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.
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