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Initial Attempt to Develop an AFP Gene Regulation Analysis System
Author(s) -
BÉLANGER L.,
COMMER P.,
HARDY K. J.,
CHIU J. F.
Publication year - 1978
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1978.tb03923.x
Subject(s) - complementary dna , polysome , microbiology and biotechnology , messenger rna , rna , biology , immunoprecipitation , reticulocyte , centrifugation , dna , gene expression , biochemistry , gene , ribosome
This is a preliminary report on methods being developed for the isolation of rat AFP messenger RNA and the synthesis of DNA complementary to this mRNA. Using Morris hepatoma 7777 as source material, AFP‐synthesizing polysomes have been isolated by indirect immunoprecipitation. The 18S Poly A + RNA (putative mRNA AFP ) recovered from these polysomes by differential centrifugation and oligo‐dT cellulose chromatography, was translated in a reticulocyte cell‐free system into a peptide immunoprecipitable by anti‐AFP sera (and not by anti‐albumin sera) and co‐migrating with native AFP on polyacrylamide gel electrophoresis. DNA complementary to the mRNA was synthesized by the use of avian myeloblastosis virus reverse transcriptase. The cDNA probe is highly homogeneous, as judged by alkaline sucrose gradient sedimentation, and yields a 1/2 C,t value of 8.5 times 10 ‐3 with its template. Preliminary hybridization experiments indicate that sequences hybridizable to the cDNA probe constitute 3.0%, 0.5%, and 0.008% of the total poly A + polysomal RNA populations of 13 day old rat liver, Morris hepatoma 7777 and adult rat liver, respectively.