B.J. Noland

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.

Methylation of lysine residues of histone fractions in synchronized mommalian cells

Abstract Synchronized cultures of mammalian cells were labeled with 14 C-methyl methionine. Labeled methionine methyl groups were incorporated into certain histone fractions, forming methyl lysine. Incorporation of labeled methyl group into histone fractions as 14 C-methyl lysine was followed through the cell cycle from late G 1 into early M . The 14 C-methyl lysine contents of fractions F2a and F3 began to rise in S and reached maxima after termination of DNA and histone synthesis, coincident with the beginning of mitosis, and began to fall by mid- M . The 14 C-methyl lysine content of fraction F2b rose to a maximum early in S , coincident with initiation of DNA synthesis, and rapidly decreased to its original unmethylated level by late S . Fraction F1 remained unmethylated during the period G 1 - M . Evidence is presented to demonstrate differential methylation of histone fractions and to substantiate differential temporal coupling of the methylation of specific histone fractions with histone and DNA biosynthesis.

Histone acetylation in synchronized mammalian cell cultures

Abstract Synchronized cultures of mammalian cells were labeled with [ 3 H]acetate. Incorporation of labeled acetate into histone fractions was followed through the cell cycle from late G 1 , through S and G 2 , into early M. The acetate contents of histone fractions F2a, F2b and F3 rose rapidly to a maximum in late S, coincident with the termination of DNA synthesis, and declined rapidly thereafter. The acetate content of histone fraction F1 rose to a maximum in mid-S, coincident with maximal DNA synthesis, and decreased rapidly thereafter. Fractions F1 and F2b lost the majority of their incorporated acetate, while fractions F2a and F3 retained approximately half their incorporated acetate. Evidence is presented for differential acetylation of histone fractions, for a temporal coupling of histone biosynthesis and acetylation and for a differential turnover of histone fraction acetyl groups.

Histone phosphorylation in synchronized mammalian cell cultures

Abstract Synchronized cultures of mammalian cells were labeled with inorganic radiophosphorus ( 32 P). Incorporation of label into histone fractions as alkali-labile 32 P-phosphate was followed through the cell cycle from late G 1 , through S and G 2 , into early M. No evidence was obtained for the incorporation of phosphorus into histone fractions F2a or F3. The phosphorus content of histone fraction F1 rose steadily during this period, with no evidence of dephosphorylation, while the phosphorus content of histone fraction F2b rose to a maximal value in mid-S and declined rapidly thereafter. Evidence is presented for differential phosphorylation and dephosphorylation of specific histone fractions. The significance of these observations is discussed.

The intracellular distribution of basic proteins in the Chinese hamster ovary cell

Abstract Basic proteins were obtained by acid extraction from whole cells and from nuclei, nucleoli, chromatin, cytoplasmic ribosomes and lysosomes of fluid cultured Chinese hamster ovary (CHO) cells. The limits of nucleoprotein dissociation ranged from pH 2.8-0.6. Basic proteins isolated from whole cells demonstrated complex electrophoretic patterns containing approximately 30 components. Basic proteins from nuclei demonstrated less complex banding patterns, while the patterns from nucleoli and chromatin qualitatively resembled those from nuclei. Lysosomal basic protein patterns resembled those of nuclei with three additional bands which were present in whole cell preparations, were observed as trace components in ribosomal preparations and were absent in nuclear basic proteins. Ribosomal basic proteins demonstrated a complex banding pattern which appeared both to share certain fractions with nuclear and lysosomal fractions and to contain a number of unique components. A comparison of relative band staining intensities among the preparations, both visually and by means of a photodensitometer, suggested for the common bands a distribution of a mixture of basic proteins among intracellular sites, the proportions of which mixture varied according to the individual locus.

Effects of ionizing radiation upon intracellular levels of soluble microtubule protein in cultured mammalian cells

Abstract Intracellular soluble microtubule protein levels were determined in cultured mammalian cells following X-irradiation. Microtubule protein levels rose along with corresponding RNA and total soluble protein levels as cells accumulated in G 2 and declined as cells resumed division. These results suggest that radiation-induced division delay of mammalian cells does not arise from a radiation-induced defect in synthesis and accumulation of microtubule protein.

Histone acetyltransferase activity in synchronized mammalian cells.

Abstract Intracellular histone acetyltransferase activity was measured in synchronized mammalian cell cultures to determine if variations in histone acetate contents previously observed in similar cultures reflected corresponding variations in levels of transferase activity. No such correlations were observed, suggesting that temporal control of histone acetylation within the cell cycle lies in another, as yet undetermined, area of cellular activity.

Histone phosphokinase levels in synchronized mammalian cells

Abstract Histone fractions F1 and F2b are phosphorylated in a specific sequence and at specific times in synchronized mammalian cells. Intracellular histone phosphokinase was measured in synchronized cell cultures to determine if differential histone phosphorylation through the cell cycle reflected varying levels of kinase activity. No direct correlations were obtained, suggesting that control of observed alterations in histone phosphorylation lies in yet another area of cellular activity.

The effects of x-irradiation on histone acetylation and methylation in cultured mammalian cells

Abstract Random populations of mammalian cells in suspension culture with generation times ranging from 16–17 hr were subjected to 1600 rads of x -irradiation. Irradiated cells displayed a postirradiation division delay period of approximately 16 hr, during which period protein accumulation continued. DNA and histone biosynthesis ceased between 10 and 12 hr postirradiation. Approximately 15% of the irradiated population completed their first postirradiation division 16 hr after irradiation and very few were able to complete a second division. The DNA, protein and histone contents of the cell population rose during the division delay period, approximating levels attained by normal cells in the G 2 portion of their life cycle. Accumulation of labeled acetate in histone fractions F1, F2b and F3 of irradiated cells paralleled control values, while accumulation of incorporated acetate into histone fraction F2a of irradiated cells was depressed below control values between 16 and 24 hr after irradiation. Accumulation of labeled methyl groups as methyl-lysine derivatives in histone fractions F2a, F2b and F3 of irradiated cells was depressed below control values 24 hr postirradiation. No methylation of the lysine residues of histone fraction F1 was observed in either control or irradiated cultures during this period. Histone acetyltransferase and methyltransferase activity levels increased during the division delay period to follow the accumulation of cellular protein in irradiated cells. However, enzyme activity increased more rapidly than total protein, reached G 2 levels at 16 hr and decreased with resumption of cell division. These observations provide confirmatory evidence that mammalian cells irradiated under these conditions are arrested within the cell cycle in G 2 or a G 2 -like state.

Histone methylkinase levels in synchronized mammalian cells

Abstract An enzyme system capable of methylating histones and histone fractions was observed and characterized in the cell-free extracts of cultured mammalian cells. Histone fractions F2 a , F2 b and F3 are methylated at specific times and in a specific sequence in synchronized mammalian cells. Intracellular histone methylkinase activity was measured in synchronized mammalian cells to determine if a temporal relationship existed between histone methylation and methylkinase levels. While fluctuations were observed in enzyme activity levels around the cell cycle, no dramatic alterations in kinase levels were seen which could be correlated with histone methylation maxima.