A novel gene of HIV-1, vpu, and its 16-kilodalton product

A 16-kilodalton protein expressed in cells producing the human immunodeficiency virus (HIV-1) was identified as the gene product of the vpu open reading frame. When expressed in vitro, the 81-amino acid vpu protein reacted with about one-third of the serum samples from AIDS patients that were tested, indicating that the vpu open reading frame is expressed in vivo as well. Introduction of a frame-shift mutation into the vpu open reading frame did not significantly interfere with expression of the major viral proteins in a transient expression system. However, a five- to tenfold reduction in progeny virions was observed after the infection of T lymphocytes with the mutant virus. These data suggest that the vpu gene product is required for efficient virus replication and may have a role in assembly or maturation of progeny virions.     

A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses

The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection.     

Expression in Escherichia coli of open reading frame gene segments of HTLV-III

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.     

Nucleotide sequence of the transforming gene of avian myeloblastosis virus

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.     

梨磷脂酰胆碱转移蛋白基因的克隆及其表达分析

据转录组测序结果,结合RACE(cDNA末端快速扩增技术)和Genome walking(染色体步移技术)获得了梨磷脂酰胆碱转移蛋白基因的完整读码框序列,其开放阅读框长为1 323 bp,编码的440个氨基酸序列与葡萄磷脂酰胆碱转移蛋白质的同源相似性达73%,其具有START domain(启动域)、ZnF_TAZ(TAZ锌指结构)、DCD(双氰胺)保守结构域,将梨磷脂酰胆碱转移蛋白基因命名为PePCTP1。PePCTP1基因与GFP融合,构建植物表达载体,洋葱表皮亚细胞定位观察结果显示,PePCTP1基因编码的蛋白质分布在细胞膜上。用激素生长素(IAA)、脱落酸(ABA)、水杨酸(SA)、赤霉素(GA)、细胞分裂素(6-BA)及NaCl诱导处理,发现与对照(CK)相比,SA和GA处理后PePCTP1基因表达量显著上升;NaCl和IAA处理后PePCTP1基因的表达量随时间逐渐上升;而ABA和6-BA处理不能增加目的基因的表达量。据此推测PePCTP1可能参与梨的逆境胁迫抗性反应。     

玉米病程相关蛋白1基因的克隆与表达分析

采用逆转录聚合酶链式反应(RT-PCR)方法从耐玉米穗粒腐病自交系R15中分离得到病程相关蛋白1(pathogenesis-related protein 1,PR1)基因的开放阅读框,命名为ZmPR1,测序结果显示该序列长为528bp,编码175个氨基酸,其蛋白质分子质量为18.7ku.利用NCBI/Blastp和Genedoc软件进行同源性比对显示,ZmPR1基因编码蛋白与水稻、小麦、拟南芥等高等植物中的PR1蛋白相似性较高,且具有相同的富含半胱氨酸蛋白(cysteine-rich secretory protein,CAP)的保守结构域.将构建的重组载体pET32a(+)-ZmPR1在宿主菌Escherichia coli BL21中经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白,在不同的诱导时间、诱导温度和IPTG诱导浓度下对诱导条件进行优化.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明,ZmPR1基因的最佳诱导条件为IPTG终浓度0.6mmol爛L-1,诱导温度28℃,且诱导时间对表达量影响不大.免疫印迹(Western blot)检测证实有39ku的融合蛋白表达,表明ZmPR1基因在大肠杆菌中已成功表达,这为蛋白纯化及单克隆抗体的制备提供了一定的基础.     

洋葱查尔酮合成酶基因的分子克隆和表达分析

查尔酮合成酶是类黄酮类物质合成的关键酶。本研究克隆了洋葱的查尔酮合成酶基因AcCHS1和AcCHS2。AcCHS1的ORF序列1182 bp,编码393个氨基酸残基;AcCHS2的ORF序列1 176 bp,编码391个氨基酸残基。在同等条件下,AcCHS2基因的表达明显高于AcCHS1。     

新基因RSTA14-44在大肠杆菌中的表达及抗体制备

RSTA14-44 基因是中国协和医科大分子生物学试验室在研究中获得的一个新基因,全长 1124 bp,可读编码框自 100bp~684 bp,编码 194 个氨基酸,预测其编码蛋白质分子量为 21.8 KDa。构建了包含 RSTA14-44 cDNA 全部编码框的 pET30a(+)-14 表达载体质粒,经过聚丙烯酰氨凝胶电泳纯化,免疫家兔制备了抗 RSTA14-44 抗体。Western免疫印迹分析表明,该抗体具有较高的反应性和特异性,适合用作 RSTA14-44 蛋白的进一步研究。     

棉花GhDHAR3基因克隆、功能序列分析及烟草的遗传转化

对棉花EST数据库进行同源搜索、比对和序列拼接,经RT-PCR从陆地棉纤维组织中扩增得到脱氢抗坏血酸还原酶基因GhDHAR3的cDNA,其开放阅读框为792 bp,编码由263个氨基酸组成的蛋白质.同源性比对分析显示GhDHAR3蛋白具有较高的保守性,进化树分析表明其与拟南芥AtDHAR3亲缘关系较近.利用软件进行蛋白...     

锯缘青蟹精氨酸激酶基因的克隆与表达

通过分子生物学方法,克隆得到锯缘青蟹（Scylla serrata）精氨酸激酶（Arginine Kinase,AK）开放阅读框基因序列.测序结果显示：该开放阅读框基因的序列长度为1 071 bp,编码356个氨基酸残基;该序列登录Genbank（GQ：851626）,序列比对结果显示,锯缘青蟹精氨酸激酶与凡纳滨对虾（Litopenaeus vannamei）精氨酸激酶的氨基酸序列同源性高达94%,与岸蟹（Carcinus maenas）、中华绒鳌蟹（Eriocheir sinensis）等甲壳类动物的精氨酸激酶也有较高的同源性,均高于90%;将克隆得到的锯缘青蟹精氨酸激酶基因与pGEX-4T-3载体连接,转化大肠杆菌BL21,经IPTG诱导得到分子质量为65 ku的融合表达蛋白（GST-AK）,经兔抗锯缘青蟹精氨酸激酶多克隆抗体的免疫印迹分析证实,GST-AK具有抗原性.     

春兰GLO基因的克隆和实时定量表达分析

采用RT-PCR结合RACE技术从春兰(Cymbidium goeringii)中分离到一个GLO基因.该基因含有一个630 bp的开放阅读框(ORF),共编码210个氨基酸.系统进化树分析显示,该基因属于B类MADS-box基因的PI/GLO家族,其编码的蛋白与其他植物PI/GLO类蛋白具有很高的同源性,命名为CgGLO(登录号HM106984).实时荧光定量表达分析表明,CgGLO主要在第二轮花器官唇瓣和花瓣中表达,在萼片、子房和叶片中表达较少,在蕊柱和根中表达量最少,这种表达模式支持了van Tunen对ABC模型的修正,也显示了CgGLO基因可能在春兰花器官以及子房的形成过程中起着重要作用.     

洋葱二氢黄酮醇-4-还原酶基因的分子克隆

鳞茎的皮色是洋葱的重要经济性状之一。洋葱的二氢黄酮醇-4-还原酶基因AcDFR1的开放阅读框为1158bp,编码385个氨基酸残基;具有5个内含子,均符合GT—AG规则。本研究为阐明洋葱皮色形成的分子机制以及开发分子标记奠定了基础。     

小麦TaLTR基因的克隆及初步表达分析

以小麦山融3号为试验材料,克隆了TaLTR cDNA序列(Low temperature-responsive RNA-bind-ing protein)。序列分析表明,TaLTR序列包含完整的ORF区,编码蛋白含有162个氨基酸,其氨基端包含一个RRM(RNA recognition domain)超家族保守的结构域,羧基端则富含甘氨酸;经半定量RT-PCR分析,表明TaLTR参与低温、干旱、盐胁迫逆境反应。     

Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI

Complementary DNA clones encoding mouse cytokine synthesis inhibitory factor (CSIF; interleukin-10), which inhibits cytokine synthesis by TH1 helper T cells, were isolated and expressed. The predicted protein sequence shows extensive homology with an uncharacterized open reading frame, BCRFI, in the Epstein-Barr virus genome, suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host.     

Expression of interleukin-10 activity by Epstein-Barr virus protein BCRF1

Cytokine synthesis inhibitory factor (CSIF; interleukin-10), a product of mouse TH2 T cell clones that inhibits synthesis of cytokines by mouse TH1 T cell clones, exhibits extensive sequence similarity to an uncharacterized open reading frame in the Epstein-Barr virus BCRF1. Recombinant BCRF1 protein mimics the activity of interleukin-10, suggesting that BCRF1 may have a role in the interaction of the virus with the host's immune system.     

Molecular cloning of odorant-binding protein: member of a ligand carrier family

Odorant-binding protein (OBP) is found in nasal epithelium, and it selectively binds odorants. Three complementary DNAs encoding rat odorant-binding protein have now been cloned and sequenced. One clone contains an open reading frame predicted to encode an 18,091-dalton protein. RNA blot analysis confirms the localization of OBP messenger RNA in the nasal epithelium. This OBP has 33 percent amino acid identity to alpha 2-microglobulin, a secreted plasma protein. Other members of an alpha 2-microglobulin superfamily bind and transport hydrophobic ligands. Thus, OBP probably binds and carries odorants within the nasal epithelium to putative olfactory receptors.     

油菜长链酰基辅酶A合成酶基因(LACS4)的电子克隆和表达分析

长链酰基辅酶A合成酶 (LACSs)把游离脂肪酸活化成为酰基辅酶A硫脂,在油脂的合成和降解途径中发挥着重要的作用.通过电子克隆的方法,发现了1个油菜(Brassica napus)LACS基因.该基因长 2 270 bp,包含2 004 bp的开放阅读框,预测编码1个含有667个氨基酸残基的蛋白质,命名该蛋白为BnLACS4,序列分析显示,BnLACS4具有典型的LACS分子特征.表达分析显示BnLACS4在成熟油菜的根、茎、叶和花中都有表达.在授粉后(DAP)35 d, 不同含油量的角果皮和种子中,BnLACS4的表达也存在差异.表达谱说明,BnLACS4可能参与油脂的生物合成以及油菜种子中的油脂积累.     

小鼠性腺特异表达基因(GSE)的克隆及初步研究

从小鼠的睾丸中克隆了GSE(gonad-specific expression gene)基因,并对该基因进行序列分析和Southern、Northern杂交分析.核苷酸序列分析表明,GSE基因的ORF长度为745 bp,编码247个氨基酸,预测蛋白质分子量为27.6 kDa;Southern杂交证明GSE可能是单拷贝的基因;Northern杂交显示,GSE基因在小鼠体细胞组织(心脏、肝脏、肾脏、大脑、骨骼肌和脾脏)中没有检测到其表达,在睾丸中表达量很高,在卵巢中表达量较低.从推断出的氨基酸序列表明,GSE蛋白在细胞中可能是一个不带有信号肽的可溶性蛋白.     

Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus

The transforming protein ofKirsten murine sarcoma virus (Ki-MuSV) is a virally encoded 21-kilodalton protein called p21 kis. The sequences encoding p21 kis were genetically localized to a 1.3-kilobase segment near the 5' end of the viral genome by assaying the capacity of a series of defined deletion mutants of molecularly cloned Ki-MuSV DNA to induce focal transformation of mouse cells. Nucleotide sequencing of a portion of this region has led to the identification of an open reading frame of 567 nucleotides coding for p21 kis protein.     

一个新的水稻TE的克隆及其与CACTA转座子结构的比较

对水稻受稻瘟病菌诱导的新因子Rim2的基因组结构进行了研究.以Rim2 cDNA片段作探针, 筛选水稻BAC文库并对其亚克隆, 获得了一个基因组DNA克隆Rim2-569.序列分析表明, Rim2-569序列具备Class 2 转座子的基本结构特征.它两端具有完整的末端颠倒重复(TIRs), 若干正向和反向的亚末端重复 (STRs) , 以及插入位点3 bp的同向重复.它TIRs上的保守序列CACTG有别于以往报道的CACTA转座子.该因子包含一个开读框, 其预测蛋白与CACTA转座子编码的TNP2、TNPD等转座酶有低程度的同源性.该因子没有能够编码类似TNP1/TNPA 的DNA结合蛋白的开读框.Southern杂交显示, Rim2因子在多个不同起源的水稻品种上广泛存在,连同检索结果,证明该家族具有很多拷贝.上述特点表明, 该因子属于一个与CACTA转座子有一定差异的新的转座子大家族.