Use of H1 histone to test the fidelity of protein biosynthesis in mouse tissues [proceedings

Abstract

Proteins that do not contain certain amino acids can be used to study the fidelity of translation (or possibility of misincorporation) if these amino acids are introduced into systems in uitro or in vivo in labelled form. However, little work has been carried out by this approach (Loftfield & Vanderjagt, 1972; Edelman & Gallant, 1977; Buchanan & Stevens, 1978), because the problem of perfect purification of the relevant protein still remains difficult. To test the fidelity of protein synthesis in vivo the HI group of histones would be an obvious choice. The sequence analysis of histones (from calf thymus, rabbit thymus and some other sources) showed that histone HI does not contain either methionine or cysteine. The solubility of histones in acids creates the unique possibility for their very high purification. Histone HI does not participate in the nucleosome structure; it has higher molecular weight than H2A, H2B, H3 or H4 histones and can therefore be easily separated from them by different met hods. However, our first attempt to use [35S]methionine incorporation during histone-Hl synthesis in rat tissues for the study of mistranslation was unsuccessful. We found that all fractions of histone H1 from rat tissues are mixed with minor rat-specific methioninecontaining subfractions (Medvedeva et al., 1975). This problem was not found for the case of chromatin from mouse tissues, where the main HI histone was methioninefree, and a methionine-containing subfraction was present in tissue-specific histoneH1° fraction only (Medvedev et a/., 1978). The method for testing the error frequency was based on the comparison between the specific radioactivity of highly purified H1 histone and radioactivity of H2A, H3 and H4 histones (one methionine residue per molecule) or H2B histone (two methionine residues per molecule) some time after groups of CBA mice of different age received injections of [35S]methionine. In our experiments two groups of mice received double injection within 20h (10mCi/40 mice in each group). Isolation of nuclei from thymus, spleen and liver was the same as described in earlier work (Medvedev et al., 1977). H1 histones were extracted with 5 % (w/v) HCIO,. Other histones were extracted by O.~M-HCI from residue chromatin. Before these extractions non-histone proteins, which can contaminate H1 histone, had been eliminated by repeated extraction of chromatin with0.35~-NaCI (Goodwin &Johns, 1973). Further purification of HI histones and the separation of H1 and HIo fractions were made by Bio-Gel P-60column chromatography. Although H1 and H1° histones from mouse liver and spleen chromatin appear as welldefined peaks during chromatography, histone H1 collected after the first fractionation is contaminated by small amount of HI0 histone mixed with an H1° histone minor subfraction. To reach higher purification the combined fractions of the histone-HI peak were dialysed, freeze-dried and used for repeated Bio-Gel column chromatography with an excess of unlabelled H1° histone separated earlier from liver chromatin of control mice. Fig. 1 shows that the second Bio-Gel fractionation moved the remaining radioactivity from the histone-HI into the histone HI0 peak. The total radioactivity of combined fractions from the second Bio-Gel elution of H1 histone from about 40 spleens was only 21 c.p.m., whereas the radioactivity of a comparable amount of H2B histone was 21 340c.p.m. The ratio of these two values (1/1016) can be used for the approximate evaluation of the fidelity of translation. If the radioactivity of HI histone represents misincorporation only, it could be assumed that a single histone H1 molecule per every 500 molecules has methionine (error frequency about lop5 for each amino acid residue). However, the radioactivity in H 1 histone is not only related to misincorporation. The failure to cleave the initiation methionine from the end of the polypeptide chain,