STUDIES ON THE BIOSYNTHESIS OF CHOLECYSTOKININ: A BRAIN-GUT POLYPEPTIDE HORMONE (GENE, SEQUENCE, EXPRESSION)
Abstract
The polypeptide hormone Cholecystokinin (CCK) is synthesized in the brain and gastrointestinal tract. In the gut it causes the release of digestive enzymes from the pancreas and bile from the gall bladder, while in the brain its function has not been clearly established. The biosynthesis of CCK was studied by isolating and characterizing CCK-mRNA from a rat medullary thyroid carcinoma which exhibited high levels of immunoreactive CCK. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst I restriction site of pBR322 via poly(dG)(.)poly(dC) tailing. Escherichia coli strain HB101 was transformed with the DNA and colonies containing CCK-cDNA were identified using the hybridization probe, d(TCCATCCANCCCATGTAGTC). The sequence of this probe was deduced from the known amino acid sequence of porcine CCK-8, Asp(.)Tyr(.)Met(.)Gly(.)Trp(.)Met(.)Asp(.)Phe(.)NH(,2). The nucleotide sequence of CCK-containing plasmids was determined and revealed that CCK is initially synthesized as a 115 amino acid precursor. Northern blot analysis of CCK-mRNA demonstrated that it is approximately 750 nucleotides in length, as is CCK-mRNA from the brain and the gastrointestinal tract. Using cDNA fragments, the rat CCK gene was isolated and characterized. It is 7 kilobases in length and interrupted by two intervening sequences. The gene has been sequenced (excluding 4 kb of intron-2) by the dideoxy method of Sanger. The promoter elements responsible for CCK gene expression have been examined by a transient expression assay. A chimeric gene involving the 5' region of the CCK gene and a promoterless structural gene for chloramphenicol acetyl transferase (CAT) was constructed. It was found that the CCK promoter is active when 144 bases upstream of the transcription initiation site is present. Expression of the CCK involves the synthesis of a 750 base mRNA from a 7 kb CCK gene. Translation of CCK-mRNA results in a preprohormone of 115 amino acids which in turn is proteolytically processed to generate several bioactive peptides, the smallest of which is a sulfated, amidated octapeptide.
Degree
Ph.D.
Subject Area
Biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.