Expression, translation, and localization of a novel, small growth hormone variant.
A novel transcript of the GH gene has been identified in ocular tissues of chick embryos. It is, however, unknown whether this transcript (small chicken GH, scGH) is translated. This possibility was therefore assessed. The expression of scGH mRNA was confirmed by RT-PCR, using primers that amplified a 426-bp cDNA of its coding sequence. This cDNA was inserted into an expression plasmid to transfect HEK 293 cells, and its translation was shown by specific scGH immunoreactivity in extracts of these cells. This immunoreactivity was directed against the unique N terminus of scGH and was associated with a protein of 16 kDa, comparable with its predicted size. Most of the immunoreactivity detected was, however, associated with a 31-kDa moiety, suggesting scGH is normally dimerized. Neither protein was, however, present in media of the transfected HEK cells, consistent with scGH's lack of a signal sequence. Similar moieties of 16 and 31 kDa were also found in proteins extracted from ocular tissues (neural retina, pigmented epithelium, lens, cornea, choroid) of embryos, although they were not consistently present in vitreous humor. Specific scGH immunoreactivity was also detected in these tissues by immunocytochemistry but not in axons in the optic fiber layer or the optic nerve head, which were immunoreactive for full-length GH. In summary, we have established that scGH expression and translation occurs in ocular tissues of chick embryos, in which its localization in the neural retina and the optic nerve head is distinct from that of the full-length protein.
Baudet
M-L
ML
Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Martin
B
B
Hassanali
Z
Z
Parker
E
E
Sanders
E J
EJ
Harvey
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D018507:Gene Expression Regulation, Developmental; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009900:Optic Nerve / Q000196:embryology / Q000502:physiology; D014176:Protein Biosynthesis; D012160:Retina / Q000196:embryology / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D013006:Growth Hormone