Analysis of mRNA populations by cDNA.mRNA hybrid-mediated inhibition of cell-free protein synthesis.
Hybridization of mRNA to its corresponding cDNA was found to specifically inhibit translation of the mRNA in vitro. Using hybridization of globin cDNA to globin mRNA as a model system, we found that equivalent amounts of cDNA were required both for the saturation of the mRNA hybridization and for complete inhibition of globin synthesis. Also, the rate of inactivation of translation was identical to the rate of hybridization and followed the predicted kinetic form. This assay has been applied to the analysis of a set of abundant mRNAs in mouse liver. Hybridization of liver mRNA with total liver cDNA in slight excess to a low C(0)t value specifically inhibited translation of several major polypeptides. Melting of the hybrids prior to translation restored synthesis of these polypeptides. Moreover, we found that different liver mRNAs are inactivated with different kinetics; the results suggest that the mRNAs for the major urinary polypeptide and for albumin are the most abundant and second most abundant, respectively, in mouse liver. The general applications of this technique are discussed.
D000418:Albumins / Q000235:genetics*; D000818:Animals; D004247:DNA; D005796:Genes; D005914:Globins / Q000235:genetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D009693:Nucleic Acid Hybridization; D010455:Peptides / Q000235:genetics* / Q000652:urine; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D000418:Albumins; D010455:Peptides; D012333:RNA, Messenger; D005914:Globins; D004247:DNA
J Biol Chem. 1977 Dec 10;252(23):8489-97
925007
Virology. 1977 May 15;78(2):522-36
867816
Proc Natl Acad Sci U S A. 1977 Oct;74(10):4370-4
270678
Cell. 1977 Nov;12(3):751-65
562718
Cell. 1976 Dec;9(4 PT 2):761-74
1017013
J Biol Chem. 1976 Dec 10;251(23):7461-7
1002695
J Mol Biol. 1973 Feb 19;73(3):329-49
4686199
Proc Natl Acad Sci U S A. 1974 Sep;71(9):3435-9
4530315
Nat New Biol. 1973 Feb 14;241(111):204-7
4512585
J Biol Chem. 1975 Jul 25;250(14):5688-95
167021
Eur J Biochem. 1973 Feb 15;33(1):192-200
4691350
J Biol Chem. 1975 Aug 10;250(15):6077-84
1150672
Nature. 1970 Aug 15;227(5259):680-5
5432063