Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing.
A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.
D000324:Adrenocorticotropic Hormone / Q000235:genetics; D000818:Animals; D002460:Cell Line; D004262:DNA Restriction Enzymes; D004744:Enkephalin, Methionine / Q000235:genetics; D004745:Enkephalins / Q000235:genetics* / Q000302:isolation & purification; D005796:Genes; D006801:Humans; D051379:Mice; D010911:Pituitary Neoplasms; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000235:genetics; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D013045:Species Specificity; D014158:Transcription, Genetic
D004745:Enkephalins; D011498:Protein Precursors; D012333:RNA, Messenger; C029992:proenkephalin; D004744:Enkephalin, Methionine; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D004262:DNA Restriction Enzymes