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Biomedical subjects

R A Walters

Publications and source records attributed to R A Walters.

At least 37 records · Page 2Linked to original sources
PubMed1977

Action of heparin on mammalian nuclei. I. Differential extraction of histone H1 and cooperative removal of histones from chromatin.

Heparin interacts strongly with the histone component of chromatin, forming heparin-histone complexes which resist dissociation by 0.2 M H2SO4. Heparin treatment of unfractionated histones isolated from nuclei of Chinese hamster cells indicates that the affinities of the histone classes for heparin appear in the order from greatest to least: (H3, H4) greater than (H2A, H2B) greater than H1. However, when isolated nuclei are treated with heparin, H1 is released from the chromatin more readily than the other four histone classes. The release of these four histones (H2A, H2B, H3, and H4) is coordinate and occurs in a highly cooperative manner, as indicated by (1) dependence of the initial kinetics of histone removal upon heparin concentration, (2) analysis of DNA and histones in the fractions obtained from differential sedimentation of heparin-treated nuclei, and (3) analysis of the products from heparin-treated nuclei by equilibrium centrifugation in metrizamide density gradients. The results suggest rapid procedures for using heparin as an agent for studying the accessibility of histones in chromatin of intact nuclei. The relationship of these results to current models of chromatin structure is discussed.

Binding Sites↗
PubMed1976

Chain elongation and joining of DNA synthesized during hydroxyurea treatment of Chinese hamster cells.

We have previously presented evidence that hydroxyurea treatment of synchronized G1 Chinese hamster cells did not prevent the entry of cells into the DNA synthetic period but that the DNA synthesized during this period (in which total DNA synthesis was severely depressed) was quite small (Walters, R.A., Tobey, R.A. and Hildebrand, C.E. (1976) Biochem. Biophys. Res. Com. 69, 212-217). In view of the reported effects of hydroxyurea on deoxyribonucleoside metabolism and possible relationship to control of DNA replication (Bjursell, G. and Reichard, P. (1973) J. Biol. Chem. 248,3904-3909 and Walters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 319, 336-347), we examined the fate of DNA synthesized during and shortly after hydroxyurea treatment to determine if this DNA exhibited any kinetic behavior which might be an indicator of aberrant synthesis. We found that, upon hydroxyurea removal, DNA grew at a linear rate of 0.98 +/- 0.12 - 10(6) dalton/min (0.98 +/- 0.12 mum/min) for about 2.3h. Beginning at 2.3 h, DNA with a molecular weight approx. 1.4 - 10(8) was very rapidly integrated into bulk DNA of greater than or equal to 3.5 - 10(8) daltons. The apparent growth rate of the 1.4 - 10(8) dalton DNA was approx. 10.6 mum/min. The data suggest that, at least for this DNA, joining into bulk DNA required one-third to one-half of the S period to begin and once begun, occurred very rapidly. The possibility of inegration of replicon clusters is considered.

Cell Division↗
PubMed1976

Effects of ionizing radiation of RNA metabolism in cultured mammalian cells. III. Specific reduction in mRNA synthesis during resumed division and plateau phases following exposure to 800 rads of x irradiation.

The cell growth response of cultured Chinese hamster ovary cells, line CHO, to 800 rads of X irradiation involves a period of division delay, followed by a period of resumed division which terminates in a plateau phase. Over 95% of the cells die eventually. No direct effects of RNA or protein metabolism are evident during the delay period. During the resumed division and beginning of plateau phases, however, a specific and relatively constant reduction in mRNA synthesis relative to messenger-like RNA and heterogenous nuclear RNA synthesis is evidenced. The ratio of mRNA to messenger-like RNA synthesis ranges from 0.8 to 0.65 during these phases. The effect is not due to altered cell-cycle distribution, and evidence is presented to indicate that it is probably not a compensatory response to the unbalanced growth that occurs during the division delay period.

Adenosine↗
PubMed1975

Lack of specific correlation of the deoxycytidine triphosphate pool level with rate of DNA synthesis.

The levels of the four deoxyribonucleoside triphosphate pools and the distribution of cells in the various phases of the cell cycle have been examined in Chinese hamster cells as thymidine, present as a regular constituent in the growth medium, was removed in stages. The results indicate that: 1. Duration of the DNA synthetic phase was lengthened when thymidine was removed from the growth medium. 2. Temporally correlated with lengthening of the DNA synthetic phase upon thymidine removal was a 7-fold increase in level of the dCTP pool, reduction in the dGTP pools, and little or no change in dATP pool. 3. Radioactive labeling procedures indicated that expansion of the dCTP pool could be completely accounted for by increased ribonucleotide reductase activity and that the dTTP pool switched from a largely exogenous thymidine source to endogenous dTTP synthesis as the extracellular thymidine concentration was reduced. 4. Deoxyuridine and thymidine were apparently transported by the same system in Chinese hamster cells, while deoxycytidine was transported by a different system. Although deoxycytidine transport was unaffected by thymidine, phosphorylation of intracellular deoxycytidine compounds to the triphosphate level was stimulated by thymidine. Cytidine transport was not significantly affected by thymidine.

Cell Line↗
PubMed1975

Sequential phsophorylation of histone subfractions in the Chinese hamster cell cycle.

Ion exchange chromatography and preparative electrophoresis were used to examine the phosphorylation of histone f1 and f3 subfractions in synchronized Chinese hamster cells (line CHO). Three discrete f1 phosphorylation events were demonstrated to occur in sequence during the cell cycle. The first event (f1G1) commenced in G1 2 hours prior to entry of cells into S phase; the second event (f1s) commenced simultaneously with initiation of DNA synthesis; and the third event (f1M) commenced when cells entered mitosis. F1M phosphorylation occurred simultaneously with the phosphorylation of histone f3 (which is not phosphorylated during G1, S, or G2). Fractionation of f1 and f3 revealed no differences in these sequential phosphorylation patterns among the various f1 and f3 subfractions, indicating that these phosphorylations are general biochemical events of the cell cycle. Phosphorylated (f1G1) was found to accumulate in cells as they traversed THEIR CELL CYCLE. F1s was phosphorylated to twice the extent of f1G1, but f1s did not accumulate in the cells as they passed through interphase. F1M was phosphorylated to about 4 times the extent of the first phosphorylated form (f1G1). A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests that (a) f1G1 phosphorylation is involved with chromatin structural changes necessary for cell proliferation; (b) f1s phosphorylation is involved with DNA replication; (c) F1M and f3 phosphorylations are involved in chromosome condensation.

Animals↗