一个人类精子发生相关新基因TSARG7的克隆和初步功能研究

精子发生是一个多基因参与的复杂的生理过程,虽然人们克隆了一些与精子发生相关的基因,但迄今为止,人们对精子发生的分子机制了解有限,因而克隆相关的新基因,并研究其在理论上和实践上的功能仍然十分重要。该文从人类睾丸cDNA文库出发,以小鼠精子发生相关基因mTSARG7基因为电子探针,得到1个与人类的精子发生相关的新基因TSARG7（GenBank登录号为AY513610）,该基因的cDNA全长为2463bp,含有12个外显子和11个内含子,定位在人类8号染色体8p11.21上。TSARG7编码的蛋白质含有456个氨基酸,分子量为56．295kDa,等电点为9．13,为胞浆中非分泌性蛋白,具有磷酸酰基转移酶（Hsc）的结构域,属于酰基转移酶家族的新成员,该家族成员具有脂质合成的功能。TSARG7和mTSARG7,TSARG7和Au041707分别具有97％的同源性。该基因在睾丸中特异表达,亚细胞定位在胞浆中表达。TSARG7 mRNA在13岁的睾丸中开始表达,并且随着精子的发生和性成熟而稳定增加,热应急实验显示该基因的表达与温度相关。综上所述,该文克隆了人的1个新基因,该基因与精子的发生和性成熟相关。     

人类高度进化保守基因hnulp1中转录抑制区段的定位

hnulp1是具有碱性螺旋-环-螺旋(bHLH)的新的一类转录因子.其C端含一个DUF654结构域,其序列在同源基因中相当保守,但该结构域功能未知.利用GAL4转录因子中的DNA结合结构域(DBD)和含有与DBD结合序列的荧光素酶报告基因(GAL4-Luc)质粒,构建了哺乳动物细胞转录因子活性分析系统,随后利用GAL4-Luc荧光素酶报告基因对5种含DUF654结构域的不同缺失片段转录抑制活性进行检测.检测结果表明,该基因DUF654结构域中从Δ228-407氨基酸区段具有强烈的转录抑制活性.该结果为进一步研究DUF654结构域的功能和hnulp1基因转录调控的机制奠定了基础.     

人类新基因ANKZF1的克隆与表达研究

心脏发育是一个非常复杂的过程,受一系列基因的精确调控.研究表明,许多锌指蛋白参与心脏的形成和疾病的发生.为了鉴定新的与心脏发育有关的人类锌指基因,运用同源基因克隆法,通过PCR技术扩增获得一个新的人类基因,其cDNA全长2459bp,其编码的蛋白由342个氨基酸残基组成(相对分子质量约为7.45kD).经国际人类基因命名委员会批准被命名为ANKZF1.该基因是哺乳动物物种特有基因.利用RTP-CR技术分析表明,该基因在胚胎的心脏、胃、肾和脑组织中有特异性的表达,提示该基因可能与心脏等组织的发育有关.     

人类新基因LACE1的克隆和特性初步分析

目的:构建人类新基因LACE1,同时对该基因进行初步的特性和功能研究.方法:通过生物信息学EST拼接技术,RT-PCR等技术,克隆出30个人类未知功能基因.利用RT-PCR技术对该基因的表达谱进行研究,同时结合绿色荧光蛋白与荧光显微镜对该蛋白的定位进行初步分析,并利用MTT初步分析该基因的功能.结果:成功克隆出30个未知功能的人类新基因,其中LACEI(Homo sapiens lactation elevated 1)是一个没有任何功能文献报道的新基因,通过生物信息学分析该基因NCBI:NM一145315.3,其cDNA全长为2 262bp,有13个外显子和12个内含子组成,主要定位于人6号染色体,该基因定位于细胞质中,MTT结果显示该基因能明显抑制细胞增值.结论:人类新基因LACEI是一个抑制细胞增值的相对保守的人类新基因.     

人纤溶酶原K1-3基因的克隆、表达和产物的纯化及抗癌活性分析

纤溶酶原K13功能区是近年发现的血管生成抑制因子,具有抑制肿瘤生长和转移的活性。以人血管生成抑制素cDNA为模板用PCR技术扩增了K13功能区的基因,DNA序列分析后克隆至质粒pPIC9K上获得重组质粒pPIC9K13,转化毕节酵母GS115,用PCR和G418法筛选高拷贝转化子,进行摇瓶发酵。SDSPAGE和Western blot分析结果证实K13基因已在GS115分泌表达,并具有免疫活性。选用30L和80L罐进行高密度发酵,甲醇诱导48h细胞密度达到OD250～300,比摇瓶提高5～6倍,表达量150200mg/L。发酵上清液经Streamline SP离子交换及PhenylSephorose疏水层析纯化,在SDSPAGE上显示一条带,纯度96%,动物实验证明纯化产物具有抗血管生成和抗肿瘤的活性。      

m6A去甲基化酶ALKBH5对大鼠S1PR3基因3′-非编码区双荧光素酶活性的影响

[目的]明确m⁶A去甲基化酶ALKBH5对含S1PR3基因3′-非编码区(3′-UTR)双荧光素酶报告基因的调控作用。[方法]以大鼠前额叶皮层脑区cDNA为模板,利用聚合酶链式反应(PCR)扩增S1PR3基因3′-UTR中含有m⁶A修饰位点的目的片段。利用重叠延伸PCR方法将三个靶序列GGACT、GGACT、AGACT中的第三位A突变为C,并将野生型S1PR3-3′-UTR片段和突变型S1PR3-mut-3′-UTR片段分别正向插入到pmiR-RB-Report^TM vector载体中。将pcDNA3.1-ALKBH5或空载体pcDNA3.1与野生型和突变型双荧光素酶报告载体分别共转染PC12细胞,并检测荧光素酶活性。[结果]成功构建了包含S1PR3基因3′-UTR野生型双荧光素酶报告载体pmiR-S1PR3-3′-UTR和突变型双荧光素酶报告载体pmiR-S1PR3-mut-3′-UTR。荧光素酶活性分析表明与空载体pcDNA3.1组相比,ALKBH5可显著降低野生型及突变型C1和C2报告载体荧光素酶的活性(P<...     

人热休克因子1对抗增殖蛋白基因启动子转录调控的研究

目的：研究人热休克因子1（hHSF1）对抗增殖蛋白（pmhibitin）动子转录活性的影响。方法：采用PCR方法扩增hHSF1全长编码序列,构建peDNA3．1（＋）-hHSFl真核表达载体,将peDNA3．1（＋）-hHSF1和人prohibitin基因启动子表达载体pGL3-prohibitin共同转染HEK293细胞,采用双荧光素酶报告基因检测系统,检测双荧光索酶活性,分析hHSF1对抗增殖蛋白基因启动子的转录调控作用。结果：成功构建peDNA3．1（＋）-hHSF1真核表达载体,荧光素酶活性测定发现hHSF1明显上调pGL3-prohibitin转录。结论：hHSF1对prohibitin具有转录调控作用。     

人类14—3—3ζ和GCH1蛋白相互作用的初步研究

目的寻找人类14-3-3ζ白的相互作用蛋白,为进一步揭示14-3-3ζ的作用机理提供线索。方法以14-3—3ζ为“诱饵”,利用酵母双杂交系统筛选人脑cDNA文库得到“猎物”蛋白,通过GSTpulldown技术和哺乳动物细胞内免疫共沉淀技术验证14-3-3ζ和“猎物”蛋白的特异性结合。结果首次利用酵母双杂交cDNA文库筛选技术发现了14-3-3ζ能够与GTP环化水解酶I（GTP cyclohydrolase1,GCHl）相互作用,并通过了体内和体外的蛋白质结合实验证实了这两个蛋白质之间的特异性相互作用。结论发现并验证了14-3-3ζ与GCH1之间的蛋白质相互作用,为进一步深入研究这两种蛋白质的功能及所引起的相关疾病的发病机理提供了新的线索。     

人纤溶酶原K1-3基因的克隆、表达和产物的纯化及抗癌活性分析

纤溶酶原K1－3功能区是近年发现的血管生成抑制因子,具有抑制肿瘤生长和转移的活性。以人血管生成抑制素cDNA为模板用PCR技术扩增了K1－3功能区的基因,DNA序列分析后克隆至质粒pPIC9上获得重组质粒pPIC9K13转化毕节酵母GS115,用PCR和G418法筛选高拷贝转化子,进行摇瓶发酵。SDS－PAGE和Western blot分析结果证实K1－3基因已在GS115分泌表达,并具有免疫活性。选用30L和80L罐进行高密度发酵,甲醇诱导48h细胞密度达到OD250－300,比摇瓶提高5－6倍,表达量150－200mg/L,发酵上清液经Streamline SP离子交换及Phenyl-Sephorose疏水层析纯化,在SDS－PAGE上显示一条带,纯度96％,动物实验证明纯化产物具有抗血管生成和抗肿瘤的活性。     

Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3γ in astrocyte

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3γ could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3γ up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3γ up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3γ up-regulation indicating that ischemia-induced up-regulation of 14-3-3γ in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway.     

Molecular cloning and characterization of a novel human kinase gene,PDIK1L

We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ’PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser locatedPDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.     

甘蓝型油菜Toc33基因编码区的分离及蛋白序列的比较

以甘蓝型油菜新鲜嫩叶为实验材料提取其总DNA,以其为模板,根据拟南芥Toc33基因编码区序列设计引物,PCR扩增甘蓝型油菜叶绿体外膜蛋白转运机器的构件蛋白基因Toc33,得到两条扩增带,测序结果显示克隆到的两个片段分别长1370bp、1490bp,将这两个片段分别命名为Bn Tpc33-1,Bn Toc33-2,序列比较发现它们之间的同源性为78％,其中外显子的同源性为96％,而内含子的同源性仅为60％。为研究Toc33与同一基因家族的Toc34基因功能间的关系,对拟南芥、油菜、诸葛菜等植物的Toc33、Toc34蛋白序列进行比较分析并构建了分子系统进化树。     

Molecular cloning of cDNAs encoding variants of meiotin-1

Meiotin-1 is a chromatin-associated protein, originally isolated from microsporocytes of Lilium longiflorum, which is found predominantly in cells undergoing meiotic prophase. Chromatin fractionation studies demonstrated that meiotin-1 has an unusual stoichiometry relative to that of histone H1 and the core histones in chromatin fibers. The protein is found less frequently than is histone H1, and appears to be distributed once every 5 to 13 nucleosomes. This distribution may approximate the number of nucleosomes per turn of the chromatin solenoid. A truncated cDNA was identified by immunoscreening of an expression library, and the cDNA was used as a hybridization probe to select a full length cDNA. Variations between the sequence of the predicted polypeptide and sequenced peptides, and variations between the amino acid composition of the protein and the deduced protein indicate that the cDNAs encode minor variants of mature meiotin-1. RNA gel blot hybridization studies reveal that the meiotin-1 mRNA is restricted to anthers in which meiosis is occurring. Computer analysis of the polypeptide deduced from the cDNA indicates that the protein begins with a region highly homologous to the conserved central globular domain of histone H1 molecules. DNA gel blotting experiments demonstrate that homologous sequences exist in the genomes of a fern, a fungus, and both mono-and dicotyledonous plants. Meiotin-1 has been evolutionarily conserved and I propose that it arose from histone H1 to fulfill a role in organizing meiotic chromatin.     

红藻氨酸致敏癫痫大鼠海马内AP-1 DNA结合活性和成分的改变

一次皮下注射惊厥剂量（７．５ｍｇ／ｋｇ）的红藻氨酸（ｋａｉｎｉｃ ａｃｉｄ,ＫＡ）诱发Ｆｉｓｈｅｒ３４４大鼠出现急性癫痫发作,７ｄ后即可形成癫痫敏感大鼠,继用Ｇｅｌ ｓｈｉｆｔ、Ｓｕｐｅｒ－ｓｈｉｆｔ和Ｗｅｓｔｅｍ ｂｌｏｔ方法测定大鼠海马内ＡＰ－１ ＤＮＡ结合活性及其组成成分。Ｇｅｌ ｓｈｉｆｔ结合显示,癫痫敏感大鼠海马内ＡＰ－１ ＤＮＡ结合活性的基础水平较对照组为高;Ｓｕｐｅｒ－ｓｈｉｆｔ实研