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ABSTRACT

Genomic DNA encoding the ovine insulin-like growth factor-I (IGF-I) gene was cloned and sequenced. The predicted amino acid sequence of the mature form of ovine IGF-I was highly homologous to that of human, rat and mouse. Analysis of the DNA sequence between exons 1 and 2 suggested the existence of an alternative 5′ exon (exon 1A) and this was confirmed by polymerase chain reaction (PCR) analysis of sheep liver mRNA. Primer extension of mRNA from exon 1A indicated a class of transcripts which initiated at a point 32 nucleotides 5′ to the Met codon of exon 1A to give a mRNA comprising exons 1A, 2, 3 and 5. In liver these transcripts co-existed with the alternative exon 1, 2, 3 and 5 mRNA form. Analysis by PCR of the 3′ terminus of liver RNA indicated heterogeneity arising from multiple polyadenylation sites; however, of the two possible alternatively spliced 3′ exons, only exon 5 could be detected.

Expression of IGF-I mRNA, as measured by a solution hybridization/RNase protection assay, predominated in the liver of the neonate and the late-gestation fetus; however, lower levels of expression were seen in multiple tissues throughout fetal and neonatal development.

ABSTRACT

The sheep insulin-like growth factor-I (IGF-I) gene encodes mRNAs containing three different 5′-untranslated sequences as a consequence of alternate splicing of leader exons. Using a combination of RNase protection and primer extension assays, we have mapped the transcriptional start sites of one of the leader exons, exon 1A. Transcription from exon 1A appeared to initiate from multiple points within a 20 bp region situated about 60 bp upstream of the exon 1A splice site. The presence of this transcript in the liver of animals treated with GH was enhanced five- to tenfold and contributed to about 95% of the total hepatic increase in IGF-I mRNA. This exon is generally expressed in a number of tissues immediately after birth; by about 4 weeks postpartum, however, expression is confined to liver. The regulation of hepatic and non-hepatic IGF-I synthesis by GH may involve different mechanisms.

ABSTRACT

Genomic DNA encoding the 5′ region of the porcine IGF-I gene was cloned and sequenced and shown to be highly homologous to that of man, rats and sheep. Two leader exons (exons 1 and 2), which are alternately spliced to exon 3 (encoding part of the mature IGF-I molecule), were identified by RNase protection analysis. In both cases, transcription initiates upstream from exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes (1 and 2) which differ in the precursor peptides predicted from their individual leader sequences.

The expression of class 1 and 2 transcripts was measured in liver and muscle RNA from two groups of 2-month-old pigs whose energy status had been manipulated within physiological limits to produce marked differences in plasma IGF-I levels and growth rates. For this purpose, RNase protection probes were developed that contained the individual leader exons 1 and 2 linked separately to the common exon 3, so that class-specific and total IGF-I gene expression could be determined in a single assay. At normal plasma IGF-I concentrations (200 ng/ml), class 1 and 2 transcripts comprised 81 and 19% respectively of total liver IGF-I mRNA, while at a lower plasma concentration (90 ng/ml) the corresponding values were 95 and 5% respectively. Although both classes of transcript declined with the decrease in plasma IGF-I, the relative drop in levels of class 2 transcripts (84%) was substantially greater than that of class 1 (54%). In longissimus dorsi, cardiac and soleus muscles IGF-I mRNA was predominantly of class 1 and did not change in response to decreased plasma IGF-I. This suggests that liver-derived endocrine IGF-I has an important function in the regulation of muscle growth and that class 2 IGF-I transcripts are more sensitive to conditions that promote optimal growth.