小鼠金属硫蛋白 Ⅰ cDNA编码序列的克隆及其在昆虫细胞中的表达

将质粒ｐＢＸ－ＭＴ上的小鼠ＭＴ－ⅠｃＤＮＡ片段切下作为模板,通过ＰＣＲ方法删除该片段的非编码序列,将编码序列克隆到质粒ｐＢＳ－ＳＫ中,经ＤＮＡ序列测定后证明其克隆序列正确．再将ＭＴ－ⅠｃＤＮＡ编码序列插入到转移载体ｐＢａｃＰＡＫ８的ＢａｍＨⅠ和ＥｃｏＲⅠ位点之间,通过磷酸钙／ＤＮＡ共转染方法将其导入昆虫细胞Ｓｆ９中,以Ｗｅｓｔｅｒｎｂｌｏｔ和ＤｏｔＥＩＡ方法对表达产物进行了检测,表达量为１ｍｇ／Ｌ     

The sequence of human serum albumin cDNA and its expression in E. coli

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.     

应用酵母单杂交系统分离水稻蜡质基因5'调控区结合蛋白的基因

在水稻蜡质基因５’上游区中一段３１ｂｐ 核苷酸序列能与水稻未成熟种子核蛋白特异结合。为了克隆这一核蛋白基因,以此３１ ｂｐ 序列构建成“鱼饵”质粒,从水稻ｃＤＮＡ文库中筛选到１３ 个阳性克隆。根据这些阳性克隆中插入ｃＤＮＡ 片段的相互杂交结果,对这些克隆进行了分组。     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.     

Cloning of chicken lysozyme structural gene sequences synthesized in vitro.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.     

扩展青霉PF898碱性脂肪酶cDNA的克隆及序列分析

扩展青霉 (Penicilliumexpansum)PF898可产生一种具有工业价值的碱性脂肪酶 (PEL) .在测定了其N端 12个氨基酸残基序列的基础上 ,通过RT PCR、5′RACE、基因克隆及序列测定 ,获得了PEL完整的cDNA序列 (GenBank登录号为AF2 84 0 6 4 ) .cDNA全长 10 5 0bp ,包括PEL编码区、3′非翻译区和部分 5′非翻译区基因的序列 .编码区cDNA由 85 5个碱基组成 ,编码 1个由 2 85个氨基酸残基组成的酶蛋白 ,其信号肽及前肽部分由 2 7个氨基酸残基组成 ,成熟肽部分由 2 5 8个氨基酸残基组成 .根据氨基酸组成推导该脂肪酶蛋白的分子量为 2 7 3kD .该脂肪酶的氨基酸序列 130～ 134位上有各类脂肪酶中普遍存在的G X S X G保守序列     

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 sequencing of a carp beta s-crystallin cDNA

The mRNAs were extracted from common carp (Cyprinus carpio) lenses, purified, reverse transcribed, dC tailed and cloned into Escherichia coli with pBR322 as vector. The cloning efficiency was around 1 X 10(7) colonies per micrograms of mRNA. A clone (pC20) was found by hybrid-arrested to contain the cDNA related to carp crystallins. However, comparison of the derived amino-acid sequence with bovine gamma-II and beta s-crystallins indicates that this carp crystallin sequence resembles closely the bovine beta s-crystallin and should be better classified as such except that this fish sequence does not contain the N-terminal 'arm' of four amino-acid residues present in bovine beta s-crystallin.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

小花棘豆(Oxytripis glabra DC)毒素基因的cDNA克隆及序列测定

在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .     

Molecular cloning of cDNA sequences coding for the major alpha- and beta-globin polypeptides of adult Xenopus laevis.

This report describes the synthesis and cloning of almost complete DNA copies of the mRNAs encoding the major alpha-globin and major beta-globin of X. laevis. Double-stranded globin cDNA was inserted into the PstI site of the plasmid pBR322 and two cloned recombinants (designated pXG6C1 and pXG8D2) were selected. These were shown to contain almost complete copies of X. laevis globin mRNA. Restriction enzyme maps were determined for each cDNA sequence using the established method of partial digestion of end labelled DNA. However, this procedure was modified such that isolation of individual DNA fragments was no longer required. Each plasmid was shown, by both hybrid arrested translation and filter selection of complementary RNA, to contain a sequence coding for one or other of the two major globin polypeptides. Sufficient DNA sequence information has been determined from each cDNA clone to demonstrate that pXG8D2 contains a beta-globin sequence and pXG6C1 contains an alpha-globin sequence.     

Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.     

Characterization of complementary deoxyribonucleic acid for human adrenocortical 17 alpha-hydroxylase: a probe for analysis of 17 alpha-hydroxylase deficiency

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.