Title

Characterization and expression of rainbow trout ({\it Oncorhynchus mykiss\/}) growth hormone family genes

Date of Completion

January 1997

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

This study of the rainbow trout (Oncorhynchus mykiss) growth hormone (GH) gene family (GH, prolactin (PRL), and somatolactin (SL)) was focused at the mRNA level in order to understand how the expression of these genes is regulated in fish. Since two GH cDNAs and one PRL cDNA have been identified previously (Agellon et al., 1988; Mercier et al. 1989), this study started from identification and characterization of rainbow trout SL cDNA. This SL cDNA encodes a polypeptide of 27 kDa which shares 53-99% amino acid identity with SLs from other fish. Different from the original hypothesis that GH, PRL and SL are pituitary specific proteins, mRNAs for these proteins are detected not only after pituitary gland formation, but also in embryos prior to pituitary gland formation and in mature oocytes. In adult fish, small amounts of GH, PRL and SL mRNAs are also present in extrapituitary tissues. Both GH1 and GH2 mRNAs can be detected in all developmental stages. However, the amount of GH1 mRNA is higher than that of GH2 in 10-day fry and 2-year old female fish. Nucleotide analysis of the GH1 gene showed that, similar to gene GH2, gene GH1 spans approximately 4.5 kb and is comprised of 6 exons and 5 introns. The 5$\sp\prime$-flanking regions of both GH1 and GH2 genes contain several Pit-1 binding sequences. Consensus sequences related to response elements for cAMP, thyroid hormone, retinoic acid, retinoid, estrogen and glucocorticoid hormones are present in the GH1 gene. All these hormone response elements, except estrogen response element, are also present in gene GH2. A rainbow trout pituitary cell line RTP-2 was established as an assay system to detect the response of these genes to hormonal factors in vitro. Additionally, two somatolactin-like protein cDNAs, SLP1 and SLP2, were also identified. These proteins contain conserved sequences and cysteines also formed in the GH family, but no hydrophobic N-terminal signal peptides. The green fluorescent protein-SLP1 fusion protein aggregates around the nucleus in the transfected cells. A CAAX box-like fragment at the C-terminus has been found to be required for the aggregation of the fusion protein. ^