Primary structure of guinea pig preprodynorphin and preproenkephalin mRNAs: multiple transcription initiation sites for preprodynorphin.
Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A((1-17)) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5' end. This sequence has high homology to the 5' flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160-190 nt 5' with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.
LaForge
K Steven
KS
Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY 10021, USA. laforgk@mail.rockefeller.edu
Nyberg
Fred
F
Kreek
Mary Jeanne
MJ
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000502:physiology; D004399:Dynorphins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004745:Enkephalins / Q000737:chemistry* / Q000235:genetics*; D006168:Guinea Pigs; D008297:Male; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000502:physiology; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry*; D024363:Transcription Initiation Site
D004745:Enkephalins; D011498:Protein Precursors; D012333:RNA, Messenger; C036448:pre-prodynorphin; D004399:Dynorphins; C033660:preproenkephalin