胆固醇损伤内皮细胞差异表达基因的生物信息学分析

目的利用差异表达基因克隆方法（抑制消减杂交）获得大量的动脉粥样硬化相关候选基因和表达序列标签后，探讨如何进行后续基因表达及功能的研究。方法利用Internet网络上的数据库及生物学分析软件对胆固醇损伤内皮细胞后获得的差异表达基因进行核酸序列和蛋白质序列分析，探索差异表达基因克隆后的研究方法和思路。结果通过电子延伸得到一个684bp的全长cDNA序列；通过核酸序列分析，该序列定位在线粒体基因组的7587位～8270位，含有一个完整的开放阅读框，编码与氧化磷酸化相关的9个亚基。对其中一个亚基细胞色素氧化酶Ⅱ（COX2）分析得知，细胞色素氧化酶Ⅱ基因编码一段25．6kDa的弱酸性的信号锚蛋白，细胞色素氧化酶Ⅱ蛋白的三维结构是一个典型的椅式结构，它含有一段疏水区域，一个跨膜结构域和一个胞质结构域。运用蛋白的进化分析，得知细胞色素氧化酶Ⅱ蛋白胞质结构域的氨基酸序列在进化过程中高度保守。结论生物信息学技术是一种高效的获取疾病相关基因信息的方法，利用生物信息学方法对细胞色素氧化酶Ⅱ基因进行分析，获得了基因及其编码蛋白的相关信息，该蛋白参与电子传递，可能与细胞的氧化应激有关。     

问号钩端螺旋体GGDEF和EAL/HD-GYP结构域蛋白生物信息学分析

目的分析问号钩端螺旋体(钩体)环二鸟苷酸(Cyclic dimeric-GMP,c-di-GMP)调节相关的GGDEF和EAL/HD-GYP结构域蛋白生物信息学结果,为后续的功能研究奠定基础.方法 PSI-BLAST方法分析编码GGDEF和EAL/HD-GYP结构域蛋白的基因;利用相应生物信息学软件对蛋白信号肽序列,跨膜序列,蛋白结构,同源性进行分析并构建目的蛋白进化树.结果 问号钩体中22个蛋白分别含有GGDEF或EAL/HD-GYP结构域,多数蛋白均有感受器结构域和跨膜区.7个具有PAS输入感受器的GGDEF结构域蛋白进化较为独立,位于单独的一个分支,其余6个GGDEF结构域蛋白则分散在不同的分支上.4个EAL结构域蛋白和2个HD-GYP结构域蛋白进化上也较为独立,分别处于同一进化分支.3个GGDEF和EAL结构域耦合的蛋白在进化关系上更趋向于PDE活性蛋白.结论 钩体具有多拷贝的合成和降解c-di-GMP相关蛋白编码基因,表明在钩体中c-di-GMP网络具有高度复杂性,同时反映出钩体对环境条件的改变具有复杂的应对机制.     

结膜吸吮线虫中富亮氨酸结构域蛋白的筛选及生物信息学分析

目的对结膜吸吮线虫分泌蛋白基因组数据进行注释分析,筛选出富亮氨酸结构域蛋白,分析预测该基因序列及其编码蛋白质的结构和功能。方法对结膜吸吮线虫基因组进行结构分析和注释,在分泌蛋白组中筛选富亮氨酸结构域蛋白基因序列,利用ExPASY、DNAstar、MEGA 7.0等生物信息学软件预测分析其编码蛋白的理化性质、抗原表位等,并进行同源序列比对分析;建立系统发育树,进行系统进化分析。结果从结膜吸吮线虫分泌蛋白组中筛选出1条含有完整编码框的富亮氨酸结构域蛋白基因序列,其长度为2 439bp,编码812个氨基酸。编码蛋白相对分子质量(Mr)为204.001 24×103,为疏水性蛋白,含一个信号肽,无跨膜区,且含有较多抗原表位,与盘尾丝虫同源序列相似性为66%。结论生物信息学分析结膜吸吮线虫富亮氨酸结构域蛋白含有抗原表位,故该蛋白及其编码基因序列可作为诊断抗原和疫苗候选分子,也为该蛋白的功能研究了提供基础数据。     

阴道毛滴虫RRas同源基因的克隆和序列分析

目的 克隆和分析阴道毛滴虫RRas同源基因，以探讨其在细胞内信号传导通路中的功能。 方法 从已构建的阴道毛滴虫cDNA表达文库中分离得到一个与人类RRas同源的cDNA克隆，用PCR扩增该cDNA克隆TvRRas相对应的基因组DNA，并对cDNA克隆及其对应的基因组DNA进行测序。利用BLASTP，RPS-BLAST和ClustalW等工具进行序列分析。 结果 TvRRas cDNA序列全长705对碱基，读码框含615对碱基，推测蛋白质序列具205个氨基酸。序列分析显示该基因的基因组DNA序列含有5′端ATG起始密码子和3′端的终止密码子，与cDNA序列完全一致，提示该基因无内含子；进一步分析表明该基因系RRas亚家族的同源基因，其氨基酸序列与人类和小鼠的RRas同源性最高（两者的一致性均为51%，相似性均为70%），同时拥有人类RRas基因高度保守的结合GTP的结构域和完全一致的效应结构域。进化树分析表明该基因与人类的RAS原癌蛋白基因分支及RRas分支聚类。 结论 获得了阴道毛滴虫RRas同源基因。     

日本血吸虫一种新腺苷酸激酶全长cDNA的克隆与功能分析

目的：对用表达序列标签（Expression Sequence Tag,EST）策略从日本血吸虫尾蚴cDNA文库中筛选出的新基因进行功能预测。方法：用NCBI站点的BLASTx及BLASTn程序对所获得的新基因序列进行同源性搜索；用NCBI BLAST站点的blast two sequence程序对同源性高的基因进行核苷酸及氨基酸水平的同源性比较；利用Motif Scan in a Protein Sequence对cDNA序列所编码的蛋白质进行结构域搜索；利用CD－Search程序对目标蛋白进行保守区域搜索。结果：发现一个与曼氏血吸虫22kDa单核苷酸激酶mRNA高度同源的日本血吸虫新基因，基因与氨基酸水平的同一性均分别为86．0％和87．0％；蛋白结构域搜索及保守区域搜索结果显示，该cDNA序列所编码的蛋白质是一种腺苷酸激酶。结论：用EST策略筛选新基因是一个可行的方法，所筛到的日本血吸虫新基因编码一种腺苷酸激酶，全长编码序列与曼氏血吸虫腺苷酸激酶mRNA高度同源。     

日本血吸虫一个新钙结合蛋白全长编码区的克隆与功能预测

目的　对用表达序列标签 (ExpressionSequenceTag ,EST)策略从日本血吸虫尾蚴cDNA文库中筛选出的新基因进行功能预测。方法　用NCBI站点的BLASTx及BLASTn程序对所获得的新基因序列进行同源性搜索 ;用NCBIBLAST站点的blasttwosequence程序对同源性高的基因进行核苷酸及氨基酸水平的同源性比较 ;利用motifscaninaProteinsequence对cDNA序列所编码的蛋白质进行结构域搜索。结果　发现一个与曼氏血吸虫尾蚴期特异性 8kDa钙结合蛋白 (Sm8)基因高度同源的日本血吸虫新基因 ,cDNA序列与氨基酸水平的同一性均为 82 % ;蛋白结构域搜索结果显示 ,该cDAN序列所编码的蛋白质具有 2个EF手钙结合位点。结论　用EST策略筛选新基因是一个可行的方法 ,所筛到的日本血吸虫新基因编码一种钙结合蛋白 ,全长编码序列与Sm8基因高度同源 ,对该基因的期特异性及免疫保护性的探讨具有重要的意义。     

CRP2蛋白的研究进展

LIM蛋白是一类富含半胱氨酸、具有一个或多个锌指结构的蛋白家族，目前发现的LIM蛋白已有60多种。它们不仅参与多种基因的转录调控，而且与许多细胞的分化和发育相关。据氨基酸序列的同源性不同，可将LIM蛋白分为四类：LIM-HD．LIM-Only，LIM-K和含一端LIM结构域的LIM蛋白。LIM-only蛋白的特点是含有一个或多个LIM结构域，     

FRG4基因全长克隆及生物信息学分析

为了对新的人突触相关蛋白FRG4进行全长克隆及生物信息学分析,从人胎肝文库PCR扩增FRG4基因全长cDNA序列,用生物信息学方法 对FRG4基因进行基因组结构分析、多序列同源性比较、跨膜区段、亲疏水性分析、功能结构域预测等.结果 表明cDNA文库基础上运用热启动PCR获得FRG4全长cDNA序列,生物信息学分析显示FRG4基因与人类的突触相关蛋白有99%同源性;定位在X染色体的短臂2区2带2亚带2次亚带;由9个外显子和8个内含子组成;无跨膜区段;有一结构域BSD;该基因编码蛋白可能为一水溶性蛋白.     

细粒棘球绦虫丙酮酸脱氢酶的重组表达及生物信息学分析

目的 目的 克隆表达细粒棘球绦虫丙酮酸脱氢酶 （EgPDH） 基因, 并对其进行生物信息学预测与分析。 方法 方法 提取细粒棘球绦虫Total?RNA并反转录成cDNA, 以此为模板扩增目的基因。将目的基因连接至pET28b构建重组质粒并转化大肠杆菌BL21 （DE3） 进行重组表达。采用SignalP4.1、 TMHMM sever v.2.0和TargetP1.1对EgPDH编码蛋白序列分别进行信号肽、 跨膜区及亚细胞定位的预测。采用SMART分析EgPDH编码蛋白结构域, 用BLASTP和GeneDoc进行EgPDH同源序列比对及保守位点分析, 并采用MEGA6软件邻接法构建系统进化树。 结果 结果 成功克隆并构建重组质粒pET28b? EgPDH, 目的基因大小约1 080 bp, 重组蛋白以可溶性形式表达。EgPDH为信号肽的分泌蛋白, 并含转酮酶结构域, 其高度保守酶活性位点分别为Glu57 、 Leu72 、 Ile86 、 Phe114 。系统进化树分析显示EgPDH与多房棘球绦虫PDH亲缘关系最近。 结 结论 论 成功克隆表达了细粒棘球绦虫EgPDH基因并进行了生物信息学预测分析, 为进一步研究该蛋白功能奠定了基础。     

日本血吸虫MBLAC1基因的克隆及生物信息学分析

目的 克隆日本血吸虫金属β内酰胺酶结构域蛋白1(Metallo-beta-lactamases domain-containing protein 1,MBLAC1)基因,并研究其编码蛋白的生物学特性。方法 以成虫虫体cDNA为模板,利用PCR技术扩增SjMBLAC1基因,并通过生物信息学技术分析该基因编码蛋白的结构特征。结果 SjMBLAC1扩增基因片段大小为711 bp,编码236个氨基酸,预测蛋白质分子量约为26 kD,理论等电点为4.84。该蛋白的第7~222位氨基酸为保守结构域,属于MBL超级家族;不存在跨膜结构域及信号肽;具有一个N-糖基化位点,在第186位氨基酸;二级结构中包含α-螺旋8.90%、β-折叠27.97%、无规则卷曲区域63.14%,推测SjMBLAC1蛋白具有9个优势B细胞抗原表位。结论 成功克隆了SjMBLAC1基因并对其编码蛋白进行了生物信息学分析,从而为开展该蛋白的生物学功能研究及筛选抗血吸虫病疫苗候选分子提供了基础。     

The evolutionarily conserved Krüppel-associated box domain defines a subfamily of eukaryotic multifingered proteins.

We have previously shown that the human genome includes hundreds of genes coding for putative factors related to the Krüppel zinc-finger protein, which regulates Drosophila segmentation. We report herein that about one-third of these genes code for proteins that share a very conserved region of about 75 amino acids in their N-terminal nonfinger portion. Homologous regions are found in a number of previously described finger proteins, including mouse Zfp-1 and Xenopus Xfin. We named this region the Krüppel-associated box (KRAB). This domain has the potential to form two amphipathic alpha-helices. Southern blot analysis of "zoo" blots suggests that the Krüppel-associated box is highly conserved during evolution. Northern blot analysis shows that these genes are expressed in most adult tissues and are down-regulated during in vitro terminal differentiation of human myeloid cells.     

The plastid genome of Cryptomonas phi encodes an hsp70-like protein, a histone-like protein, and an acyl carrier protein.

The plastid genome of Cryptomonas phi, a cryptomonad alga, contains three genes that have not previously been found in any organellar genome. Each of these genes encodes a functional class of organellar gene product not previously reported. The first gene, dnaK, encodes a polypeptide of the hsp70 heat shock protein family. The predicted amino acid sequence of the DnaK protein is 54% identical to that of the Escherichia coli hsp70 protein (DnaK), 50-53% identical to that of two nucleus-encoded mitochondrial hsp70 proteins, and 43-46% identical to that of several eukaryotic cytoplasmic members of the hsp70 protein family. The second gene, hlpA, encodes a polypeptide resembling bacterial histone-like proteins. The predicted amino acid sequence of the HlpA protein is 25-53% identical to that of several bacterial histone-like proteins, and the identity increases to 39-76% over a conserved region corresponding to the long arm that binds DNA. The third gene, acpA, encodes an acyl carrier protein, which is a key cofactor in the synthesis and metabolism of fatty acids. Its predicted amino acid sequence is 36-59% identical to that of eubacterial and plant chloroplast (nucleus-encoded) acyl carrier proteins.     

The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains

The recent characterization of the prokaryotic Cys₂His₂ zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified ≈300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys₂His₂ zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros_56–142C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys₂His₂ coordination, in Ros homologues can either exploit a CysAspHis₂ coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.     

Gastric DNA-binding proteins recognize upstream sequence motifs of parietal cell-specific genes.

Polymerase chain reaction amplification of cDNA from pig gastric mucosa demonstrated the presence of zinc-finger proteins called GATA-GT1, GATA-GT2, and GATA-GT3, each having zinc-finger sequences similar to previously characterized GATA-binding proteins. Subsequently, full-length cDNAs of GATA-GT1 and GATA-GT2 were obtained from rat stomach. The zinc-finger domains of GATA-GT1 and -GT2 were 66-86% identical on the amino acid level with each other and with other GATA-binding proteins. Potential protein kinase phosphorylation sites were present in the zinc-finger region. In contrast, regions outside the zinc fingers shared significantly lower similarities. GATA-GT2 was found to bind to the upstream sequence of the H+/K(+)-ATPase beta gene and to a sequence containing the GATA motif. GATA-GT1 and -GT2 were expressed predominantly in the gastric mucosa and at much lower levels in the intestine (GATA-GT2, also in testis), their tissue distributions being distinct from those of GATA-1, -2, or -3. These results clearly suggest that GATA-GT1 and GATA-GT2 are involved in gene regulation specifically in the gastric epithelium and represent two additional members of the GATA-binding protein family.     

Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins.

We have designed three zinc-finger proteins with different DNA binding specificities. The design strategy combines a consensus zinc-finger framework sequence with previously characterized recognition regions such that the specificity of each protein is predictable. The first protein consists of three identical zinc fingers, each of which was expected to recognize the subsite GCG. This protein binds specifically to the sequence 5'-GCG-GCG-GCG-3' with a dissociation constant of approximately 11 microM. The second protein has three zinc fingers with different predicted preferred subsites. This protein binds to the predicted recognition site 5'-GGG-GCG-GCT-3' with a dissociation constant of 2 nM. Furthermore, selection experiments indicate that this is the optimal binding site. A permuted version of the second protein was also constructed and shown to preferentially recognize the corresponding permuted site 5'-GGG-GCT-GCG-3' over the non-permuted site. These results indicate that earlier observations on the specificity of zinc fingers can be extended to generalized zinc-finger structures and realize the use of zinc fingers for the design of site-specific DNA binding proteins. This consensus-based design system provides a useful model system with which to study details of zinc-finger-DNA specificity.     

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.     

Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described; however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A subsequent database search revealed that the amino acid sequence of a lysine-rich C-terminal segment of HP0508 is identical to the C terminus of HP0863. Recombinant proteins expressed from HP0508 and HP0863 bound plasminogen specifically and in a lysine-dependent manner. We designate these genes pgbA and pgbB, respectively. These proteins are expressed by a variety of H. pylori strains, have surface-exposed domains, and do not inhibit plasminogen activation. These results indicate that pgbA and pgbB may allow H. pylori to coat its exterior with plasminogen, which subsequently can be activated to plasmin. The surface acquisition of protease activity may enhance the virulence of H. pylori.     

Isolation and characterization of a Xenopus laevis C protein cDNA: structure and expression of a heterogeneous nuclear ribonucleoprotein core protein.

The C proteins are major components of heterogeneous nuclear ribonucleoprotein complexes in nuclei of vertebrate cells. To begin to describe their structure, expression, and function we isolated and determined the DNA sequence of Xenopus laevis C protein cDNA clones. The protein predicted from the DNA sequence has a molecular mass of 30,916 kDa and is very similar to its human counterpart. Although mammalian genomes contain many copies of C protein sequence, the Xenopus genome contains few copies. When C protein RNA was synthesized in vitro and microinjected into stage-VI Xenopus oocytes, newly synthesized C proteins were efficiently localized in the nucleus. In vitro rabbit reticulocyte lysate and in vivo Xenopus oocyte translation systems both produce from a single mRNA two discrete polypeptide species that accumulate in a ratio similar to that of mammalian C1 and C2 proteins in vivo.