牛Irak-1基因的电子克隆及生物信息学分析

电子克隆提供了一种利用基因组数据库克隆新基因全长cDNA序列的策略。利用小鼠Irak-1基因编码序列(NM_008363)为种子序列进行电子克隆获得了牛Irak-1基因完整编码序列。然后,用生物信息学方法分析了该基因的结构,微卫星位点,密码子偏性和氨基酸的同源性等。结果表明:该基因cDNA全长2 645bp,无内含子,最大开放阅读框2 157bp,编码718个氨基酸,与小鼠的同源性为77%。     

克隆特异基因的几种新方法

从基因组中克隆特异基因是当前分子遗传学、发育生物学、基因工程和基因组图谱分析等领域中的重要研究课题。归纳各种已建立的克隆方法主要有两种策略 ,一是定位克隆 ,二是消减杂交或差异展示。本文主要对一些克隆特异基因的新方法作一简要介绍。1　外显子设陷法真核细胞的基因中大多含有较长的内含子序列 ,在形成成熟的 m RNA过程中 ,这些内含子需经拼接除掉。拼接位点两侧的核苷酸序列是保守的 ,其中 5′端保守的 AG/ GTRAGT(R为嘌呤核苷酸 ,/为剪切位点 )提供拼接供位序列 ,3′端保守的 NCAG/ G(N为任意核苷酸 )提供拼接受位序列…     

草鱼腺苷酸基琥珀酸裂解酶克隆及序列分析

腺苷酸基琥珀酸裂解酶(Adenylosuccinate lyase,ADSL)是嘌呤核苷酸合成过程中的关键酶.研究以草鱼(Ctenopharyngodon idellus)肠道cDNA文库为基础,应用PCR、RT-PCR和RACE技术,成功获得了草鱼肠道组织腺苷酸基琥珀酸裂解酶基因的cDNA全长和基凶组DNA全长.该基因全长1584 bp,包含一个1449 bp的开放阅读框,编码482个氨基酸,与其他脊椎动物比对显示,其序列具有较高的保守性.草鱼腺苷酸基琥珀酸裂解酶基因组DNA由13个外显子和12个内含子组成,其外显子拼接位点非常保守.遵循GT-AG原则.     

生物信息学辅助定位及延伸辐射诱导未知表达序列标签

研究辐射诱导的基因表达调控对于认识细胞对辐射损伤的应激反应有重要意义.在低剂量辐射诱导新基因RIG1表达序列标签(expression sequence tag,EST)片段的基础上,通过非克隆cDNA文库和RACE(rapid amplification of cDNA end)技术获得了其3′末端.依据实验得到的这两段EST序列所提供的信息,通过生物信息学分析将RIG1基因初步定位在20号染色体.对20号染色体RIG1区基因组序列进行外显子扫描,发现预测的外显子正好与实验得到的EST相吻合.利用预测的外显子设计特异引物,成功地克隆了RIG1基因全长序列.同时,对20号染色体RIG1区的生物信息学分析表明,在RIG1基因的上游存在启动子区,从而确定了RIG1基因的基因组序列.因此,通过生物信息学辅助设计实验,快捷地定位及延伸了未知EST片段RIG1,基本完成了RIG1的全基因、基因组序列及染色体定位研究.     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

生物信息学辅助定位及延伸辐射诱导未知表达序列标签

研究辐射诱导的基因表达调控对于认识细胞对辐射损伤的应激反应有重要意义.在低剂量辐射诱导新基因RIG1表达序列标签(expression sequence tag,EST)片段的基础上,通过非克隆cDNA文库和RACE(rapidamplification of cDNA end)技术获得了其3′末端.依据实验得到的这两段EST序列所提供的信息,通过生物信息学分析将RIG1基因初步定位在20号染色体.对20号染色体RIG1区基因组序列进行外显子扫描,发现预测的外显子正好与实验得到的EST相吻合.利用预测的外显子设计特异引物,成功地克隆了RIG1基因全长序列.同时,对20号染色体RIG1区的生物信息学分析表明,在RIG1基因的上游存在启动子区,从而确定了RIG1基因的基因组序列.因此,通过生物信息学辅助设计实验,快捷地定位及延伸了未知EST片段RIG1,基本完成了RIG1的全基因、基因组序列及染色体定位研究.     

成人视网膜假定蛋白基因ARHP的克隆及生物信息学分析

从UniGene库中选取编号为BG2 2 2 62 4来自人鼻咽组织的表达序列标签 (EST )序列 ,联网到NCBI调用Blast服务器分析 ,发现该EST序列是一个代表新基因的未知序列 .利用Blast检索GenBank的nr数据库和EST数据库 ,构建EST重叠群 ,联网到NCBI的ORFfinder服务器 ,分析发现该EST重叠群具有完整的阅读框架 .分别在cDNA序列阅读框架的起始密码子和终止密码子的两侧设计引物 ,以人胎脑cDNA文库为模板 ,进行PCR扩增 ,测序确定该基因的cDNA全长序列 .该基因cDNA序列全长为 1672bp ,阅读框架位于第 3 0 4～ 1557位之间 ,编码由 417个氨基酸组成 ,分子质量为 46 58ku的蛋白质 ,其理论 pI为 4 2 1.将蛋白质序列通过NCBI的Blast服务器进行序列相似性分析 ,发现该基因编码的蛋白质和成年小鼠视网膜未知蛋白 (BAB3 2 2 14 )同源 .经与国际人类基因组命名委员会协商定名为成人视网膜假定蛋白 (adultretinahypotheticalprotein ,ARHP) ,GenBank登录号为AY174896.生物信息学分析表明 ,该蛋白质可能为一参与转录调控的核蛋白 .ARHP基因定位在染色体 5q3 5,跨越 3 5163bp ,含 4个外显子和 3个内含子 .在基因的 5′非翻译区有 2个CpG岛     

家蚕MLP基因的克隆及其结构分析

利用生物信息学的方法快速获得家蚕MLP (Muscle LIM protein, MLP)基因cDNA电子序列, 经RT-PCR生物验证正确, 登录GenBank (No. DQ311195)。MLP基因cDNA长2 327 bp, ORF全长1 485 bp, 编码产生494个氨基酸。该MLP基因组DNA含有11个外显子, 10个内含子, 所有内含子/外显子边界都符合典型的GT/AG剪切模式。MLP基因编码的蛋白富含Gly (14.4%), 分子量约为53.03 kDa, 等电点(PI)为8.29。通过BLAST分析发现该基因编码的家蚕肌肉LIM蛋白, 含有5个保守的LIM结构域, 家蚕的另一种LIM蛋白(AAR23823)含一个LIM结构域, 两者可能是通过可变剪切产生; 后者可能通过竞争作用调节前者在肌细胞中的功能。MLP的克隆为进一步研究其体内功能奠定了基础。     

人组织型纤溶酶原激活剂突变体微小基因的构建

tPA基因全长约36kb,至少由13个内含子分隔为14个外显子。根据tPA的第一、二外显子的编码情况,考虑建立从第二至第六外显子序列在内的tPA微小基因。即将tPA的部分基因组序列与LAtPA cDNA的序列在第六外显子的NarI位点处相连。     

中华鳖gp130基因全长cDNA克隆及生物信息学分析

目的:从中华鳖脾脏、肝脏、肠道构建的SMART cDNA文库中克隆中华鳖gp130基因cDNA全长。方法:采用RACE法克隆中华鳖gp130基因全长cDNA,并对其进行生物信息学分析。结果:中华鳖gp130基因cDNA全长3806 bp,对应基因组全长73 252 bp,包含16个内含子及17个外显子。中华鳖gp130与鸟类、哺乳类相比均有保守的共线性关系。该基因编码由927个氨基酸残基构成的序列,二级结构主要包括α螺旋、延伸链、无规则卷曲、β转角,三级结构包含配体结合、跨膜结构域、纤维链接蛋白结构域等。系统进化分析显示与鸟类首先聚类,其次是哺乳类,最后是两栖类与鱼类。同时以人GP130蛋白为模型构建了中华鳖GP130蛋白的3D结构模型,一致性为58.32%。结论:获得了中华鳖gp130基因全长并做了生物信息学分析,为深入研究GP130及相关信号通路提供了基础。     

Cloning and developmental regulation of α1 acid glycoprotein in swine

A cDNA clone of porcine alpha₁ acid glycoprotein (α₁AGP) has been isolated and sequenced. Sequence homologies between porcine, human, and rat indicate that porcine α₁AGP is similar in structure to the rat and human proteins. RNA blots from days 40, 60, 80, and 110 fetal, newborn, and adult livers showed that α₁AGP mRNA is relatively abundant throughout fetal development, particularly at the later stages and in the newborn; there is a rapid decline in abundance following birth. From birth to 3 days of age, there is a three- to four-fold decline in abundance, and α₁AGP mRNA is approximately 100 times less abundant in the adult liver than in that of perinatal pigs. Southern blots showed that α₁AGP is probably a single-copy gene. The isolation of a cloned cDNA for porcine α₁AGP provides a tool to investigate the molecular mechanisms involved in the developmental regulation of the gene and to correlate changes in gene expression during development with fetal growth and well being.     

人胎肝唾液酸转移酶基因的克隆及测序

根据已克隆的唾液酸转移酶的保守区的序列,以人胎肝ｍＲＮＡ为模板扩增出１５０ｂｐ的片段并测序。其中一个片段（ｓ３８）与已克隆的唾液酸转移酶的活性中心有５７％～９７％的同源性。根据ｓ３８的序列合成寡核苷酸并标记后用作探针筛选人胎肝ｃＤＮＡ文库。从文库中分离了一个编码α２,３唾液酸转移酶的ｃＤＮＡ。该ｃＤＮＡ序列含一个编码３４０个氨基酸的开放读框,推导的氨基酸序列与人颌下腺Ｇａｌβ１,３ＧａｌＮＡｃα２,３唾液酸转移酶相同,与猪颌下腺α２,３唾液酸转移酶有８３２％的同源性。表明从人胎肝ｃＤＮＡ文库中分离的ｃＤＮＡ所编码的蛋白为Ｇａｌβ１,３ＧａｌＮＡｃα２,３唾液酸转移酶     

The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th α-helical repeat from chicken brain α-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th α-helical repeat from chicken brain α-spectrin). An extensive Φ-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.     

Distinguishing specific and nonspecific interdomain interactions in multidomain proteins

Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.     

20周人胎脑cDNA文库的构建

为了研究舰船有害气体、噪声、磁场等特殊环境下人脑特异性基因表达的变化情况 ,构建了一个 2 0周人胎脑cDNA文库 ,实验从胎脑组织中抽提总mRNA后 ,经过一系列酶反应后合成cDNA ,分级分离柱除去小片段后克隆到λgt1 0载体 ,转染宿主菌E .coli.C6 0 0hf1后文库的包装效率为 4 .6× 1 0 6pfu/μg ,cDNA平均插入片段大于 1 .2kbp     

Lactose- and Heparin-Inhibitable Agglutinin from Human Fetal Brain

An agglutinin activity which was sensitive to lactose and heparin was estimated during prenatal brain development. The agglutinin showed higher specific activities in cerebral cortex and midbrain. There was an increase in lectin specific activity in all the brain regions with development. In addition to brain, other fetal organs also showed the presence of developmentally regulated agglutinin. Cerebral cortical agglutinin was purified by Sepharose CL-6B gel filtration and asialofetuin- and heparin-Sepharose affinity chromatography. Purified agglutinin was strongly inhibited by lactose, asialofetuin, and heparin. It showed no requirement for divalent cations and was maximally active at pH 8.0. Electrophoretic characterization showed the aggregate nature of the agglutinin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave subunit molecular weights of 58,000, 45,000, and 24,000.     

Neocortical Cholinergic Enzyme and Receptor Activities in the Human Fetal Brain

In the human fetus, obtained postmortem at estimated gestational ages of 8-22 weeks, biochemical activities of cortical choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were comparable to those of adult brain tissue. In contrast cholinergic receptor binding, including muscarinic M1 and M2 subtypes (measured by displacement of [3H]N-methylscopolamine with, respectively, pirenzepine and carbachol) and [3H]nicotine (putative nicotinic) binding were undetectable before 13-14 weeks and even at 22 weeks were substantially (three- to fourfold) below the respective adult values. Cortical ChAT activity decreased significantly with gestational age whereas binding to the three receptors, including the proportion M1/M2, increased significantly. AChE was present at all ages investigated as the two molecular monomeric (G1) and tetrameric (G4) forms. The proportion of G4, which was much more soluble in fetal compared with adult cortex, increased approximately threefold. Histochemically AChE, although intense in the nucleus of Meynert, was generally confined to subcortical white matter at early fetal developmental periods, appearing later in the cortex localized to nerve fibres and occasional cell bodies. These observations suggest that during the second trimester of human fetal development, cortical cholinergic function may be preceded by relatively high ChAT activity and paralleled not only by increasing receptor binding but also by a proportional increase in the tetrameric form and histochemical reactivity of AChE.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.