Complementary DNA cloning and analysis of gene structure of pyruvate kinase from Drosophila melanogaster

Screening a lambda-gt10 cDNA library of Drosophila melanogaster using a hum an pyruvate kinase (Pyk) cDNA clone (pCJ11) and a rat pituitary Pyk cDNA cl one (pCJ22) as probes, we isolated 2 cDNA clones, cDMPK15 and cDMPK06. Comp lete nucleotide sequencing of the two cDNA clones (GenBank AF061507) reveal ed that they encompassed the coding region of pyruvate kinase cDNA (1602 bp ; 533 amino acids + TAA) flanked by a 5' untranslated region of 240 bp and a 3' untranslated region of 253 bp. An alignment of the deduced amino acid sequence from the Pyk cDNA clones with those of PK from other organisms ind icated that the amino acid residues constituting the active sites have been highly conserved. In addition, the overall positional identity between the sequence of the "Drosophila" enzyme and those from other sources was 42%-6 3%. Polymerase chain reaction was applied to amplify the genomic DNA fragments from the Pyk gene of D. melanogaster. These overlapping amplicons, which co vered the complete coding region of Pyk, were further sequenced using cycle -sequencing with an ABI Prism 377 DNA sequencer. A total of 3447 bp of the nucleotide sequence (GenBank AF062478) was determined from these amplicons. By comparing these sequences with the sequence of Pyk cDNA clones isolated , 4 exons were identified of 282, 1390, 157, and 266 bp in length. The intr ons identified all contained the consensus 5'- and 3'-splicing sites (GT-BG ). RT-PCR analysis was performed to determine the number of species of the Pyk transcript in adults of D. melanogaster. The observation that only a si ngle amplicon appeared in each amplification suggests that a single Pyk tra nscript is expressed in adults of D. melanogaster, and might imply that the re is only 1 Pyk gene in D. melanogaster.