GENETICS OF PHOSPHOFRUCTOKINASE STRUCTURE & FUNCTION

在目前的支持下（AM31676），我们已经完成了最 该项目的重要组成部分，克隆和表征 两个磷酸果糖激酶基因;一个来自兔肌肉（RMPFK）， 另一个来自B。嗜热脂肪菌（BsPFK）。 的主要目标 该建议是寡核苷酸定向诱变， 策略性地选择这两种蛋白质中的残基，以阐明 对催化功能、变构调节和 结构完整性。 (1)BsPFK基因的表达及突变 基于 我们已经获得了序列信息，该基因将被亚克隆 在用于表达诱导型载体中，以及在M13噬菌体形成位点中 如上所述在预选位点进行定向诱变。 的 将研究突变蛋白质的酶活性、变构 调节和物理特性。 (2)RMPFK cDNA的构建、表达及突变 该基因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片段 将被已知具有独特限制性的核酸内切酶切割 在cDNA中的位点，并将使用DNA连接酶连接在一起。 将如此构建的全长cDNA克隆到诱导型质粒中。 用于在E.在M13中进行亚克隆 用于定点诱变的噬菌体。 研究中心的理由 突变如上所述。 将分析突变蛋白质 用于酶活性、变构控制和物理生化 行为 (3)完成RMPFK基因中未完成的序列： 确定了所有22个外显子和65%的内含子的DNA序列 RMPFK基因全长17 kbp。 TATA的序列 box、cap位点、polyadenylation信号位点和40%的内含子 仍有待完成。 我们将通过以下方式完成这些任务： 染色体步移、DNA测序和计算机分析 数据

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.