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Genetic control of biosynthesis, structural assembly and activity expression of eukaryotic pyruvate dehydrogenase

真核丙酮酸脱氢酶生物合成、结构组装和活性表达的遗传控制

基本信息

批准号：
63580156
负责人：
KOIKE Masahiko
金额：
$ 1.09万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63580156/
关键词：
pyruvate dehydrogenase (PDH)alpha_2beta_2 subunit structure PDHalpha, beta cDNAs PDHalpha, beta mRNAs biosynthesis PDHalpha gene PDHbeta gene nucleotide sequences

项目摘要

1. The cDNAs encoding human pyruvate dehydrogenase (PDH)alpha and beta subunits were isolated from a HeLa cell and human foreskin fibroblast cDNA libraries. They spans 1,363 base pairs (alpha) and 1,557 base pairs (beta), respectively, and encoded alpha precursor of 392 amino acid residues (Mr 43,414) and beta precursor of 359 residues (Mr 39,046), respectively. The amino acid sequences deduced from two cDNAs were highly homologous with those of mature porcine PDHalpha and beta. Two cDNA inserts subcloned into expression vector pTZ18R synthesized mRNAs identical with HeLa cell PDHalpha and betamRNAs in in vitro transcription system. Large scale synthesis of each subunit protein have been attempted by using two cDNAs constructed into prokaryotic expression vectors.2. Genomic clones encompassing the entire gene for human PDHalpha has been isolated from a leukocyte genomic library. The PDHalpha gene spans 17,082 base pairs and is composed of 11 exons and 10 introns. A total of seven Alu repeats were found in five introns. The entire nucleotide sequence of the gene has been determined and typical consensus promoter sequences in the 5'-flanking region were found. Primer extension analysis indicated that transcription start site is a thymine residue 124 bases upstream from ATG codon in exon 1.3. Genomic clones encompassing the entire gene for human PDHbeta has been isolated from a leukocyte genomic library constructed into lambdaEMBL3. The clone was characterized by restriction enzyme analysis, extensive DNA sequencing and primer extension analysis. The PDHbeta gene spans 18 kilobase pairs and is composed of 10 exons and 9 introns. Two Alu repeats were found in two introns. The 5'-flanking region of the gene contains CAAT box and 3 Spl binding sequences. The transcription was initiated with a adenine residue 132 bases upstream from the translation initiation codon in exon 1.

1. 从HeLa细胞和人包皮成纤维细胞cDNA文库中分离到编码人丙酮酸脱氢酶(PDH) α和β亚基的cDNA。它们分别跨越1363个碱基对（α）和1557个碱基对（β），分别编码392个氨基酸残基的α前体（Mr 43414）和359个氨基酸残基的β前体（Mr 39046）。从两个cdna推导出的氨基酸序列与成熟猪的PDHalpha和β高度同源。两个cDNA插入子亚克隆到表达载体pTZ18R中，在体外转录系统中合成了与HeLa细胞PDHalpha和betamRNAs相同的mrna。通过将两个cdna构建成原核表达载体，已尝试大规模合成每个亚基蛋白。从白细胞基因组文库中分离出包含人类PDHalpha全部基因的基因组克隆。PDHalpha基因跨越17082个碱基对，由11个外显子和10个内含子组成。在5个内含子中共发现7个Alu重复序列。确定了该基因的全核苷酸序列，并在5'侧区发现了典型的一致启动子序列。引物延伸分析表明，转录起始位点是位于1.3外显子ATG密码子上游124个碱基的胸腺嘧啶残基。从构建成lambdaEMBL3的白细胞基因组文库中分离出包含人pdhβ整个基因的基因组克隆。通过限制性内切酶分析、广泛的DNA测序和引物延伸分析对该克隆进行了鉴定。pdhβ基因跨越18千碱基对，由10个外显子和9个内含子组成。在两个内含子中发现两个Alu重复序列。该基因的5'侧区包含CAAT box和3个Spl结合序列。转录始于翻译起始密码子上游132个碱基的腺嘌呤残基，位于外显子1上。