GENETICS OF PHOSPHOFRUCTOKINASE STRUCTURE & FUNCTION

磷酸果糖激酶结构的遗传学

• 批准号: 3230261
• 负责人: Simon H Chang
• 金额: $ 8万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3230261
• 关键词: 6 phosphofructokinase; Bacillus stearothermophilus; chemical structure function; complementary DNA; computer data analysis; endonuclease; genetic regulation; genetic transcription; molecular genetics; mutagens; nucleic acid sequence; oligonucleotides; plasmids; polyadenylate

Under the current support (AM31676), we have accomplished the most important part of the project, the cloning and characterization of two phosphofructokinase genes; one from rabbit muscle (RMPFK) and the other from B. stearothermophilus (BsPFK). The major goal of this proposal is the oligonucleotide directed mutagenesis of strategically selected residues in these two proteins to elucidate sites critical for catalytic function, allosteric regulation and structural integrity. (1) Expression and mutagenesis of BsPFK gene. Based on the sequence information we have gained, this gene will be subcloned in an inducible vector for expression, and in M13 phage for site directed mutagenesis at pre-selected sites as described above. The mutated proteins will be studied for enzymatic activity, allosteric regulation and physical properties. (2) Construction, expression and mutagenesis of RMPFK cDNA: Our results on the DNA sequence of this gene have enhanced our capability to construct a full-length RMPFK cDNA. We will approach this task by reverse transcription, oligonucleotide synthesis and an in vitro intron deletion technique. The cDNA pieces produced will be cleaved with endonucleases known to have unique restriction sites in the cDNA and will be linked together using DNA ligase. The full-length cDNA so constructed will be cloned in an inducible plasmid vector for expression in E. coli host and subcloned in M13 phage for site directed mutagenesis. The rationale for sites of mutation is as described above. Mutated proteins will be analyzed for enzymatic activity, allosteric control and physical biochemical behavior. (3) Completion of the unfinished sequence in RMPFK gene: We have determined the DNA sequence for all 22 exons and 65% of the introns of RMPFK gene which is 17 kbp in length. The sequences for TATA box, cap site, polyadenylation signal site and 40% of the introns remain to be completed. We will complete these tasks by chromosomal walking, DNA sequencing and computer analysis of the data.

在当前的支持下（AM31676），我们取得了最大的成就 项目的重要部分，克隆和表征 两个磷酸果果因子酶基因；兔子肌肉（rmpfk）和 另一个来自B. stearothrophilus（BSPFK）。 主要目标 该建议是寡核苷酸的定向诱变 这两种蛋白质中策略性选择的残留物以阐明 对催化功能，变构调节和 结构完整性。 （1）BSPFK基因的表达和诱变。 基于 我们获得的序列信息，该基因将被亚克隆 在诱导载体中进行表达，在M13噬菌体中 如上所述，在预选位点的定向诱变。 这 将研究突变的蛋白质以进行酶促活性，变构性 调节和物理特性。 （2）RMPFK cDNA的结构，表达和诱变：我们的 该基因的DNA序列的结果增强了我们的 构建全长RMPFK cDNA的能力。 我们将接近 通过逆转录，寡核苷酸合成和 一种体外内含子缺失技术。 产生的cDNA片 将被已知具有独特限制的测核酸核酸内切酶切割 cDNA中的位点，并将使用DNA连接酶连接在一起。 如此构造的全长cDNA将克隆在可诱导的 用于在大肠杆菌宿主中表达的质粒载体并在M13中亚克隆 位点定向诱变的噬菌体。 地点的基本原理 突变如上所述。 将分析突变的蛋白质 用于酶活性，变构控制和物理生化 行为。 （3）在RMPFK基因中完成未完成的序列：我们有 确定所有22个外显子的DNA序列和65％的内含子 RMPFK基因的长度为17 kbp。 塔塔的序列 盒子，帽位点，聚腺苷酸信号位点和40％的内含子 保持待完成。 我们将通过 染色体步行，DNA测序和计算机分析 数据。