Biomedical subjects

R L Seale

Publications and source records attributed to R L Seale.

27 records · Page 2Linked to original sources

PubMed1977

Chromatin replication in vitro. Properties of a HeLa nuclear system.

An in vitro HeLa chromatin replication system was developed and characterized. Purified nuclei synthesized DNA from replication forks which were undergoing chain elongation at the time of cell fractionation. DNA replication in isolated nuclei was deficient in DNA maturation functions; the Okazaki fragments synthesized in vitro accumulated and were ligated to high-molecular-weight DNA with low efficiency. The chromatin protein components of nuclei incubated in the DNA replication assay conditions were investigated for displacement, degradation, and exchange. Displacement of total nuclear protein during in vitro incubation occurred to the extent of 3%, and involved only the nonhistone nuclear proteins. Degradation was not detectable, assayed both by loss of acid-soluble radioactivity and by protein electrophoretic patterns in polyacrylamide gels. No detectable protein was exchanged from chromatin to exogenous DNA.

Cell Nucleus↗

PubMed1976

Studies on the mode of segregation of histone nu bodies during replication in HeLa cells.

Two models were tested for the mode of distribution of histone nu bodies at the replication fork. The replication fork was labeled by brief incubation of cells with 3H-thymidine. Nuclei were isolated and digested with low levels of micrococcal nuclease, and the kinetics of cleavage of the pulse-labeled chromatin DNA were compared to the kinetics of clevage of perental chromatin DNA. In chromatin labeled for 30 sec to 10 min, the rate of cleavage of the pulse-labeled region into monomeric nu body-sized units exceeded the rate of cleavage of parental chromatin by a factor of 2, but did not approach the predicted value of 5-6 for random segregation. This value dropped to 1.6 in 15 min and was equivalent to perental chromatin in 20 min labeling experiments. DNA synthesized in the presence of cycloheximide was also digested at twice the rate of parental chromatin DNA. A Poisson analysis of the kinetics of cleavage by micrococcal nuclease further confirmed these observations. The predicted difference in the rate of production of monomeric, dimeric, and trimeric deoxyribonucleoprotein units was very similar to the experimental values of both total chromatin and nascent chromatin. Thus the nu body spacings in newly replicated chromatin closely approximate those in parental chromatin. These results agree well with a conservative or nondispersive model of nucleosome distribution in which the proteins are associated with one of the two daughter chromosomes during replication.

Chromatin↗

PubMed1976

Temporal relationships of chromatin protein synthesis, DNA synthesis, and assembly of deoxyribonucleoprotein.

Chromatin assembly has been investigated in terms of the sites on DNA where newly synthesized chromatin proteins associate. Chromatin from cells labeled with [14C]-BrdUrd and [3H]lysine was fixed with formaldehyde and resolved in CsCl gradients. By varying the spacing of the labeling intervals of the two isotopes so as to encompass all possible periods in S-phase, the association of labeled, newly synthesized proteins on newly synthesized (BrdUrd-substituted) or preexisting chromatin DNA was determined. In all experiments it was found that newly synthesized chromatin proteins predominantly associated with nonreplicating DNA. Possible mechanisms by which cells recycle preexisting chromatin proteins to restore the protein content of newly synthesized DNA are discussed.

Binding Sites↗

PubMed1975