Title

Developmental expression of insulin-like growth factor and insulin receptor family member messenger RNAs in rainbow trout, Oncorhynchus mykiss

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Genetics|Biology, Zoology|Agriculture, Fisheries and Aquaculture

Degree

Ph.D.

Abstract

The appearance of insulin-like growth factor I (IGF-1) and II (IGF-II) mRNA was studied during rainbow trout embryonic development. Three forms of IGF-I mRNA and one form of IGF-II mRNA were detected in total RNA from embryos isolated from a staged series. A competitive RT-PCR assay was developed and used to quantitate the levels of IGF-I and IGF-II mRNAs. The absolute number of molecules of IGF-I mRNA/μg total RNA in rainbow trout embryos was shown to be significantly lower than the absolute number of molecules of IGF-II mRNA/μg total RNA both during early and late embryonic development. ^ The importance of the IGF type I and insulin receptor in embryonic growth has been demonstrated by genetic analysis of IGF type I and insulin receptor null mutants in mice and chemical mutagenesis in Drosophilia. Since very little is known about the nature and structure of IGF type I and insulin receptors in teleosts, the cloning of rainbow trout IGF type I and insulin receptor cDNAs was undertaken. Two rainbow trout IGF type I receptor (rtIGFR) cDNAs were isolated by a 5 rapid amplification of cDNA ends (RACE) method and confirmed as separate genes by genomic Southern blot hybridization. Three rainbow trout insulin receptor (rtIR) cDNAs were isolated by screening a cDNA library and confirmed as separate genes by genomic Southern hybridization. A high degree of amino acid identity was observed between rtIGFRs and rtIRs and their human homologs. ^ Developmental and tissue-specific polyadenylation was observed in various tissues from juvenile and adult rainbow trout and in rainbow trout embryos from cleavage to feeding stages for two forms of rtIGFR and two forms of rtIR mRNAs. A relative quantitative RT-PCR assay was used to determine the steady state levels of two forms of rtIGFR and three forms of rtIR mRNAs in various tissues from juvenile and adult rainbow trout and in rainbow trout embryos from cleavage to feeding stages. These results suggest a complex expression pattern of IGF type I and insulin receptor mRNAs throughout the life history of a partially tetraploid fish. ^