Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101. The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E. coli. A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined. To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture. The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined. Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH. Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard. RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary. The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3′-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons. The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively. Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains. Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH. Similarities and differences were also noted in the predicted secondary structure of sGH and pGH.
ASJC Scopus subject areas
- Animal Science and Zoology