一个新的人类乙酰转移酶基因hNATL的克隆与表达谱研究

从小鼠cDNA序列入手,依据同源分析的电子克隆方法得到一个包含N末端乙酰转移酶结构域的人类新基因hNATL(Human NAT Like)。hNATL的cDNA长1803bp,编码区621bp,Genbank 登陆号AY632082。hNATL编码的蛋白长206个氨基酸残基,包含有N乙酰转移酶结构域。hNATL 基因定位于人染色体17q25．2区,包括6个外显子和5个内含子。基因表达汇编分析结果显示, hNATL在人脑和生殖腺中高表达。Northern杂交显示,在成年小鼠中hNATL在心脏,脾脏和卵巢中出现表达。整体原位杂交的结果显示,hNATL特异表达于E7．5和E8．5的小鼠胚胎脑部和HH10期鸡胚胎的头部。上述结果提示,hNATL对胚胎期脑的发育和成体中重要器官行使正常功能发挥十分重要的作用。     

大肠杆菌N~α-乙酰基转移酶RimI对人胸腺素α原N末端乙酰化修饰的影响

目的:研究大肠杆菌Nα-乙酰基转移酶RimI对人胸腺素α原(ProTα)N末端乙酰化修饰的影响。方法:在大肠杆菌中共表达ProTα与大肠杆菌Nα-乙酰基转移酶RimI,同时设置只表达ProTα的对照菌,分别提取纯化ProTα,利用HPLC检测其发生乙酰化修饰的程度。结果:ProTα的乙酰化修饰发生在翻译后水平,有无RimI的过表达、诱导时间的长短以及这两个因素之间的交叉效应均对ProTα的乙酰化程度有影响,其F值分别为53.8、89.22及16.28,P值均小于0.0001;无论有无RimI过表达,乙酰化的ProTα所占比例在诱导6 h后逐渐升高(P<0.0001);在过表达Ri-mI的菌株中,乙酰化的ProTα在诱导12 h后占37.4%,而在无RimI过表达的菌株中占64.3%;RimI对ProTα乙酰化的抑制作用从诱导6 h后开始显现(P=0.0007)。结论:在大肠杆菌中,过量的Nα-乙酰基转移酶RimI可抑制人胸腺素α原的乙酰化修饰。     

茶树CsASMT基因的克隆和表达分析

该研究在茶树转录组测序的基础上,以茶树品种‘舒茶早’为试验材料,采用SMART^TM RACE技术,克隆了茶树褪黑素合成途径中关键限速酶N 乙酰基 5 羟色胺甲基转移酶基因（^CsASMT）的cDNA全长序列。^CsASMT基因序列全长1 282 bp,其中包含1 065 bp完整开放阅读框（ORF）,编码354个氨基酸,预测等电点5.37,蛋白分子量38.98 kD。系统进化分析表明,CsASMT与珠美海棠（Malus zumi）ASMT9的亲缘关系最近。将目的基因分别与pET 32a(+)和pMal c2X载体重组,然后再转化入原核表达菌株BL21(DE3)中,pET 32a（+）/CsASMT在28 ℃用0.5 mg/mL IPTG诱导6 h后,以包涵体蛋白的形式表达,而pMal c2X/CsASMT经诱导后在上清中可获得分子量为79 kD大量可溶性融合蛋白。实时定量 PCR 分析表明,茶尺蠖取食抑制CsASMT基因表达;褪黑素、ABA、MeJA和SA均能够诱导CsASMT基因显著上调表达。该研究结果为N 乙酰基 5 羟色胺甲基转移酶功能研究奠定了一定的基础。     

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。     

大豆酪氨酸氨基转移酶基因的克隆与表达分析

采用电子克隆与实验克隆相结合的方法获得了大豆酪氨酸氨基转移酶基因的cDNA序列,GenBank登录号为DQ003328.序列分析结果表明,该cDNA序列含有一个编码425个氨基酸的完整的开放读码框,5′非翻译区具有多个同框终止密码子,3′端具有3个加尾信号和polyA尾巴.启动子区除含有通用核心元件外,还含有许多与光反应有关的作用元件.氨基酸序列比对和系统发育分析结果显示,不同物种之间酪氨酸氨基转移酶的氨基酸序列同源性较高.电子表达分析和RT-PCR组织表达分析结果表明,该基因的表达量与组织中叶绿体含量具有很高的关联,强光逆境能够上调该基因的表达.     

火把梨谷胱甘肽S-转移酶基因的克隆与表达

依据火把梨编码谷胱甘肽S-转移酶(GST)的EST序列设计基因特异引物,采用快速扩增cDNA末端技术,从云南火把梨中克隆到一个新的GST基因的全长cDNA序列。该基因被命名为PpGST(GenBank登录号为HQ889136)。PpGST全长cDNA为1 177bp,具有130bp 5′-UTR、696bp ORF以及351bp 3′-UTR,编码含231个氨基酸的蛋白质。与已知植物GSTs家族成员间的氨基酸序列聚类分析将PpGST聚为zeta类GST。RT-PCR分析显示,PpGST在火把梨光照的果皮和没有光照的果皮中大量表达,并且表达强度不受光照时间的影响,而在幼嫩叶片中没有表达。研究结果暗示在果皮中大量表达的PpGST可能参与维持火把梨果实发育过程中的氧化还原平衡及应答逆境胁迫。     

一个新的人类睾丸特异基因的cDNA克隆和表达分析

运用“数据库消减杂交”(DigitalDifferentialDisplay)方法筛选人类睾丸特异表达新基因 ,获得了有差异显示的代表新基因的克隆重叠群 ,挑选其中一个克隆重叠群HS .12 9794进行多组织RT PCR ,初步证实该重叠群在人睾丸中有高表达。然后从包含该重叠群的IMAGE克隆出发 ,采用生物信息学的方法快速克隆了一个人类新基因的全长cDNA序列 ,其全长 2 4 30bp ,开放阅读框为 6 76～ 12 4 8bp ,定位于 3p2 1 1,编码由 190个氨基酸组成 ,分子量为 2 0 4 17 8Da ,等电点为 5 2 3的一个偏酸性蛋白质 ,该蛋白与已知蛋白质无明显同源性。克隆实验验证该基因阅读框完全正确 ,半定量RT PCR进一步显示该基因在人不同发育时期的睾丸及精子细胞中有表达 ,推测其可能与精子生成相关 ,暂命名为SRG5 (TestisSpermatogenesisRelatedGene 5 ,SRG5 ) (GenBank登录号 :AY2 2 1117)。SRG5基因的成功克隆表明 ,“数据库消减杂交”与实验验证相结合的技术路线是一种快捷高效的发现更多人类功能新基因的新策略。     

板栗咖啡酸氧甲基转移酶基因CmCOMT的克隆及原核表达

该研究根据板栗(Castanea mollissima Bl.)cDNA文库分析得到EST序列,采用RT-PCR技术,克隆板栗咖啡酸氧甲基转移酶(caffeic acid O-methyltransferase,COMT)基因全长cDNA(CmCOMT),分析其编码蛋白的相关信息并进行原核表达研究,为板栗木质素合成关键酶基因CmCOMT的生物学功能研究与应用奠定基础。结果表明:(1)CmCOMT基因(GenBank登录号为KU365322)具有一个1 098bp开放阅读框(ORF),共编码365个氨基酸,推测蛋白分子质量为39.684 9kD,理论等电点为5.83,具有植物SAM依赖甲基转移酶的典型特征。(2)CmCOMT核苷酸序列及其编码氨基酸序列与垂枝桦(Betula pendula)和白桦(Betula platyphylla)的相应序列一致性均在90%以上;同源建模表明,CmCOMT的3D模型与苜蓿(Medicago sativa)MsCOMT的蛋白结构相似,推测其可能与MsCOMT具有相似的功能;系统发育树分析显示,CmCOMT与其他植物COMTs具有相同的进化祖先,与桦木科植物物种亲缘关系最近。(3)SDS-PAGE电泳分析表明,CmCOMT蛋白最佳诱导表达条件为0.3mmol/L IPTG在25℃下诱导6h,蛋白分子量约为44kD,其主要以可溶性蛋白的形式存在。     

一个新的人乙酰化酶MAK3的克隆、表达和纯化

在对乙酰GNAT家族进行研究分析的过程中,找到一个人GNAT家族的新成员MAK3。MAK3含有一个保守的乙酰化酶结构域,并且不同物种中的MAK3的蛋白质序列高度保守。通过半定量和定量RT—PCR检测了在不同组织中MAK3的mRNA水平。并且把MAK3分别克隆到了真核和原核表达质粒中,通过免疫印迹技术证实了MAK3的表达。还进一步用亲和层析的方法成功纯化了细菌中表达的MAK3,并对纯化后的MAK3的酶活性进行了测定。     

一新种的兔内皮素B受体基因克隆和全序列分析

198 8年 ,Ｙａｎａｇｉｓａｗａ和其同事在培养的猪主动脉内皮细胞上清中分离到能对离体的猪冠状动脉产生收缩作用的多肽即内皮素 (Ｅｎｄｏｔｈｅｌｉｎ) ,简称为ＥＴ[1] 。ＥＴ不仅具有强大的缩血管作用 ,而且是体内多种细胞的促分裂剂 ,对心血管、肾脏、呼吸、消化、神经、生殖、内分泌各系统均有作用 ;更为重要的是 ,在多种疾病中 ,它又作为一种致病因子参与疾病的发生 ,具有重要的病理生理意义。ＥＴ只有与靶细胞上特异的ＥＴ受体结合 ,启动一系列细胞内信息传递系统 ,其中主要是磷酸肌醇系统 ,调节胞内钙浓度 ,才得以发挥其效应。在人…     

一种新的兔内皮素B受体基因克隆和全序列分析

Endothelin(ET)is the most potent mammalian vasoconstrictor identified to data. As a pathogenic factor,ET is involved in the genesis of many diseases. In this study, a pair of primers was designed and synthesized according to the human ETB receptor gene (hETBR)sequence. A 394bp of DNA fragment was amplified by polymerase chain reaction(PCR) and labeled with a-32p-CTP using Random Primer-Labeling method. With this probe, rabbit lung cDNA library was screened by in situ hybridization and 11 positive clones were identified. Sequencing result showed that a complete reading frame of rabbit ETB receptor(rETBR)cDNA could be produced from three positive clones of eleven. By a series of subcloning,a recombinant plasmid including the 1326 bp of rETBR coding sequences, named pBlu Script-rETBR, was constructed. The deduced amino acid sequence indicated that the rETBR is 441 residues in length, with an expected molecular mass of approximately 49.44 kD. N-terminal 18 residues is the potential signal peptide (Score＝11.11)and therefore the molecular mass of mature rETBR is 47.65 kD with 423 amino acid residues. Analysis of the rETBR hydropathy profile indicates the presence of seven hydrophobic regions, putative transmembrane domains. Potential N-glycosylation sites are the 60th and the 118 th. The structure exhibits a significant sequence and topographical similarity with G protein-coupled receptors.     

Cloning and Nucleotide Sequence of the Alcohol Acetyltransferase II Gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7

The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery lager yeast have one ATF2 gene, while S. bayanus has no similar gene.     

人肿瘤坏死因子受体I死亡域融合蛋白基因的克隆与表达

通过设计４个引物进行重叠ＰＣＲ,由此克服了人肿瘤坏死因子受体Ｉ死亡域与氯霉素乙酰转移酶（ＣＡＴ）的融合蛋白基因（ＤｄＬｃａｔ）。该融合蛋白基因经测序,证明与设计的序列相同。构建成的重组表达质粒ｐＬＴ１０ＤｄＬｃａｔ转化大肠杆菌后发酵,ＩＰＴＧ诱导２ｈ,ＳＤＳ－ＰＡＧＥ测定ＤｄＬｃａｔ蛋白质的分子量为３９ｋＤ。Ｗｅｓｔｅｒｎ印迹实验进一步作了鉴定。表达产物大部分为包涵体。ＤｄＬｃａｔ经Ｑ－Ｓｅｐｈａ     

Cloning,Characterization and Primary Function Study of a Novel Gene,Cymg1,Related to Family 2 Cystatins

Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags (ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation. Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags(ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation.     

多发性骨髓瘤细胞中一个表达上调基因的克隆与分析

根据GenBank收录的多发性骨髓瘤细胞株 (ARH 77)表达上调ESTAF4 2 5 30 0设计引物 ,运用RT PCR检测了 5例多发性骨髓瘤患者及 4例正常人骨髓细胞中该EST的表达水平 .Northern印迹杂交分析该EST在多种组织中的表达 .进一步利用该EST作探针 ,筛选ARH 77cDNA文库 ,获得全长cDNA克隆 ,对该序列进行了分析 .结果显示 ,该EST在多发性骨髓瘤患者骨髓细胞中亦有较高的表达 ,而在正常人骨髓细胞中低表达 .经测序证实 ,该cDNA全长为 4 5 2bp(GenBank收录号 :AF4 87338) .预测其编码一个 5 7个氨基酸的小分子量蛋白质 ,属于与DNA复制有关的解旋酶 引物酶基因家族的新成员 .该基因在多发性骨髓瘤细胞中表达上调 ,其表达水平的改变可能与多发性骨髓瘤的发生与发展有关     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice 10 kD Prolamin

Using PCR technique, two prolamin genes from Oryza sativa var. indica (cv. Guanglu′ ai) and O. sativa var. japonica (cv. Zhonghua 8) were amplified and cloned. The prolamin gene contained 525 base pairs and encoded 134 amino acid residues. The two genes cloned from two different rice cultivars exhibited 100% homology and were highly homologous with the 10 kD prolamin gene in other rice species amountin an homology ranging from 96.6% to 100%. The deduced amino acid sequence shared 34.2% homology with that of maize 10 kD prolamin. As for dicots, only two types of storage protein shared some homology with rice 10 kD prolamin. One was from Brazil nut and the other from castor bean. Analysis on the signal peptide of rice 10 kD prolamin showed that it shared higher homology with that of storage proteins in some monocots such as maize, sorghum and oat. No similar sequence was found in dicots. The gene sequences of "Guangluai” and "Zhonghua 8” 10 kD prolamin would appear in EMBL data-base under the accession number L36604 and L36605 respectively.