Date of Award

1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

Two rabbit phosphofructokinase (PFK) genomic clones were isolated from a lambda Charon 4A rabbit liver DNA library. A 180-bp rabbit muscle PFK cDNA encoding 60 amino acids in the C-terminal region of RM-PFK was used as a probe. By using the shotgun subcloning approach, we cloned, sequenced and characterized two rabbit PFK genomic clones. The two clones overlapped and represented a total length of 17 kb which encoded the entire RM-PFK protein. The organization of this gene was elucidated by restriction mapping and DNA sequencing. It contains 22 exons, ranging in size from 45 to 190 bp, separated by 21 introns of 78-3,500 in length. This gene can be divided into two nearly homologous halves, the N- and C-halves. Each half is composed of almost an equal number of exons. The 22 exons code for 779 amino acid residues with a calculated molecular mass of 84,975 daltons. Exons XV and XVI together code for the 30 amino acid residues which were left as an unidentified gap in the published primary structure for this enzyme. Sequence analysis showed that 74% of the bases at the third position of the codons in the coding exons are either G or C. When overlaid on the amino acid sequence of the protein, most of the introns are located between or near the ends of the secondary structural elements. However, introns are not located at analogous positions in the two protein-coding halves of the gene. Sequence homologies between bacterial and rabbit muscle PFKs and between the amino- and carboxyl-terminal halves of the latter suggest that the mammalian enzyme evolved from a prokaryotic progenitor by gene duplication and divergence. A cDNA clone encoding one-quarter of the RM-PFK sequence was sequenced. The nucleotide sequence of the cDNA agrees with the determined RM-PFK genomic sequence. In addition, the amino acid sequence deduced from the cDNA is identical to the RM-PFK sequence previously reported by Poorman et al.

Pages

148

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