Molecular cloning, characterization and expression analysis of adenylosuccinate lyase gene in grass carp (Ctenopharyngodon idella).
Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. In the present study, we have constructed a grass carp (Ctenopharyngodon idella) intestinal cDNA library that has over 2.3 × 10(5) primary clones. An expressed sequence tag (EST) of grass carp adenylosuccinate lyase (gcADSL) gene was screened from this library. Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA sequence, which contains a 1,446 bp open reading frame encoding 482 amino acids about 54.552 kDa. The deduced amino acid sequence shares high homology with its vertebrate counterparts, which shares 94% similarity with zebrafish, 81% with African clawed frog as well as chicken, 77% with human and 76% with mouse. This gcADSL genomic sequence, consisted of 13 exons and 12 introns, is 8,557 bp in size. Real-time quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill. In western blotting analysis, His(6)-tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an anti-His(6)-tag monoclonal antibody but also by an anti-human ADSL polyclonal antibody, indicating immunological crossreactivity occurs between grass carp and human ADSL protein. 1,082 bp 5'-flanking region sequence was cloned and analyzed.
Yuan
Tian
T
School of Life Sciences, Sichuan University, 610064 Chengdu, People's Republic of China.
Gu
Ji-Rui
JR
Gu
Wen-Bo
WB
Wu
Jiang
J
Ge
Shao-Rong
SR
Xu
Heng
H
D024506:5' Flanking Region / Q000235:genetics; D000264:Adenylosuccinate Lyase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002347:Carps / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D016678:Genome / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D004247:DNA; D000264:Adenylosuccinate Lyase