Title

Molecular Characterization of Insulin-Like Growth Factor-I (IGF-I) In Channel Catfish, (Ictalurus punctatus)

Date of Award

2004

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Shiao Wang

Advisor Department

Biological Sciences

Abstract

Insulin-like growth factor-I (IGF-I) is the principal growth regulator in bony fishes. The channel catfish IGF-I gene was cloned and sequenced to help determine the endocrine regulation of growth in this agriculturally important species. Partial cDNA sequence, missing exon 1 and part of exon 2, was obtained by 5 ' - and 3' -RACE experiments using degenerate primers designed from highly conserved vertebrate IGF-I sequence. Primers designed from channel catfish were used to isolate two clones from a channel catfish BAC library. Primer walking on the BAC clones produced sequence through 640 by upstream of the initiator methionine. Exon 2 contained a 100 by region with 81% GC content with the potential for a stable secondary structure. Full-length cDNA was amplified by RT-PCR and the product was cloned into pCR-TOPO4Blunt. However, full-length cDNA clones were only created by transformation into recombination-deficient DH10B strain E. coli because GC-rich portions of exon 2 were excised in clones transformed into TOP10 cells. The genomic sequence revealed a putative TATA box 506 by upstream of the initiator methionine. The catfish IGF-I coding region consisted of 477 nucleotides, containing five exons interrupted by four introns. Channel catfish IGF-I encodes a 159 amino acid pre-propeptide. Protein sequence comparisons demonstrated that channel catfish IGF-I shares 88.2% identity with ubiquitously expressed Thai catfish IGF-I, 77.4% identity with zebrafish, and 54.9% identity with human IGF-I. A CT-microsatellite repeat in intron 1 of IGF-I was found to be highly polymorphic in channel catfish families and allowed for the linkage map position of the IGF-I gene to be determined. Channel catfish IGF-I is located in linkage group U19 approximately 17.3 cM in distance from the IpCG0011 locus.