Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs.
The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 [35S]methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.
D000818:Animals; D002250:Carbon Radioisotopes; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008099:Liver / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008767:Methylmercury Compounds / Q000494:pharmacology; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D013462:Sulfur Radioisotopes; D013950:Thymus Gland / Q000378:metabolism*
D002250:Carbon Radioisotopes; D008767:Methylmercury Compounds; D011506:Proteins; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D011061:Poly A; D012313:RNA; D008715:Methionine; C020771:methylmercury hydroxide