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

利用人類及大白鼠丙酮酸鹽激酶的cDNA當探針篩選黃果蠅λ-gt10 cDNA圖書館，我們得以分離出二個cDNA菌株，分別是cDMPK15及cDMPK06。其核甘酸序列顯示此二cDNA菌株包含黃果蠅丙酮酸鹽激酶基因的密碼區域（1602鹹基對：533個胺基酸+TAA）加上240鹹基對的5端未轉譯區域及253鹹基對的3端末轉譯區域。其胺基酸序列和其它生物的丙酮酸鹽激酶的胺基酸序列比較顯示構成活性中心區域的胺基酸殘基具有高度保守性。除此之外，其胺基酸序列和其它生物的丙酮酸鹽激酶的胺基酸序列的同源性則是42－63%。聚合酶連鎖反應被應用去複製黃果蠅丙酮酸鹽激酶基因片段。這些DNA片段合起來包括整個丙酮酸鹽激酶的密碼區域則以ABI 377核酸自動定序儀進行直接定序。共3477個鹹基對被定序出。和cDNA序列比較結果4個表現子則被決定出，它們分別是282，1390，157及266鹹基對長。內轉子則由於它們未出現在cDNA序列中以及全部含有保守的5端及3端切割區域（GT-AG）。RT-PCR則是被用來決定黃果蠅成蟲丙酮酸鹽激酶轉錄物的種類，而從結果中每一個聚合酶連鎖反應僅複製出一條複製物建議在黃果蠅成蟲中可能只有一條丙酮酸鹽激酶轉錄物，此外更進一步暗示只有一個丙酮酸鹽激酶基因在黃果蠅中。

關鍵字

丙酮酸鹽激酶；基因選株；反轉錄聚合酶連鎖反應；黃果蠅

並列摘要

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.

並列關鍵字

Pyruvate kinase ； cDNA ； Cloning ； RT-PCR ； Drosophila melanogaster

國際替代計量

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

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