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R T Simpson

Publications and source records attributed to R T Simpson.

At least 91 records · Page 5Linked to original sources
PubMed1976

Mapping DNAase l-susceptible sites in nucleosomes labeled at the 5' ends.

We have used lambda-32P-ATP and polynucleotide kinase to label stoichiometrically the 5' ends of DNA in intact isolated HeLa cell nucleosomes. DNA is not nicked or degraded during the modification reaction. We have used these modified nucleosomes to study both the distribution and the relative availability of sites within the nucleosome which are susceptible to digestion by DNAase l. The results show that the nucleosome contains a potential cleavage site every 10 nucleotides, with the exception of the site 80 nucleotides from the 5' end. Favored cleavage sites are located 20, 40, 50, 100, 120, and 130 nucleotides from the 5' end; sites 30 and 110 nucleotides from the 5' end are strongly disfavored, while the potential site 80 nucleotides from the 5' end is virtually never cleaved. These findings provide constraints for models of histone-DNA interactions within the chromatin subunit.

Adenosine Triphosphate↗
PubMed1976

Histones H3 and H4 interact with the ends of nucleosome DNA.

Isolated HeLa cell nucleosomes (core particles) were labeled at the 5'-termini of their DNA with 32P using [gamma-32P]ATP and polynucleotide kne by sequential methylation, depurination, Schiff base formation, and reduction with sodium borhydride. After digestion of the noncrosslinked DNA by DNase I and venom phosphodiesterase, histones were separated by gel electrophoresis and those crosslinked to the 5'-termini were identified by 32P-autoradiography. Histones H3 and H4 occur with equeal frequency as the nearest protein neighbors to the end of the DNA in nucleosomes. Histone arrangements within the core particle compatible with these results are discussed.

Base Sequence↗
PubMed1976

Chemical evidence that chromatin DNA exists as 160 base pair beads interspersed with 40 base pair bridges.

Digestion of rat liver nuclei by an endogenous endonuclease generates double-stranded DNA fragments which are initially about 205 base pairs long, as reported previously by Hewish and Burgoyne. As digestion proceeds, the average size of these fragments is reduced to about 160 base pairs. Electrophoresis under denaturing conditions shows that these DNA fragments contain single strand nicks at ten base intervals. Fifteen bands, 10-150 bases, are clearly resolvable. DNA Fragments of 160 to 200 nucleotides are not resolved as distinct species. The results suggest that the chromosomal subunit contains both a 160 base-pair DNA segment, in a conformation susceptible to single strand nicking at ten base intervals, and a forty base-pair DNA segment in a conformation more uniformly susceptible to endogenous endonuclease activity. This chemical evidence agrees with morphological observations suggesting that chromatin has a "bead and bridge" structure.

Animals↗
PubMed1976

Preparation and physical characterization of a homogeneous population of monomeric nucleosomes from HeLa cells.

We describe a method of isolating a homogeneous population of "trimmed" monomeric nucleosomes from Hela cells. These nucleoprotein particles contain a 140 +/- 5 base pair length of DNA and have a histone/DNA ratio of 1.2. They lack H1 and contain equal amounts of the four smaller histones. The DNA contains no single strand nicks. The particles sediment with an S20,w of 11S in D2O density gradients. After formaldehyde fixation, they band at a density of 1.4370 in neutral CsCl. Digestion of nucleosomes with either micrococcal nuclease or DNase I generates the same pattern of DNA fragments observed when intact nuclei are digested. Circular dichroism spectra indicate that the 280 nm positive ellipticity maximum of nucleosomes is about one-half that of chromatin. In the presence of 6 M urea, nucleosomes sediment with an S20,w of 6S, have a multiphasic thermal denaturation profile, and exhibit a circular dichroic spectrum nearly identical to that of B-form DNA. Our yield of purified nucleosomes (10-15% of the input DNA) is similar to the yields of other methods; our nucleosome population is substantially more homogeneous than those previously reported.

Chromatin↗
PubMed1974

Distribution of RNA polymerase binding sites in fractionated chromatin.

Calf-thymus chromatin was fractionated by ion-exchange chromatography on ECTHAM-cellulose and sucrose gradient sedimentation. [ECTHAM-cellulose is a cationic adsorbent prepared by coupling tris(hydroxymethyl)aminomethane to cellulose with epichlorohydrin.] The capacity of these fractionated chromatins to support RNA synthesis by DNA-dependent RNA polymerase of Escherichia coli was examined, using procedures that permit measurement of binding site frequency. Unfractionated calf thymus chromatin has 5-10% as many binding sites as protein-free DNA. By combination of the two fractionation methods, chromatin samples were obtained containing as few as 2% and as many as 47% of the number of binding sites found on protein-free DNA.

Amino Alcohols↗