Expressed sequence tag analysis of reproductive hormones and muscle proteins and analysis of the alpha-actin promoter in channel catfish, Ictalurus punctatus

The objective of this research was the molecular cloning of gonadotropin cDNAs for potential applications to artificial spawning; characterization of expressed sequenced tags (ESTs) for application in functional genomics studies; and isolationg and characterization of muscle-specific gene promoter for tissue-specific gene expression in muscles.;Complementary DNA (cDNA) encoding the channel catfish (Ictalurus punctatus) conadotropin (GTH) beta subunits I and II were cloned and sequenced, which encode 132 and 140 amino acid protein, respectively. Both the GTH-Ibeta and the GTH-IIbeta were highly induced during ovulation after injection of carp pituitary extract, suggesting their regulation is different from that of the alpha subunit. The catfish GTH-Ibeta was highly similar to GTH-IIbeta and luteinizing hormone, suggesting that the teleost GTH I may be the ancestor for both FSH and LH in evolution.;One hundred and two randomly picked cDNA clones were analyzed from the channel catfish muscle cDNA library. Gene expression of the unique ESTs was comparatively studied in young and adult catfish muscles. Significant differences were observed for aldolase, myostatin, myosin light chain, parvalbumin, and one unknown gene. While myosin light chain and an unknown (CM192) were down-regulated in the mature fish muscle, the aldolase, myostatin, and parvalbumin were significantly upregulated in the mature fish muscle.;The alpha-actin gene of channel catfish has a similar organization as those from other vertebrate species. The variable upstream regions of the alpha-actin gene including a TATA box, a CAAT box, three E-boxes, and a CArG box were fused to the reporter gene chloramphenicol acetyl transferase (CAT). Transfection of the clones into C2C12 cells indicated that all of these motifs are required for transcriptional activities. The channel catfish alpha-actin gene is associated with two distinct short interspersed repetitive elements (SINE) that were designated as Mermaid and Merman. Approximately 9,000 copies and 1,200 copies of the Mermaid and Merman elements exist per hapoid channel catfish genome, respectively. Both the Mermaid and the Merman elements were found to be frequently associated with gene sequences, mostly those of aquatic animals, suggesting their evolutionary origin in association with aquatic organisms and their function in shaping the evolution of genomes in aquatic animals.