Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen.

DNA extracted from hepatitis B virus Dane particles has been cloned in bacteria using a plasmid vector. A full-length clone has been examined by restriction endonuclease analysis, and the nucleotide sequence of an 892-base pair fragment from cloned hepatitis B viral DNA encoding the surface antigen gene is reported. The amino acid sequence deduced from the DNA indicates that the surface antigens is a protein consisting of 226 amino acids and with a molecular weight of 25,398. The portion of the gene coding for this protein apparently contains no intervening sequences.     

Structure of the pro alpha 2 (I) collagen gene

Fifty-four kilobase pairs (kbp) of cloned chicken DNA containing the entire 38-kbp pro alpha 2 (I) collagen gene have been isolated and characterized. DNA sequence analysis of a select 4 kbp of the gene has precisely described 14 exons which comprise one-third of the sequences encoding the triple-helical domain of the collagen protein. These exons range in size from 45 to 108 base pairs (bp), are all multiples of the 9 bp that code for the repeating triplet, Gly-X-Y, and have an average size of 70 bp. About 50 introns interrupt this gene. Nevertheless, introns do not separate the coding sequences for the ends of the central triple-helical structural domain and the ends of the propeptide domains.     

马铃薯天冬氨酸蛋白酶抑制剂基因5′上游调控区的分离和序列分析

利用单一特异引物聚合酶链反应技术，将马铃薯ＤＮＡ中天冬氨酸蛋白酶抑制剂家族一成员的５′上游区分离，并克隆进ｐＵＣ１８质粒ＳｍａＩ位点中．用３２Ｐ中标记的单一特异引物──ＰｒｉｍｅｒＡ为探针，经斑点杂交得一阳性克隆．ＤＮＡ测序获得含有马铃薯天冬氨酸蛋白酶抑制剂基因５′上游区的插入物的核苷酸序列．从这序列结构中见到ＴＡ富含区，并发现典型的调控序列ＴＡＴＡ和ＣＡＡＴ．此上游序列与国外所得到的天冬氨酸蛋白酶抑制剂的上游区相比较在序列和ＴＡＴＡ及ＣＡＡＴ盒的位置上有着很大的不同．     

Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata

Biological nitrogen fixation requires the nitrogenase enzyme complex, ATP, and a strong reductant. Klebsiella pneumoniae contains 15 linked nitrogen fixation (nif) genes, three of which, nifH, nifD and nifK have been sufficiently conserved in evolution that cloned K. pneumoniae nifHDK DNA will hybridize to DNA sequences from every nitrogen-fixing bacterium examined to date, including the purple, non-sulphur bacterium Rhodopseudomonas capsulata, in which one complete nifHDK operon has been mapped. Using cloned K. pneumoniae nifHDK DNA we report here that R. capsulata contains multiple copies of the genes for nitrogenase components. Two regions containing sequences homologous to all three nif structural genes have been identified, and mutations in one region produced a Nif- phenotype. Nif+ pseudorevertants were derived from these mutants, some of which retained the original mutation suggesting that some of the extra nif gene sequences can be functionally activated.     

嗜热菌EPT3硫酸酯酶基因的克隆及生物信息学分析

利用筛选培养基筛选到一株产硫酸酯酶的深海嗜热菌EPT3,通过16SrDNA分析,将该菌株归属为Geobacillussp．EPT3．以菌株EP＇13的基因组DNA为模板,使用硫酸酯酶引物进行PCR扩增,将目的基因克隆至pUCm—T载体后进行测序．测序结果表明,克隆基因的大小为1956bp,预测编码651个氨基酸残基．对该基因编码蛋白质进行了生物信息学分析,结果表明,该蛋白质序列与其他菌株来源的硫酸酯酶具有很高的相似性,提示本研究克隆的基因编码硫酸酯酶．该硫酸酯酶的理论分子质量为75．1ku,理论等电点为6．90．采用同源建模法建立了GeobaciUussp．EPT3硫酸酯酶的三维结构模型,为球状结构．     

Nucleotide sequence of chicken c-myb complementary DNA and implications for myb oncogene activation

Avian myeloblastosis virus (AMV), like other acute transforming viruses, arose by recombination between its helper virus and host cellular sequences. The latter sequences, termed v-myb, are responsible for the oncogenic properties of the virus. AMV causes acute myeloblastic leukaemia in chickens and transforms a specific class of haematopoietic cells in vitro, but does not induce morphological transformation of cultured fibroblasts, suggesting that only a restricted target-cell population is responsive to its transforming gene product. The normal cellular counterpart of v-myb, c-myb, is highly conserved and is present in all vertebrate and some invertebrate species examined. DNA rearrangements and altered expression of the myb oncogene have been reported in mouse lymphoid tumours and human myeloid and colon tumours. The mechanism of activation of the cellular proto-oncogenes is thought to involve the structural alteration of the coding regions that result in either the synthesis of an altered gene product or the enhanced expression of a proto-oncogene caused by alterations in its regulatory elements. To distinguish between these two mechanisms, we have cloned and sequenced the chicken c-myb complementary DNA and compared it with that of v-myb sequences. We demonstrate that during the transduction of the cellular sequences and/or viral passage a substantial portion of the coding region of the c-myb gene has been lost from both the 5' and 3' ends, resulting in the generation of a truncated gene product that mediates the transforming function of the virus.     

盐透析体外组装核小体及检测方法

核小体是构成真核生物染色质的基本结构单位,体内研究核小体及染色质结构受到诸多因素限制,体外重构核小体结构是研究与核小体及染色质结构相关课题的一种重要的方法手段.实验将ES1,CS1以及601DNA序列克隆到载体中,通过PCR大量扩增回收得到目的DNA条带,表达纯化了4种组蛋白且装配成组蛋白八聚体,在盐透析的条件下组装形成核小体结构,利用EB染色以及Biotin标记的方法分析检测了形成核小体的效率.结果显示,在盐透析的条件下,可以有效的组装形成核小体结构,而且随着组蛋白八聚体与DNA的比例增加,核小体的形成效率显著提高.本实验为核小体定位、染色质重塑及组蛋白变体等表观遗传学以及结构生物学领域的研究奠定一定的基础.     

Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus

Proviruses cloned from rat cells infected with mouse mammary tumour virus, a B-type retrovirus regulated by glucocorticoid hormones, show the structural features of transposable elements: short inverted repeats conclude long direct repeats at the ends of viral DNA, and short sequences of cellular DNA are duplicated during integration and flank each provirus. The integrative mechanism joins a precise site in viral DNA to non-homologous sites in host DNA.     

A general method for site-directed mutagenesis in prokaryotes

The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.     

hTR基因反义表达质粒构建及序列分析

从人的脐带血管内皮细胞中提取基因组 DNA,通过 PCR方法扩增得到了人端粒酶RNA成分 ( human telomere RNA,h TR)的基因片段 ,扩增产物经酶切克隆到逆转录病毒载体p LNCX上 ,构建了 h TR基因的反义表达质粒 .序列分析结果表明 ,PCR扩增得到的 h TR基因的 DNA序列与所发表的序列完全一致 .构建的反义表达载体中的目的基因正确地反向插入到逆转录病毒载体 p LNCX的克隆位点上 ,构成了 h TR基因的反义表达质粒     

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.     

Molecular cloning and nucleotide sequence of a transforming gene detected by transfection of chicken B-cell lymphoma DNA

A transforming gene detected by transfection of chicken B-cell lymphoma DNA has been isolated by molecular cloning. It is homologous to a conserved family of sequences present in normal chicken and human DNAs but is not related to transforming genes of acutely transforming retroviruses. The nucleotide sequence of the cloned transforming gene suggests that it encodes a protein that is partially homologous to the amino terminus of transferrin and related proteins although only about one tenth the size of transferrin.     

Kas基因启动子区的克隆及功能初步分析

利用TAIL-PCR技术,克隆到了与辣椒素合成有关的胎座特异表达基因——3-酮酯酰．ACP合成酶基因（Kas）上游400bp的调控区域．将其全长片段与GUS基因连接构建植物表达载体并转化烟草．GUS组织化学染色表明,克隆到的440bp片段具有启动子活性．对该片段进行序列分析发现,在起始密码子ATG上游存在2个TATA-box,分别为-316～-311位的TATAAA和-224～-219位的TATAAA;在TATA-box上游还存在1个位于-378～-374处的CAAT-box,序列为CCAAT．该研究旨在为利用基因调控辣椒素的生物合成,提高辣椒果实中的辣椒素含量奠定基础．     

灯盏花ITS2基因克隆及RNA二级结构预测

利用PCR方法扩增灯盏花ITS2基因并克隆,获得ITS2基因序列.用生物软件对ITS2序列进行分析并构建系统进化树和RNA二级结构,得到灯盏花的ITS2核苷酸序列为205 bp,基于ITS序列飞蓬属11种共分为5支,分支与地理分布情况基本一致.ITS2 RNA二级结构在飞蓬属种间表现基本一致,而TS2区结构表现出属间差异.利用r DNA ITS区序列一结构和二级结构相结合达到鉴定药用植物灯盏花分类的目的.     

水稻热激蛋白基因（HSP70）的PCR法快速分析

通过比较几个已知ＨＳＰ７０的ｃＤＮＡ顺序，用ＰＣＧＥＮＥ软件中的ＰＣＲＰＬＡＮ程序设计出一对简并引物，然后从水稻（广陆矮４号）总ＤＮＡ中扩增出ＨＳＰ７０的基因片段并将其克隆到ｐＢＬＵＥＳＣＲＩＰＴ载体中。在完成了对克隆的序列测定之后，用ＰＣＧＥＮＥ中有关程序对其进行同源分析，并对其他已知的ＨＳＰ７０作了一些分类及分子进化方面的探讨。     

Isolation of a feline leukaemia provirus containing the oncogene myc from a feline lymphosarcoma

The molecular mechanism by which feline leukaemia virus (FeLV) induces lymphoid malignancy in domestic cats is largely unknown. Using the insertional activation model of c-myc by avian leukosis virus in the induction of bursal lymphoma in chickens, we have now characterized the c-myc locus in feline tissues and investigated the possibility that FeLV may also insert within feline c-myc. We used the 1.5 kilobase (kb) PstI fragment of MC29 v-myc in Southern blot analysis to characterize the structure of the c-myc locus in the DNA of 31 naturally occurring feline lymphomas. Analysis of a cloned c-myc gene from one lymphoma demonstrated that sequences homologous to v-myc occupy 2.6 kb of feline DNA in which a putative intron of 0.5 kb separates sequences homologous to the 5' and 3' exons represented in avian v-myc. We also observed in the DNA of this lymphoma tumour-specific fragments homologous to v-myc. Characterization of these molecularly cloned myc-hybridizing fragments revealed the presence of at least two identical FeLV proviruses 5.5 kb in length, each containing long terminal repeats enclosing a spliced version of the feline myc gene.     

牦牛肥胖基因和肿瘤坏死因子α基因的序列分析

肥胖基因(ob gene)是近年来刚被克隆的新基因，该基因的表达产物瘦蛋白(Leptin)具有调节体重、影响动物生长和繁殖率等一系列生物学功能．肿瘤坏死因子-α(Tumor Necrosis Factor-α，TNF-α)是一种主要由活化单核巨噬细胞和T淋巴细胞产生的多效性细胞因子，时TNF-α基因的基础及应用研究已成为近年来动物抗病育种的研究热点之一，本文克隆及测序分析了牦牛上述两个基因的部分片段。结果表明：ob基因大小为1773bp，TNF-α基因为1160bp，通过genebank中的blastn比较分析牦牛ob基因和TNF—α基因与普通牛种的相应基因同源性都高达98％。     

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.     

人血管生长素基因的化学合成与克隆

采用固相亚磷酸胺法化学合成并克隆人血管生长素基因，该基因全长４２３ｂｐ，其中３６９ｂｐ的结构基因全部选用大肠杆菌偏爱的密码子，在结构基因上游设置了适合原核非融合蛋白表达的核糖体结合位点的Ｓ－Ｄ序列。采用两级退火一步拼接法成功克隆全长基因，该基因适合在多种融合或非融合原核表达载体中表达。