通黄牛繁殖则季节性不明显. 比较分析它们三者的AA-NAT序列, 从而进一步研究它们繁殖的季节性. 采用RT-PCR技术,从2个公藏黄牛个体的松果体组织总RNA中分别克隆到AA-NAT基因的表达序列, 并对其进行生物信息学分析. 结果表明:藏黄牛AA-NAT基因的表达序列全长为624bp, 开放阅读框(ORF)为621 bp, 编码207个氨基酸, 预测其表达蛋白分子量为52.3005kDa, 等电点为5.06, 不具有信号肽, 有两个具有较高可能性的跨膜区, 存在4个O连接糖基化位点, 而不存在N连接糖基化位点, 有3个潜在的Ser和3个Thr磷酸化位点. 藏黄牛该基因表达序列核苷酸及编码氨基酸与亲缘关系较远的牦牛一致, 而与亲缘关系较近的普通黄牛却有一定差异, 这可能与繁殖的季节性有关.     

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.     

盐生杜氏藻Ugd基因的cDNA克隆及序列分析

作者对不同物种Ugd基因的同源序列进行相似性分析后设计一对兼并引物，利用RTPCR技术获得一条200bp左右的片段，测序分析显示其同芋头(Colocasia esculenta)的Ugd基因的编码区有71．7％的相似性．然后再以此片段为模板设计引物，通过RACE技术获得盐生杜氏藻Ugd基因的全长序列．经克隆测序作blastx分析发现其同芋头(Colocasia esculenta)、大豆(Soybean)、水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)的Ugd基因有78％到81％的同源性．     

Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA

A synthetic fowl plague virus (FPV) haemagglutinin gene has been cloned in bacteria and the complete sequence of the RNA gene deduced. It is 1,742 nucleotides long and the mRNA codes for 56.3 amino acids in an uninterrupted sequence. The nature of some of the important domains in the haemagglutinin has been established, and their structure is discussed in relation to their function. Extensive amino acid sequence homologies exist between FPV and human influenza haemagglutinins.     

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.     

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.     

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.     

Altered nucleotide sequences of a translocated c-myc gene in Burkitt lymphoma

The nucleotide sequence of a translocated c-myc gene in a Burkitt lymphoma reveals multiple base changes in the coding region. Twenty-five base changes, generating 16 codon alterations, were found in the first coding exon; no changes occur in the second coding exon. These changes are probably the result of somatic mutations that occurred during and after translocation, and may contribute to oncogenesis by allowing synthesis of an altered c-myc gene product.     

Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

The complete nucleotide sequence of the transforming gene of a mouse sarcoma virus has been determined. It codes for a protein of 374 amino acids. The nucleotide sequence of the junctions between a murine leukaemia virus and cellular sequences leading to the formation of the viral transforming gene have also been elucidated. The viral transforming sequence and its cellular homologue share an uninterrupted stretch of 1,159 nucleotides, with few base substitutions. The predicted amino acid sequence of the mouse sarcoma virus transforming gene was found to share considerable homology with the proposed amino acid sequence of the avian sarcoma virus oncogene (src) product.     

生物信息学技术克隆并分析新基因STRF7

为进一步研究信号转导相关的新基因片段BE644250,采用生物信息学方法克隆基全长cDNA,并分析了其ORF,电子表达谱,染色体定位等,之后对全长序列进行了实验验证。电子延伸（contig)获得了729bp的延伸产物,含一个典型的74aa的ORF,命名为STRF7。与已知蛋白无明显同源性,部分地相似于人的源框蛋白CDX－4和酵母的转录调节子ADR6,属一新发现的基因;RT－PCR从IL－6刺激后的U937中克隆了STRF7基因,基序列与电子延伸结果安全一致,进一步的分析显示STRF7在多种组织中表达并定位于第6号染色体上,上述结果显示,STRF7是一个新基因,编码含74aa的蛋白,并且是一个潜在的转录因子。     

红砂促分裂原活化蛋白激酶MAPK基因cDNA片段克隆及序列分析

研究了参与非生物胁迫的促分裂原活化蛋白激酶MAPK基因在红砂抗旱中的作用.通过实验克隆得到了红砂MAPK基因550 bp的cDNA片段,与已知的植物MAPK基因的相应片段表现出较高的同源性(最高达80.5%).MAPK基因在红砂叶片和茎中无组织特异性表达,且随着干旱胁迫程度的加剧其表达量增加,说明MAPK基因在红砂抗旱中起重要作用.     

Molecular cloning and characterization of a cotton phosphoen01pyruVate carboxylase gene

Phosphoenolpyruvate carboxylase(PEPC)plays diverse physiological functions during plant development.In this study,a new phosphoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton(Gossypium hirsutum CV.zhongmian 35)by RACE-PCR.The cloned eDNA 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 cotton 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 μM)and lower Vmax(8.0 μmol min-1mg-1)compared with the C3 PEPCs previously reported.     

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.     

Organization and sequence studies of the 17-piece chicken conalbumin gene

The conalbumin gene has been cloned and shown to consist of at least 17 exons approximately 60-200 base pairs long. The DNA sequence upstream from the region coding for the 5' end of the mRNA shows similarities with sequences present in homologous positions in other genes. High and low frequency repetitive sequences are found both upstream from the conalbumin gene and within one intron.     

Molecular cloning and expression of the gene for a human D1 dopamine receptor

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.     

Molecular identification of a human DNA repair gene following DNA-mediated gene transfer

Although it has long been evident that the response of eukaryotes to DNA damaging agents is determined by the effectiveness of a variety of DNA repair systems, there is little detailed knowledge of the nature of these systems or the genes which control them. In humans, a number of hereditary conditions, including xeroderma pigmentosum, ataxia telangiectasia and Fanconi's anaemia, exhibit increased sensitivity to a variety of DNA damaging agents and a predisposition to cancer, suggesting a defect in some aspect of DNA repair. This report describes the identification of a human DNA repair gene following DNA-mediated gene transfer into Chinese hamster ovary (CHO) mutant cells, that like xeroderma pigmentosum cells, are sensitive to a variety of DNA damaging agents and are defective in the initial incision step of DNA repair. The resulting transformants exhibit normal resistance to DNA damaging agents and independent transformants demonstrate a common set of human DNA sequences associated with a human DNA repair gene. These observations provide the basis for the isolation and characterization of the human genes responsible for DNA repair.     

Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes

A putative diversity segment of immunoglobulin heavy-chain genes (D segments) has been identified 700 base pairs 5' to JH1 DNA on the germ-line genome of the mouse. This 10-base pair D segment is flanked by two sets of sequences related to (SEE FORMULAR IN TEXT) which are possible recognition sites for a recombinase. The spacer separating the heptamer and the nonamer is 12 base pairs long on both sides of the D segment. As the space separating the two signal sequences in VH DNAs and JH DNAs is 23 +/- 1 base pairs long, the two recombinations required for creation of a complete immunoglobulin VH gene, a VH--D joining and a D--JH joining, follow a 12/23-base pair spacer rule. Allelic exclusion is discussed with respect to D segments.