黄花苜蓿SKIP同源基因cDNA的克隆与生物信息学分析

本研究利用RT-PCR方法从野生黄花苜蓿中克隆得到一个编码pre-m RNA剪切因子SKIP同源基因的c DNA片段,命名为Mf SKIP。对克隆片段的序列分析表明,该c DNA片段全长2 256 bp,包含一个1 836 bp的开放阅读框,预测编码一条含611个氨基酸的多肽,分子量约为69.1 k D,理论等电点8.78。对蛋白保守结构域的分析显示Mf SKIP具有SNW/SKIP蛋白特有SKIP的S-N-W-K-N序列,与水稻SKIP(NP_001048184.1)同源性高达85%。生物信息学分析表明,Mf SKIP属于定位在细胞核内的不含跨膜结构域且不含信号肽的亲水性蛋白,二级结构主要由无规则卷曲结构组成。系统发育分析表明,野生黄花苜蓿Mf SKIP蛋白与蒺藜苜蓿(Medicago truncatula)的Mt SKIP、鹰嘴豆(Cicer arietinum)Ca SKIP亲缘关系较近。本研究成功克隆野生黄花苜蓿Mf SKIP基因全长c DNA片段,为研究pre-m RNA剪切因子在黄花苜蓿响应非生物逆境胁迫应答中的作用奠定基础,并为抗逆牧草新品种培育提供候选基因。     

新型小鼠淋巴组织特异性肌动蛋白结合蛋白相关序列cDNA克隆

 应用抑制性差减杂交技术 ( SSH)克隆两种不同小鼠胸腺基质细胞的差异表达基因 ,获得新基因片段 C55.通过 Gen Bank检索及 RT- PCR扩增出一个全长 1 .4kb的 c DNA.杂交分析认为它是一个完整的 c DNA序列 .c DNA序列分析表明 ,它拥有一个 636bp的开放读码框架 ,编码 2 1 2个氨基酸 .同源序列比较发现 ,它编码一个肌动蛋白相关蛋白的新成员 ,该序列与多种已知的肌动蛋白相关蛋白 SM2 2 α及其同源蛋白在氨基酸水平上有 62 %～ 95%的同源性 .Northern杂交分析显示 ,该基因 m RNA转录本在两种不同胸腺基质细胞中的表达存在显著差异 . RT- PCR分析显示 ,该基因特异表达于小鼠淋巴相关组织中 ,而在非淋巴组织中无表达 .     

用差异显示法从人胎脑基因文库分离一个编码序列

人１８周、２２周胎儿脑、肝肾组织总ｍ ＲＮＡ 用ＤＤＲＴ－ＰＣＲ显示出差异的条带,回收胎脑和肝肾特异性表达的４８７条电泳条带．其中某些条带用３种组织的ｃＤＮＡ 探针作点杂交,筛选只对胎儿脑总呈阳性的ＤＮＡ 片段．以其中某一条带ＤＮＡ 为探针,从胎儿脑ｃＤＮＡ 文库筛选阳性克隆,得到ＧＣ５８．经Ｎｏｒｔｈｅｒｎ 杂交和ＤＮＡ 测序,表明它是人脑表达的序列,与数据库中ＫＩＡＡ０５１５有同源性,并编码一个有２７４个氨基酸的蛋白质,该蛋白质序列尚未见报道．探讨了ＤＤＲＴ－ＰＣＲ的条件和假阳性问题．     

虎纹捕鸟蛛毒腺cDNA文库的构建

从 2 5只虎纹捕鸟蛛 (Selenocosmia huwena)中得到约 0 .6g左右的毒腺 ,从中提取出5μg m RNA.以此 m RNA为模板 ,经反转录合成双链 c DNA.再经包装成 c DNA文库 .文库的滴度达到 3.2× 1 0 7pfu/m L     

用两步PCR法克隆全长cDNA

采用两步 PCR法成功地克隆了一个全长的 c DNA.首先 ,用差式分析法克隆得到差别表达的 c DNA片段 ,再分别用这些片段内部的特异序列及 c DNA两端不同接头的序列为引物进行第一步 PCR扩增 ,得到差别 c DNA片段的上游和下游序列 .然后 ,根据第一步 PCR扩增得到的上游和下游序列设计基因特异的引物进行第二步 PCR,从而得到全长的 c DNA.     

人Leptin的基因克隆及其在大肠杆菌中的表达

用 RT- PCR自脂肪细胞 RNA扩增人瘦素 (leptin)基因的 c DNA片段 (约 50 0 bp)并克隆至克隆载体 p SK(+) ,获得重组质粒 p SK- OB,DNA序列分析显示获得的 Leptin基因和文献报道一致 .用限制酶 Eco R 和 Bam H 从 p SK- OB切下并插入原核表达载体 p BV2 2 0的相应限制酶位点 ,转化大肠杆菌 DH5α.转化菌株经 42℃诱导 ,SDS- PAGE检测和 Western印迹鉴定 ,获得高水平重组人瘦素的表达菌株 ,其表达量达菌体总蛋白的 30 %以上 .     

家蝇卵黄蛋白基因的克隆与结构序列分析

家蝇卵黄蛋白基因编码的卵黄蛋白是家蝇胚胎发育的重要营养来源 .根据 3种家蝇卵黄蛋白cDNA保守序列设计引物 ,用PCR技术从家蝇基因组DNA中扩增到大小为 76 8bp的mdYP1基因的部分DNA片段 .经地高辛标记成特异性探针 ,从构建的家蝇基因组文库中筛选出一个阳性克隆 ,并从该克隆中分离到大小为 3991bp的mdYP1基因组基因 .序列分析显示 ,该基因组序列含有约1 6kb的 5′ 上游区和 1 0kb的 3′ 下游区 ,编码区由一个 6 1bp的内含子和大小分别为 2 2 2bp和10 2 8bp的 2个外显子组成 .5′ 上游区含有典型的CAAT TATA盒 .     

PS-2基因的克隆及其在肝癌中的表达

利用荧光差异显示技术比较了正常肝、肝硬化和肝癌组织 m RNA的表达 ,1 4个有差异的条带通过 Northern blot分析表明其中 9个为阳性 .令人感兴趣的是一个～ 5 0 0 bp的 c DNA片段 ,它在正常肝和肝硬化中低表达 ,在肝癌组织中高表达 .通过测序 ,发现该片段与 PS- 2 ( presenilin- 2 )基因有 94 %的同源性 .PS- 2基因的突变与早发性阿尔茨海默氏症有关 ,但在肝癌发生中的作用未明 .也许 PS- 2基因的上调涉及到肝癌发生的分子机理     

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     

抗菌肽Diptericin cDNA的克隆及在E.coli中的融合表达

 从果蝇成虫中抽提总 RNA,RT- PCR扩增编码伏蝇素 diptericin的 c DNA,克隆并测定了全序列 .将该 c DNA亚克隆到融合表达载体 p MAL- CR1上 ,转化大肠杆菌 BL2 1菌株 ,进行了高效的可溶性融合表达 .通过离子交换层析纯化融合蛋白 ,经 Xa因子酶切后得到的重组 diptericin具有抗菌活性 .     

Molecular cloning and nucleotide sequence of human pancreatic prechymotrypsinogen cDNA

The cDNA clone encoding human prechymotrypsinogen was isolated from a human pancreas cDNA library and its nucleotide sequence was determined. The sequence consists of a 16 bp 5' non-coding region, a 789 bp amino acid coding region and a 60 bp 3' non-coding region. The predicted product consists of 263 amino acids, including 18 amino acids for a signal peptide and 15 amino acids possible for an activation peptide. Southern blot analyses using the cloned cDNA as a probe revealed that human genomic DNA carries at least two genes that are related to chymotrypsinogen.     

cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.     

The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA.

A cellular src (c-src) cDNA clone was isolated from a chicken embryonic brain cDNA library and characterized by DNA sequence analysis. Comparison with the published sequence of a chicken genomic c-src clone indicated that the brain cDNA clone contained an 18-base-pair insertion located between exons 3 and 4 of the c-src gene. The six amino acids encoded by the insertion caused an alteration in the electrophoretic mobility of the c-src gene product similar to that of the structurally distinct form of the src protein detected in neuronal cultures.     

Isolation and characterization of a human novel RAB (RAB39B) gene

Rab proteins are small-molecular-weight GTPases that control vesicular trafficking in eukaryotic cells. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel Rab protein, which showed 74.2% identity with previously isolated Rab39A at the amino acid level. RAB39B was expressed in a variety of human tissues and located in human chromosome Xq28. It consisted of two exons spanning 3764 bp of human genomic DNA.     

Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X

A 1.5-kb cDNA (FX) encoding full-length human coagulation factor X was isolated from a human fetal liver cDNA library. The identity of the insert in a selected phage lambda clone was confirmed to be FX by nucleotide (nt) sequence analysis and restriction mapping. This FX cDNA clone contained 1467 bp of coding sequence, no 5'-untranslated sequence, a short 3'-untranslated sequence of 10 nt and a poly(A) tail at the 3'-end. The FX cDNA was inserted into a mammalian expression vector and transfected into COS-1 monkey kidney cells. Media from transfected cells showed evidence of factor X antigen and, following addition of Russel's viper venom factor X activator, enhanced amidolytic activity toward a synthetic peptide rho-nitroanilide substrate. Immunoprecipitation with an anti-factor X monoclonal antibody of [35S]methionine-labeled cell-conditioned media showed evidence of polypeptides of 74, 55, and 17 kDa, as determined by SDS-PAGE followed by autoradiography. Together, these results indicate that an active factor X can be successfully expressed in a recombinant DNA expression system. This approach will allow the systematic structure/function investigation of this important blood-clotting enzyme.     

Cloning and characterization of a novel Krüppel-like zinc finger gene, ZNF268, expressed in early human embryo

With the aim of identifying genes involved in early human embryonic development, we have isolated a cDNA clone representing a novel human zinc finger gene ZNF268 from 3 week old human embryo cDNA library using a differential hybridization strategy. The complete cDNA sequence of ZNF268 contained an open reading frame of 2841 nucleotides that encodes a 947 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and 24 C-terminal zinc fingers. Northern blot analysis showed that ZNF268 mRNA is mainly expressed in 3-5 week old human embryos suggesting it could play certain roles in the embryogenesis. The gene consists of six exons spanning about 22 kb of genomic DNA. According to the genomic sequence from the HTGS database, the ZNF268 gene is assigned to human chromosome 5.