人类TECTB基因的电子克隆

用小鼠和鸡的β-tectorin基因（Tectb)的编码区序列对NCBI数据库进行Blastn比较,得到一个相似性很高的人的gDNA序列（GenBank:AL157786),用GENSCAN、MZEF程序和Blast 2 sequence程序分析AL157786,推测AL157786中包含一个编码区由10个外显子构成的基因－TECTB基因。人TECTB基因的开放阅读框为990bp,推测编码329个氨基酸。人TECTB基因与小鼠的Tectb基因在900bp有88.1%的一致性,在329个氨基酸有94.2%的一致性。用Electronic-PCR将人TECTB基因定位于10q25。     

毛竹抗逆锌指蛋白基因cDNA克隆与序列分析

根据水稻抗逆相关锌指蛋白基因的保守区序列设计引物,以毛竹基因组DNA和cDNA为模板,采用PCR方法,成功扩增出1个含有完整阅读框架的cDNA序列,长度为495bp,序列无内含子,共编码164个氨基酸,将其命名为PeZFP基因(GenBank登录号:FJ472953)。氨基酸序列(GenBank登录号:ACL01101)的分析结果表明,PeZFP与其他锌指结构蛋白有较高的同源性,同水稻锌指结构抗逆蛋白OSIAP1序列相似性高达87.7%,且其序列C端具有典型的AN1类型的锌指结构Cx2-4Cx9-12Cx2Cx4Cx2Hx5HxC,在N端具有典型的A20类型锌指蛋白结构,推测此PeZFP为植物抗逆OsIAP1类似基因,在功能上与毛竹抗逆性相关。     

小麦维生素E基因TaHGGT-7AL的克隆与表达分析

从小麦( Triticum aestivum L.)品种‘科农199’籽粒中克隆出维生素E基因 TaHGGT-7AL 及其另外两个拷贝,通过生物信息学分析,对其序列结构特征及蛋白序列的系统发育关系进行了初步研究。结果显示: TaHGGT- 7AL 基因编码区长1227 bp,共编码408个氨基酸;TaHGGT-7AL与另外两个拷贝的序列一致性为95. 45%。TaHGGT-7AL蛋白序列具有9个α-螺旋,该序列与禾本科HGGT蛋白的同源性在58. 7%~ 98. 5%之间。3个 TaHGGT 基因分别位于小麦基因组的7AL、7BL和7DL染色体上,均具有与膜相关的UbiA异戊烯基转移酶家族的保守结构域和一个转运肽。系统进化分析结果表明,TaHGGT-7AL与禾本科植物的亲缘关系较近。qRT-PCR分析结果显示, TaHGGT-7AL 只在小麦颖壳和籽粒中表达,且在花后13 d籽粒的表达量最高。在ABA、4℃低温、干旱及黑暗胁迫处理下, TaHGGT-7AL 表达量上调;NaCl处理24 h后,该基因表达量升高,表明 TaHGGT-7AL 可以对非生物胁迫产生响应。     

亚麻中雄性不育基因同源序列MS2-F的克隆和表达分析

用同源序列克隆法从亚麻中克隆了雄性不育基因同源序列MS2-F cDNA(登陆号:EU363493).该cDNA全长1 91lbp,包含一个1 608 bp的ORF,编码535个氨基酸.推导的蛋白质序列中包含2个雄性不育保守区:NAD结合区域和雄性不育C-末端区域.该基因与油菜和拟南芥雄性不育基因的一致性分别为59.65%和59.16%,为花蕾特异表达基因,推测在亚麻花粉发育过程中与脂酰辅酶A还原酶有相似功能.MS2-F cDNA对应的gDNA大小为2 696 bp(登陆号:EU365361),含有8个内含子和9个外显子.     

内蒙古白绒山羊TSC2基因克隆与表达模式分析

旨在克隆内蒙古白绒山羊TSC2基因cDNA并分析其特性及基本表达模式.利用RT-PCR分段克隆TSC2基因cDNA片段并测序,将得到的cDNA各片段核苷酸序列拼接后获得绒山羊TSC2基因编码区全长序列(HQ684023)并进行生物信息学分析.半定量RT-PCR方法检测TSC2基因在不同组织中的表达特异性.结果表明内蒙古白绒山羊TSC2基因cDNA编码区核苷酸序列为5184 bp,包含了编码1727个氨基酸残基的全长ORF.核苷酸序列与牛、猪、马、大熊猫、犬、恒河猴、人、小鼠及大鼠的同源性分别为97％、90％、89％、88％、87％、87％、87％、86％和86％.NCBI CDD程序预测该基因编码的蛋白质有一个Tuberin结构域和一个Rap-GAP结构域;Psite程序分析有5个N糖基化位点、2个cAMP和cGMP依赖蛋白激酶磷酸化位点、16个蛋白激酶C磷酸化位点、25个酪蛋白激酶磷酸化位点.PSORT程序预测其定位于胞内体膜.TSC2基因在内蒙古白绒山羊的睾丸、脑、肝脏、肺、乳腺、脾和肾脏等组织中都有表达,mRNA丰度在睾丸中较高,乳腺中较低.     

内蒙古白绒山羊VEGF164基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊血管内皮生长因子(vascular endothelial growth factor,VEGF164)基因并分析其基本表达模式。采用RT-PCR技术克隆基因,将得到的基因cDNA序列及其编码的氨基酸序列进行生物信息学分析。利用半定量RT-PCR方法进行组织表达检测。获得了内蒙古白绒山羊VEGF164基因编码区cDNA全长序列,扩增片段全长573 bp,包含了完整的ORF,编码190个氨基酸残基。核苷酸序列与绵羊的VEGF164(EU857623.1)基因同源性为99%,相应的氨基酸序列同源性为99%。SMART程序分析表明,ORF编码的蛋白质具有信号肽序列及血小板衍生和血管内皮生长因子家族(PDGF,VEGF)结构域。Psite程序分析表明,有1个蛋白激酶C磷酸化位点,4个酪蛋白激酶磷酸化位点。ProtComp Version 9.0程序分析将其定位于细胞外。RT-PCR检测表明,VEGF164基因在绒山羊脑、心脏、睾丸、胰腺、脾、肾和肺组织中均有表达。     

与绵羊KAP6-1相似的6个绒山羊全长cDNA的克隆与序列分析

用SMART^TM技术构建了绒山羊体侧部皮肤组织的cDNA文库,随机挑选克隆提取质粒,用M13正向测序引物对插入片段进行测序,得到636个cDNA序列。与GenBank数据库比对,有41个序列与绵羊角蛋白关联蛋白(Keratin Associated Protein,KAP6-1)cDNA高度同源。41个序列可归为6个不同的cDNA,GenBank登陆号分别为AY310749、AY310750、AY310751、AY310752、AY310753和AY310754。与绵羊KAP6-1基因组基因比较,推测这6个序列为全长cDNA,分别为编码82、84、71、71、83和83个氮基酸的碱性蛋白质,甘氮酸和酪氮酸的含量大于60％,它们之间核苷酸序列的一致性大于55．4％,读码框氮基酸序列的一致性大于79．8％。与其他物种KAP6s比较,绒山羊的6个eDNA和绵羊的KAP6-1 cDNA的序列一致性最高,为81．9％～98．8％,不同物种KAP6-1之间氮基酸序列一致性大于50％。     

蒙古绵羊 tnp1基因cDNA全编码区的克隆及序列分析

过渡蛋白1基因(tnp1)是圆形精子细胞特异表达的基因.绵羊tnp1基因的DNA序列至今尚未报道.为了开展绵羊圆形精子细胞标记基因的研究,根据其他物种tnp1基因cDNA的保守序列设计引物,从成年蒙古绵羊睾丸中提取总RNA,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp1基因cDNA全编码区.该基因cDNA 长246 bp,包含一个168 bp的ORF,编码含有54个氨基酸的多肽链.DNA序列测定结果与牛的核苷酸序列比对,同源性为94.0%.绵羊tnp1基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础.     

小麦维生素E基因TaHGGT-7AL的克隆与表达分析

从小麦（Triticum aestivum L.）品种‘科农199’籽粒中克隆出维生素E基因TaHGGT-7AL及其另外两个拷贝,通过生物信息学分析,对其序列结构特征及蛋白序列的系统发育关系进行了初步研究。结果显示：TaHGGT-7AL基因编码区长1227 bp,共编码408个氨基酸;TaHGGT-7AL与另外两个拷贝的序列一致性为95.45%。TaHGGT-7AL蛋白序列具有9个α-螺旋,该序列与禾本科HGGT蛋白的同源性在58.7%~98.5%之间。3个TaHGGT基因分别位于小麦基因组的7AL、7BL和7DL染色体上,均具有与膜相关的UbiA异戊烯基转移酶家族的保守结构域和一个转运肽。系统进化分析结果表明,TaHGGT-7AL与禾本科植物的亲缘关系较近。qRT-PCR分析结果显示,TaHGGT-7AL只在小麦颖壳和籽粒中表达,且在花后13 d籽粒的表达量最高。在ABA、4℃低温、干旱及黑暗胁迫处理下,TaHGGT-7AL表达量上调;NaCl处理24 h后,该基因表达量升高,表明TaHGGT-7AL可以对非生物胁迫产生响应。     

铁皮石斛蔗糖磷酸合成酶基因的克隆与原核表达

应用同源序列克隆法克隆了铁皮石斛蔗糖磷酸合成酶(SPS)基因cDNA全长,并进行了原核表达分析,为进一步研究该基因的时空表达、功能分析及多糖合成机理提供理论依据。结果表明:(1)铁皮石斛SPS基因cDNA全长3 502bp,编码区3 186bp,GenBank登录号JF423929。该基因编码1 061个氨基酸,与文心兰的SPS基因氨基酸序列的一致性最高为93%,与其他科植物SPS基因的氨基酸序列的一致性均高于60%。(2)原核诱导表达结果显示,SPS基因在大肠杆菌中的重组蛋白分子质量约为118.7kD,其表达与序列分析推测的结论一致。(3)生物信息学分析表明,铁皮石斛SPS基因的二级结构包括了螺旋、β-折叠和无规则卷曲,是非跨膜结构的亲水性不稳定蛋白,有2个功能结构域,分别是蔗糖合成功能域及糖基转移功能域。     

Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes

The human R-ras gene was isolated by low-stringency hybridization with a v-H-ras probe. The predicted 218 amino acid R-ras protein has an amino-terminal extension of 26 residues compared with H-ras p21, and shows 55% amino acid identity; conserved domains include the p21 GTP-binding site and the carboxy-terminal membrane localization sequence. R-ras has at least six exons, with the position of the first intron conserved relative to the Drosophila ras64B and Dictyostelium ras genes; there is no similarity in the exon-intron structure of the R-ras gene and of the mammalian H-, K-, and N-ras proto-oncogenes. Cloned mouse R-ras cDNAs exhibit 88% nucleotide and 94.5% predicted amino acid identity to human R-ras. Human R-ras was localized to chromosome 19, a site different from ras p21 genes. Mouse R-ras is syntenic with c-H-ras on chromosome 7.     

Molecular Cloning and Chromosomal Localization of Human Salt-Tolerant Protein

We have isolated a novel human cDNA coding for human salt-tolerant protein (HSTP), that is a homologue of the rat salt-tolerant protein (STP) and may contribute to salt-induced hypertension by modulating renal cation transport. The nucleotide sequence (1988 bp) of the HSTP cDNA contains an open reading frame encoding a polypeptide comprising 545 amino acids, two residues fewer than the rat STP cDNA. The predicted amino acid sequence exhibits 92% identity to that of the rat protein. HSTP contains predicted coiled-coil domains and Src Homology 3 domain, and shows a high degree of identity to CIP4 (Cdc42 target protein) and human Trip 10 (thyroid-hormone receptor interacting protein). We have mapped the HSTPgene to human chromosome 19 by fluorescence in situhybridization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cloning and functional characterization of Neisseria gonorrhoeae tonB, exbB and exbD genes

Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of sources including transferrin (TF) and lactoferrin (LF). To gain insight into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild-type DNA that repaired the defect in tlu mutants, which are unable to take up Fe from either TF or LF despite exhibiting apparently normal ligand binding to the receptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged in tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26–32% identity with TonB proteins of nine other bacteria. The 663 bp ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32–38% and 24–36% identity, respectively, with ExbB and ExbD proteins of three other bacteria. Thus, the sequence comparison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of the E. coli tonB , exbB and exbD genes. An insertional mutation in the tonB homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to take up ⁵⁵Fe from TF and LF. The tonB mutation did not prevent the utilization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid-phase binding assay was largely unaffected by the tonB mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on the TonB system.     

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.