Abstract
Insulin-like growth factor I plays a major role in cellular growth by mediating many of the actions of growth hormone. In mammals, IGF-I gene expression is complicated, as the single-copy gene is transcribed and processed into multiple mRNAs that encode at least two peptide precursors. Recent cDNA cloning studies have suggested that in chickens the organization of the gene might be simpler, and that comparative mapping might define essential features of IGF-I that are common to vertebrates. To achieve this goal, we have cloned and characterized the chicken IGF-I gene. The chicken gene is more compact than its mammalian homologues. It is composed of 4 exons which are distributed over 50 kilobases (kb) of chromosomal DNA and are transcribed and processed into mRNAs of 1.9 and 2.6 kb. As in the rat and human genes, the mature 70-amino acid IGF-I molecule is encoded within exons 2 and 3, while exons 1 and 4 contain NH2- and COOH-terminal extension peptides, respectively, and untranslated sequences. Both ends of the chicken gene are structurally heterogeneous. IGF-I mRNAs were found to have two regions of polyadenylation which mapped to exon 4 and were separated by approximately 0.7 kb. At the 5' end, several transcription initiation sites were identified within a 74-nucleotide portion of exon 1 that was found to be highly conserved between chicken and mammalian IGF-I genes. To test promotor function, chimeric genes were constructed linking fragments of chicken IGF-I 5'-flanking DNA to a promoterless reporter plasmid. Hybrid genes containing at least 0.6 kb of IGF-I enhanced expression of luciferase after transfection into SK-N-MC cells, a human line that synthesizes IGF-I mRNA. These observations represent the first thorough characterization of an IGF-I gene and provide an impetus for discerning the mechanisms by which IGF-I expression is regulated during growth, development, and differentiation.
Highlights
70-amino acid IGF-Imolecule is encoded within exons I gene has been characterized in its entirety [5,6,7], the struc
Insulin-like growth factor I (IGF-I) mRNAs were As part of a goal to determine threole of IGF-I in vertebrate found to have two regions of polyadenylation which growth and development,we recently reported the analysoifs mapped to exon 4 and were separatedby -0.7 kb
At a chicken IGF-I cDNA whose nucleotide sequence predicted the 5’ end, several transcription initiation sites were a mature protein that was identical in 60/70 amino acids to identified withina 74-nucleotide portionof exon 1 that rat and human IGF(-1I0).The cDNA detected a predominant was found to be highly conserved between chickaennd IGF-ImRNA of2.6 kbin liver RNA, indicating that the mammalian IGF-I genes
Summary
Total RNA and "'P-labeledchicken IGF-I-specificantisense RNA probes. Protected fragments were separated on 6% polyacrylamide, 8.3 M urea gels and were visualized by autoradiography. Reaction products were digested with restriction enzymes whose sites were contained within the primeriss,olated after fromachicken chromosomallibrarywith human IGF-IA cDNA probes and subsequently identified overlapping fragments by chromosomal walking. For isolation of cDNAs containing the 5' end of chicken IGF-I mRNAs, theanalogous "5' RACE" procedure was followed [24].After hybridized to human and rat IGF-I probes and have been designated exons. The gene extends for nearly 50 kb from the beginning of exon 1 to the end of exon 4 and is approximately two-thirds the length of rat IGF-I [6].Within synthesis of the first cDNA strand using a specific oligonucleotide the gene are 7 regions which contain repetitive elements (Rep complementary to a region within exon 2 of the gene and tailing of the 5' terminus with terminal deoxynucleotidyl transferaseand dATP, PCRwas performed successively with nested oligonucleotides complementary to regions within exon 1 of the chicken IGF-I gene (see Fig. 4). DNA was digested,isolated, subcloned, identified, and in Fig. l), asdetermined by hybridization of "'P-labeled chicken chromosomal DNA to Southern blotosf the X recombinants. Repetitive elements (Rep) were localized by hybridization of X clones with 32P-labeled chicken chromosomal DNA
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