Modified nucleosides and 5'-end groups in purified mouse immunoglobulin light chain mRNA and rabbit globin mRNA...

The borohydride reaction has been used to investigate modified nucleosides and end groups in purified immunoglobulin light chain mRNA and rabbit globin mRNA. 1. The light chain mRNA was isolated from the microsomal fraction of MOPC 41A mouse myelomas, which secrete kappa chains, by two cycles of oligo(dT) cellulose chromatography and glycerol gradient centrifugation. The 12 S mRNA was active in a Krebs II ascites cell-free system and appeared to be homogenous as judged by gradient centrifugation, polyacrylamide gel electrophoresis in 98% formamide and fingerprint analysis of 125I-labelled mRNA. 2. End group labelling of the light chain and globin mRNAs by oxidation with periodate and reduction with boro[3H]hydride showed that the RNAs have a 5'-terminal 7-methyl guanosine in 5'-pyrophosphate linkage with the next nucleoside. 3. To detect any modified residues in the interior of chains, nucleosides in complete digests of the mRNAs were converted by the borohydride reaction to 3H-labelled nucleoside trialcohols, which were fractionated by two dimensional chromatography (the Randerath technique). The light chain mRNA was found to contain N6-methyl adenosine (1 mole) but the rabbit globin mRNAs lacked this nucleoside. Deficiencies in this technique for analysis of minor constituents in large RNAs were noted.