The Separation and Identification of α- and β-Globin Messenger Ribonucleic Acids

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The size heterogeneity of the mouse and rabbit globin messenger ribonucleic acids (mRNAs) purified by oligodeoxythymidylate-cellulose has been studied by polyacrylamide gel electrophoresis. The globin mRNAs migrate as a diffuse band on sodium dodecyl sulfate 6% polyacrylamide gels with no separation of the α and β messengers. The diffuse migration pattern is partly due to secondary structure effects as electrophoresis on 4% polyacrylamide gels in the presence of formamide partially resolves two bands in the 9 S RNA region. Increasing the concentration of polyacrylamide to 10% further resolves these two bands. The difference in molecular weight between the bands is greater than that expected on the basis of a difference in the coding sequence of 15 nucleotides between the α- and β-globin mRNAs. To identify the bands, α-globin mRNA was isolated from the rabbit reticulocyte post-ribosomal supernatant fraction and subjected to electrophoresis along with total rabbit globin mRNA on the 10% polyacrylamide-formamide gels. The α-globin mRNA migrated with the faster moving band of the total mRNA preparation, thus identifying this band as the α-globin mRNA and, by inference, the slower migrating band as the β-globin mRNA. Using mRNA preparations enriched in one or the other of the messengers, it was shown that the mouse α- and β-globin mRNAs migrate identically with those of rabbit. The identity of the two bands in mouse mRNAs was further established by determining their ability to direct the synthesis of α- and β-globin chains in a cell-free protein-synthesizing system. The ratio of β-globin to α-globin synthesized by the mRNA isolated from the more slowly migrating band was 4.3, whereas the ratio for the faster migrating band was 0.32. The ratio of β- to α-mRNA in different messenger preparations was obtained by scanning the formamide gels at 260 nm and quantitating the area under each peak. The globin mRNAs from both mouse and rabbit polysomes have β:α ratios of 1.1 to 1.2, whereas mRNA prepared from total postmembrane supernatant has a β:α ratio of 0.92.