Vol. 38 No. 3, 1999

Complementary DNA Cloning and Analysis of Gene Structure of Pyruvate Kinase from Drosophila melanogaster

Yi-Chih Chien*, Yu-Jing Zhu and Chun-Mei Chuen

Department of Biology, National Changhua University of Education, Changhua, Taiwan 500

Yi-Chih Chien, Yu-Jing Zhu and Chun-Mei Chuen (1999) Screening a λ- gt10 cDNA library of Drosophila melanogaster using a human pyruvate kinase (Pyk) cDNA clone (pCJ11) and a rat pituitary Pyk cDNA clone (pCJ22) as probes, we isolated 2 cDNA clones, cDMPK15 and cDMPK06. Complete nucleotide sequencing of the two cDNA clones (GenBank AF061507) revealed 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 indicated 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%-63%.
Polymerase chain reaction was applied to amplify the genomic DNA fragments from the Pyk gene of D. melanogaster. These overlapping amplicons, which covered 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 introns identified all contained the consensus 5'- and 3'-splicing sites (GT-AG). 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 single amplicon appeared in each amplification suggests that a single Pyk transcript is expressed in adults of D. melanogaster, and might imply that there is only 1 Pyk gene in D. melanogaster.

Key words: Pyruvate kinase, cDNA, Cloning, RT-PCR, Drosophila melanogaster.

*Correspondence: Tel: 886-4-7232105 ext. 3411. Fax: 886-4-7611156. E-mail: chien@cc.ncue.edu.tw