A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

Ribosomal proteins from the yeast Saccharomyces cerevisiae were separated, on a preparative scale, by ion-exchange h.p.l.c. Proteins from the small and large ribosomal subunits were resolved, respectively, into 33 and 23 peaks, and most of the proteins present in these peaks were identified by using one- and two-dimensional gel electrophoresis. Several of the peaks appeared to contain a single protein uncontaminated by other species. Ribosomal proteins were also separated by using reverse-phase h.p.l.c. Analysis of the peaks resolved indicated that the order of elution for the proteins of both ribosomal subunits is, in certain cases, different for each of the two h.p.l.c. techniques used. Thus a combination of the two chromatographic methods employed here has the potential to facilitate the rapid and preparative separation of each of the proteins present in yeast ribosomes.

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants.

A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.

Changes in histone acetylation during the development of rabbit bone marrow erythroid cells.

The acetylation of histones in rabbit bone marrow erythroid cells was investigated by measuring the incorporation of labelled acetate into erythroblasts which were separated by velocity sedimentation at unit gravity into five fractions corresponding to different stages of development. Histone acetylation decreased during erythroid development in concert with a decline in DNA and histone synthesis. Some acetylation persisted after condensation of the nucleus and cessation of DNA synthesis in late orthochromatic cells. This residual acetylation may be related to the low level of transcription which is still present at this stage. Sodium butyrate increased the acetylation of histones 2- to 7-fold, with the greatest stimulation occurring in the most immature cells. The general decline in acetylation of histones during erythroid cell development was similar in the presence and absence of butyrate.

The nonhistone proteins of developing mammalian erythroid cells.

Anaemic rabbit bone marrow cells, labelled with [35S]methionine, were separated at unit gravity. Chromatin was isolated from these cells and proteins were separated from DNA, using urea/salt extraction. Two-dimensional gel electrophoresis of the nonhistone proteins showed that these proteins appeared to change quantitatively but not qualitatively, with one important exception, as cell development proceeded. The one protein that did change had a molecular weight of approximately 20,000 and was very basic. This protein was synthesised at low levels in the early cells, but its synthesis was seen to increase at the polychromatic stage of cell development, just prior to nuclear condensation. Treatment of bone marrow cells with sodium butyrate was shown to increase the synthesis of this protein in the early cells.