Paul Byvoet

The distribution and turnover of labeled methyl groups in histone fractions of cultured mammalian cells

Abstract Random cultures of mammalian cells were labeled with [14C-Me]-methionine. Cells were harvested either directly after labeling or after varying periods of further growth in unlabeled medium. Harvested cells were fractionated into cytosol, nucleoplasm, histone, and residual protein fractions. The distribution of radiomethyl activity among the amino acids of each fraction was determined by hydrolysis and ion-exchange chromatography, while the distribution of volatile radiomethyl components was determined by hydrolysis and gas chromatography. The specific activities of methylated lysine residues were found to be considerably higher in histones than in any other fraction, while the nucleoplasm and residual proteins contained methyl arginine at higher specific activities than did the histones. Among the histone fractions, only F2b was found to contain methylated arginine. Among the fractions, only histones were found to contain methanol as their main volatile component after brief alkaline hydrolysis. A comparison of the half-lives of histone fractions and their methyl lysine and methyl arginine components indicated that histone methylation was irreversible.

Metabolic integrity of deoxyribonucleohistones

To study the relative turnover rates of histones and DNA in various tissues, rats were simultaneously injected intraperitoneally with [5-125I]iodo-2′-deoxy-uridine to label the DNA and L [U-14C]lysine to label the histones. At various times after administration, animals were killed and lysine-rich and other histones as well as DNA from various tissues isolated. After determining the specific activities (cts/min/mg) of isolated materials, turnover rates were calculated from the first-order decay curves plotted by the method of least squares. The ratios t½ (days) lysine-rich histones/t½ DNA as well as the ratios t½ “other histones”/t½ DNA were found to be close to unity for various rapidly growing rat tissues and tumors. Long-term experiments with liver showed that neither ratio deviated largely from unity. These data indicate that histones, or at least the bulk of histones, and DNA turn over at approximately the same rates, which would appear to be evidence for metabolic integrity of the deoxyribonucleo-protein complex.

Differences in turnover between histones and their acetyl N-terminal groups.

Abstract 1. 1. Previous studies in this laboratory have shown that after individual labeling of histones and deoxyribonucleic acid of various normal and neoplastic tissues the first-order decay rates of their respective specific activities were approximately similar. In the present studies, the turnover of histones was compared with that of their acetyl N-terminal groups. 2. 2. The results showed that the decay rates of the specific activities differed depending on whether they were based on [14C]- or [3H]acetate. The ratios of t 1 2 histones (with respect to [14C]alanine) over t 1 2 histones (with respect to [3H]acetate) for rat liver, spleen, thymus, Walker carcinosarcoma, and Jensen sarcoma were 60; 22.3; 18.8; 12.2 and 20.0, respectively. The N-terminal acetyl groups in Novikoff hepatoma histones appeared to turnover at an extremely slow rate, with a ratio of 1.8. 3. 3. As incorporation of radioactive acetate into the acetyl groups of lysine-rich histones was approximately 10–20% of that into the combined arginine- and slightly lysine-rich fractions, turnover of acetyl groups in the former histone fraction was assumed to be much slower than in the latter. Implications of these findings with respect to the possible function of histone acetylation in information transfer from DNA, are discussed.

Determination of nucleic acids with concentrated H-2-SO4. II. Simultaneous determination of ribo- and deoxyribonucleic acid.

Abstract A method is deseribed for simultancous determination of RNA and DNA. After reaction with concentrated H 2 SO 4 , DNA is determined from the absorption at 470 mμ and RNA from that at 320 mμ, after applying a correction for the absorption due to DNA at the latter wavelength. In some liver and kidney RNA preparations contaminating carbohydrates appeared to interfere with the reaction. These substances could, however, be removed by treating the RNA with 2-methoxyethanol. Optimal conditions for the reaction are described.

Uptake of label into methylated amino acids from rat tissue histones after in vivo administration of [Me-14C] methionine

Abstract 2, 24 and 48 h after administration of [ Me - 14 C] methionine to Novikoff hepatoma bearing rats, histones were isolated from thymus, liver and tumor and hydrolysed. Chromatograms of the basic amino acids in the hydrolysates revealed ninhydrin peaks of ϵ - N -methyllysine, 3-methylhistidine and ω - N -methylarginine. The relative distribution of radioactivity among the methylated amino acids was constant over the entire experimental interval, and differed in the three tissues studied. It appears therefore that at least in this respect histones exhibit tissue specificity.

Intercalating agents as probes of the spatial relationship between chromatin components

Summary Specific histone lysine residues are methylated by a chromatinbound methyltransferase in cell nuclei incubated with S-adenosylmethionine, as monitored by uptake of radiomethyl. The stimulation of this process by aromatic cations via intercalation into the DNA double helix suggests a valuable new approach to investigating DNA-histone-nonhistone protein interactions in intact chromatin.

CMR studies of protein modification. Progressive decrease in charge density at the ε-amino function of lysine with increasing methyl substitution

Abstract Progressive methyl substitution at the e-amino group of lysine results in a progressive downfield shift of the signals from the e and N -methyl carbon atoms in the Carbon-13 nmr spectra of the resulting e- N -methylated lysine derivatives. The results are consistent with the view that the greater the number of e, N -methyl groups on protein basic aminoacid residues, the greater the affinity of these residues for anionic species, e.g. as in histone-DNA interactions.

Determination of nucleic acids with concentrated H2SO4. 1. Deoxyribonucleic acid.

Abstract A simple reaction described by Staron et al. (1) for the determination of DNA was studied, and found to be useful. A few modifications were introduced.

Structural Modifications of Histones in Cultured Mammalian Cells

The widespread association of histones with nucleic acids and the ability of histones to influence both in vivo and in vitro transcription reactions have led to the suggestion that histones may function as direct regulators or as intermediate moderators of gene expression (for reviews see Phillips, 1964; Busch and Steele, 1964; Murray, 1965; Hnilica, 1967; Stellwagen and Cole, 1969). Modification of the nucleoprotein complex, brought about through modification of functional groups of histone molecules, might bring about alterations of chromatin structure and eventually result in gene repression or derepression (Allfrey, 1966; Ord and Stocken, 1968; Allfrey et al., 1964). Histones may also participate in the geometric structuring of the nucleoprotein complex, perhaps in preparation for division, and histone structural alterations may be related to this function (Tidwell et al., 1968). In either case, there should exist a temporal relationship between histone structural alterations and related events. The purpose of this study was to search for such relationships within the cell cycle.