Quantitation and localization of globin messenger RNA in rabbit reticulocyte lysate.
A sensitive and quantitative method is described for the determination of globin mRNA distribution in rabbit reticulocyte lysate. The method uses high resolution sucrose density gradient centrifugation followed by [5'-3H]polyuridylate hybridization to poly(A)-mRNA in gradient fractions. Polyadenylate, purified globin mRNA, and ribonuclease-treated lysate are used to standardize the hybridization assay. It is demonstrated that changes of mRNA and ribosomal distribution do not affect quantitation of the total mRNA localization and Met-tRNAf which suggest that the monitoring of Met-tRNAf binding alone may not be sufficient to assess the mechanisms of control which affect the initiation of protein synthesis.
D000818:Animals; D005914:Globins / Q000096:biosynthesis*; D008715:Methionine; D009693:Nucleic Acid Hybridization; D011061:Poly A; D011188:Potassium / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000097:blood* / Q000302:isolation & purification; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012260:Ribonucleases
D012333:RNA, Messenger; D011061:Poly A; D005914:Globins; D012343:RNA, Transfer; D008715:Methionine; D012260:Ribonucleases; D011188:Potassium