阴道毛滴虫Rab6鸟苷三磷酸酶同源基因的cDNA克隆和序列分析

Rab鸟苷三磷酸酶是细胞膜转运机制的关键调节分子,与原虫的分泌功能密切相关。本研究旨在克隆和分析阴道毛滴虫Rab6鸟苷三磷酸酶同源基因,以便进一步探讨其功能。作者从阴道毛滴虫cDNA表达文库中分离出2个Rab6鸟苷三磷酸酶同源基因的cDNA克隆,其中1个cDNA序列长658对碱基,读码框含597对碱基,推测蛋白质序列具198个氨基酸。序列分析表明该氨基酸序列与Rab6a蛋白亚家族的同源性最高。另一cDNA序列长764对碱基,读码框含657对碱基,推测蛋白质序列具218个氨基酸。序列比对分析显示该氨基酸序列与Rab6b蛋白有较高的同源性。2个氨基酸序列都拥有Rab鸟苷三磷酸酶家族的所有保守结构域、特异性RabF结构域和典型的异戊烯化结构域。进化树分析提示毛滴虫的这2个基因系Rab6家族的同源基因,在进化上更接近原虫和单细胞的酵母Rab6亚家族。序列分析还显示这2个基因都无内含子,其基因组DNA序列与其cDNA序列完全一致。     

原生动物阴道毛滴虫的酵酶长寿保障基因LAG1同源基因克隆和序列分析

为了探讨寄生性原虫阴道毛滴虫细胞生长和衰老相关基因,作者从阴道毛滴虫的cDNA表达文库中分离出一具910碱基对的cDNA克隆,其编码框长834 bp,推测蛋白质序列有277个氨基酸.序列分析显示该克隆与酵酶长寿保障基因LAG1同源性最高,其氨基酸序列中含有LAG1及其同源基因保守的Lag1结构域和TLC结构域以及6个跨膜螺旋区和一个C-未端的内质网保留信号域.因此推测该克隆系酵酶LAG1的同源基因,该基因可能参与阴道毛滴虫的神经酰胺生物合成以及调解细胞的生命期或细胞衰老.该基因的基因组DNA与其cDNA序列一致,提示该基因序列中可能无内含子.     

日本三角涡虫热休克蛋白90 基因克隆及其表达模式

目的 克隆日本三角涡虫热休克蛋白90（Djhsp90）基因,并对其在不同应激条件下表达模式进行分析。方法 采用PCR技术克隆日本三角涡虫hsp90 cDNA全长序列和基因组DNA序列,利用生物信息学软件对其序列进行分析,采用实时荧光定量聚合酶链反应(Real-time PCR)技术检测其在再生、饥饿和热激过程中的表达模式。 结果 Djhsp90 cDNA全长2354 bp,含有2148 bp的开放阅读框（ORF）,编码715个氨基酸,含有真核生物HSP90家族蛋白的5个标签序列和末端高度保守序列MEEVD。基因组DNA测序表明,该基因仅含有1个内含子（48 bp）,内含子的5’-和３’-剪切位点符合经典的“GT-AG”法则。涡虫切断后1d Djhsp90的表达量显著增加,2d达到高峰,3d后逐步下降到正常水平。持久饥饿过程中Djhsp90的表达量维持在较高水平。提高培养温度能显著诱导Djhsp90的表达,且涡虫头部对热效应最敏感。 结论 我们成功克隆了日本三角涡虫hsp90 基因,并证实切割、饥饿和热激能诱导其表达上调。     

一个新的阴道毛滴虫Ras同源cDNA克隆的分离及其特征分析

Ras超家族是由一群具有保守氨基酸基序的GTP结合蛋白组成,作为双向分子开关对细胞的多种功能具有调节重要的作用。本文报告分离得到一个新的阴道毛滴虫Ras cDNA克隆,命名为TvRasC。TvRasC cDNA编码一个194氨基酸的蛋白,与其它Ras亚家族成员蛋白序列的一致性在46 %到52 %之间。序列分析表明,该蛋白序列拥有Ras亚家族保守的结合GTP结构域。进化树分析发现该基因与人类的Rit基因位于同一亚群。由于Rit家族成员不含有CAAX异戊烯化基序,而TvRasC在羧基末端有一个典型的CAAX异戊烯化基序,因此TvRasC不属于Rit家族成员。在TvRasC的效应结构域内,与位于HRas第33位的天冬氨酸对应的氨基酸残基是天冬酰胺,有研究表明这种变化将大大削弱Ras基因家族与下游效应分子Raf的结合能力。因此我们预测TvRasC可能只有低水平的GTP酶活性,这种替换有可能使Raf失去与Ras结合域相互作用,并可能导致有活性有丝分裂蛋白激酶没有刺激效应。有关TvRasC在寄生性阴道毛滴虫原虫中的作用有待进一步研究。     

大鼠巨噬细胞环加氧酶-2剪接异构体mRNA片段的分离和鉴定

目的：分离和鉴定大鼠巨噬细胞环加氧酶-2剪接异构体mRNA片段，并分析其可能意义。 方法： 采用RT-PCR和测序分析从大鼠巨噬细胞扩增、分离环加氧酶-2剪接异构体cDNA片段，并推导环加氧酶-2剪接异构体氨基酸序列，利用生物信息软件对氨基酸序列进行分析。 结果： 大鼠肺和血管平滑肌组织及腹腔巨噬细胞均发现COX-2目的条带（253 bp），但腹腔巨噬细胞则出现了一条新的电泳带（422 bp），经克隆和测序证实，该条带除目的条带的COX-2 DNA的第7、8外显子序列外，还包含第7、8外显子间的内含子序列，即保留的内含子（169 bp）。根据阅读框推测，在保留的内含子第21-23碱基出现终止密码。 结论： 首次发现大鼠腹腔巨噬细胞中存在COX-2基因的选择性剪接异构体(Genbank accession number:BU500100)，其生物学意义有待进一步研究。     

阴道毛滴虫黏附蛋白ap33基因克隆及其表达

通过提取阴道毛滴虫mRNA ,逆转录合成cDNA ,PCR得到预期长度片段后 ,将其克隆到pUCm-T载体中进行PCR、酶切及测序分析 ,并与GeneBank中核苷酸序列进行同源性分析。再将其克隆至表达载体PET-32a。重组子用酶切、PCR和测序鉴定后 ,转化大肠杆菌并以异丙基 -B- D- 硫代半乳糖苷 (IPTG)诱导表达。从阴道毛滴虫临床分离株中扩增出 92 7bp的ap33的基因片段 ,构建重组质粒PET- 32a (+) - ap33;IPTG诱导后 ,SDS -PAGE显示表达产物的大小约 5 0kDa。     

大鼠大脑组织中谷氨酸脱羧酶GAD67基因的克隆和序列测定

目的克隆大鼠脑组织中谷氨酸脱羧酶GAD67基因.方法采用RT-PCR方法,大鼠脑组织中的mRNA逆转录成cDNA,再以cDNA为模板,扩增谷氨酸脱羧酶GAD67基因片段,克隆入T载体,并经序列测定.结果 RT-PCR法扩增出一特异产物与预期长度1795bp相符,T载体克隆测序与大鼠谷氨酸脱羧酶GAD67 100%同源.结论采用RT-PCR和T载体技术获得了大鼠脑组织中的谷氨酸脱羧酶GAD67基因克隆,为该基因的体外表达打下基础.     

用逆转录—聚合酶链反应法对类胰岛素生长因子cDNA的扩增

逆转录-聚合酶链反应(RT-PCR)法是将PCR方法与逆转录法结合应用,可快速高效地扩增某一基因的全cDNA。类胰岛素生长因子-I(IGF-I)在人染色体中只有一个基因,全长超过45kb,并有内含子,其cDNA却只有几百个bp,为了表达得到IGF-I,本文利用RT-PCR方法,从胎肝提出的混合mRNA中,筛选并扩增了IGF-I的cDNA。经过分子克隆与测序,证明不需制备胎肝cDNA库,即可快速获得IGF-I的cDNA。     

西尼罗I型病毒亚克隆的构建及其鉴定

目的构建西尼罗病毒基因组的cDNA亚克隆，为西尼罗病毒全长cDNA克隆的研发和应用打下基础。方法根据基因组中限制性酶切位点的分布设计4对引物，QIAamp RNA抽提试剂盒提取西尼罗病毒培养细胞BHK-21的总RNA，长片段RT-PCR技术扩增目的基因片段。将目的基因片段与pMD18-T载体进行连接，转化人感受态细胞E.coli DH5α，进行蓝白斑筛选后得到阳性克隆。将重组质粒进行PCR和测序鉴定。结果获得的目的基因片段经PCR和测序结果表明西尼罗病毒功能基因正确地克隆进入载体。结论成功获得西尼罗I型病毒功能基因的cDNA亚克隆，可应用于西尼罗病毒全长cDNA克隆的制备。     

阴道毛滴虫两个Sir2同源基因的克隆和功能

目的探讨寄生性原虫阴道毛滴虫细胞生长和衰老的相关基因。方法从阴道毛滴虫的cDNA表达文库中分离出两个与酵母沉寂信息调节因子（Sir2）有较高同源性的cDNA克隆,分别命名为TvSir2和TvSir2-like,它们的编码框分别长915bp和1116bp。结果序列分析显示这两个cDNA克隆与酵母Sir2同源性很高,其氨基酸序列中含有Sir2p及其同源蛋白三个特征性保守结构域。分别从这两株cDNA克隆中扩增出表达片段植入表达载体pET-41a,转化宿主菌E.coli BL21并用IPTG（isopropylthio-β-D-galactoside）诱导表达到大量融合蛋白。用亲和层析法纯化的融合蛋白分别免疫豚鼠,获得的抗血清用Western-blot法识别到滴虫虫体全蛋白中大小为34000Mr和42000 Mr的条带。免疫荧光法检测TvSir2和TvSir2-like蛋白位于细胞核外的内质网和高尔基复合体区域。结论TvSir2和TvSir2-like克隆是酵酶Sir2的同源基因,为TvSir2和TvSir2-like在模式生物阴道毛滴虫的功能研究奠定了基础。     

A multi-copy gene encodes a potentially protective antigen in Brugia malayi

A genomic library of Brugia malayi was constructed and screened by hybridization with a cDNA clone corresponding to a potentially protective antigen of 63 kDa. The antigen is encoded by a multicopy gene family. Five distinct gene copies were isolated and one was characterized in detail by nucleotide sequence analysis. An apparent pseudogene was also characterized. The organization of genes encoding the antigen is typical of higher eukaryotes in exon/intron organization although the introns have an unusually high A+T content (75%). Organization of the genomic sequence along with S1 nuclease and primer extension analyses indicate that a short untranslated exon is spliced to the 5' end of the mRNAs encoding the antigen.     

Genetic variation at a splicing branch point in intron 9 of the low density lipoprotein (LDL)-receptor gene: a rare mutation that disrupts mRNA splicing in a patient with familial hypercholesterolaemia and a common polymorphism

Mutations in the coding sequence, splice junctions or promoter of the gene for the low density lipoprotein (LDL) receptor are known to be the underlying cause of familial hypercholesterolaemia (FH), but mutations of this type cannot be identified in all patients with a clinical diagnosis of FH. We show here that minor sequence changes elsewhere in introns can be deleterious. A minor rearrangement 30 bp upstream from the junction of intron 9 with exon 10 was detected as a heteroduplex in amplified genomic DNA from one out of 300 heterozygous FH patients. The mutation destroys the only consensus sequence for a splicing branch point in intron 9 and analysis of mRNA from cells from the patient showed that it causes retention of intron 9 or, more rarely, in the use of cryptic splice sites in exon 10. The effect of the mutation on mRNA splicing was confirmed by analysis of mRNA in cells transfected with LDL-receptor mini-gene constructs expressing exons 9 and 10, together with the normal or mutant intron 9. A common C/T polymorphism within this branch point in intron 9 of the LDL-receptor gene does not affect mRNA splicing in vitro and is not associated with significant differences in mean plasma cholesterol concentration in a healthy population.      

DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame

Summary The 5,969 by (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region. This gene contained a 2,499 by subgroup IB and a 1,306 by subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s. The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences. All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns. We detected a new family of intronic ORFs comprising seven P. anserina introns, several N. crassa introns, as well as the T4td bacteriophage intron. Sequence similarities to intron-encoded endonucleases were noteworthy. The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA.     

A gene encoding a hydrophobin, fvh1, is specifically expressed after the induction of fruiting in the edible mushroom Flammulina velutipes

A cDNA clone of the gene fvh1 was previously isolated from a preprimordial cDNA library made from the basidiomycete Flammulina velutipes. Sequence analysis showed that fvh1 encoded for a hydrophobin, a small fungal protein usually secreted by filamentous fungi. FVH1 had a highly conserved arrangement of eight cysteine residues, a putative N-terminal signal sequence and a hydropathy pattern characteristics of class I hydrophobin. A genomic fvh1 clone was isolated from a F. velutipes genomic DNA library and sequenced. Several putative promoter elements and three small introns of fvh1 were identified in this clone. Southern blot analysis of genomic DNA showed that fvh1 was a single copy gene. Northern analysis indicated that fvh1 was specifically and abundantly expressed in mycelia after the induction of fruiting and during fruit body initiation. It was not expressed in mycelia before the induction of fruiting or in mature fruit bodies.     

Structure and chromosomal localization of the human 2',3'-cyclic nucleotide 3'-phosphodiesterase gene

Human brain cDNA clones for the myelin associated enzyme 2' 3' -cyclic nucleotide 3' -phosphodiesterase (CNPase) have been isolated and sequenced. The only 5' untranslated region (UTR) sequence found was that of a human CNPII mRNA, with no direct evidence for a CNPI mRNA. Human CNPase cDNAs were used to isolate genomic clones containing the human CNPase gene which is 9 kb long. Four exons were identified, separated by three introns, and the sequence of each exon and intron/exon boundary has been established.
The polymerase chain reaction (PCR) was used to detect the presence of the human CNPase gene in DNA from a panel of rodent/human somatic cell hybrids. By this means the human CNPase gene was mapped to chromosome 17. In situ hybridization of a human CNPase genomic clone to metaphase chromosomes further localized this gene to chromosomal band 17q21.     

Cloning and sequence analysis of the circadian clock genes period and timeless in Aedes albopictus (Diptera: Culicidae)

The genes period (per) and timeless (tim) are core components of the circadian clock that regulates a wide range of rhythmic biochemical, physiological, and behavioral processes in prokaryotes and eukaryotes. We used degenerate polymerase chain reaction (PCR) and Rapid Amplification of cDNA Ends (RACE) to clone and sequence the entire cDNAs of both the per and tim genes in Aedes albopictus (Skuse). We also used the 5' end of the Ae. albopictus per cDNA to identify previously unannotated sequence coding for the N-terminal region of the PERIOD protein in Aedes aegypti L. We sequenced genomic DNA of one mosquito from each of three geographically distinct populations (New Jersey, Florida, and Brazil), and identified three introns in the per gene and eight introns in the tim gene. Phylogenetic analyses and comparison of functional domains support the orthology of the newly identified per and tim genes. Analysis of nonsynonymous to synonymous substitution rates indicates that both the per and tim genes have evolved under strong selective constraint subsequent to the divergence ofAe. albopictus and Ae. aegypti. Taken together, these results provide resources that can be used to investigate the molecular genetics of circadian phenotypes in Ae. albopictus and other culicids, to perform comparative analyses of insect circadian clock function, and also to conduct phylogeographic analyses using single-copy nuclear introns.     

Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis

Penaeidins are members of a special family of antimicrobial peptides existing in penaeid shrimp and play an important role in the immunological defence of shrimp. Here, we report one penaeidin with a putative isotype newly cloned from fleshy prawn Fenneropenaeus chinensis. The penaeidin open reading frame encodes a 79 amino acid peptide while two exons and an intron were identified within the 1126bp genomic sequence of Fenchi-penaeidin 5. Phylogenetic analysis and sequence comparison with other known penaeidins suggest the new gene belongs to a novel subfamily of penaeidins and the two isoforms were named Fenchi-penaeidin 5-1 and 5-2, respectively. Fenchi-penaeidin 5 mRNA was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in heamocytes, heart, gill, intestine and ovary. Bacterial challenge resulted in mRNA up-regulation, inducing expression in hepatopancreas and stomach. Fenchi-penaeidin 5-1 was also expressed in Pichia pastoris, and recombinant Fenchi-penaeidin 5-1 exhibited activities against Gram-positive and -negative bacteria and fungi.