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Biomedical subjects

R L Rill

Publications and source records attributed to R L Rill.

53 records · Page 3Linked to original sources
PubMed1981

Staphylococcus aureus protease. A probe of exposed, nonbasic histone sequences in nucleosomes.

The digestion of histones in chicken erythrocyte nucleosome cores and chromatin by Staphylococcus aureus protease was examined. This protease cleaves specifically at acidic residues and prefers Glu-X bonds under the conditions used. Only 1 of 24 glutamic acids and 2 of 13 aspartic acids among all four core histones are located in basic, NH2-terminal tails; hence, the protease is a highly specific probe of exposed nonbasic sequences. The protease readily degraded H1, H5, and H3, moderately degraded H2b, and only slightly degraded H2a and H4 in nucleosomes and nucleosome cores. Electrophoresis of core histone fragments from limited digests showed that most glutamic acids were inaccessible, but at least five sites in nonbasic sequences were readily cleaved. Tentative assignments of these fragments based on comparisons with products from limited digests of pure histones suggested that most accessible sites in nucleosome cores occur in H3. The most probable sites of H3 cutting are glutamic acids at positions 51, 60, 73, 94, and 97. At least one site in H2b, probably the equivalent of glutamic acid 105 in the calf H2b sequence, was accessible. No sites in H2a and H4 appeared highly accessible. H5 was readily cleaved at a site near the NH2 terminus. These data substantiate other evidence that nonbasic core histone sequences are located primarily in the nucleosome interior, but that H3 binds to the ends of core DNA and thereby is partly exposed on the upper and lower surfaces of the disk-shaped core.

Amino Acid Sequence↗
PubMed1981

Carbon 13 spin-lattice relaxation, linewidth, and nuclear Overhauser enhancement measurements of nucleosome length DNA.

Carbon 13 NMR data were obtained at four magnetic fields for double-stranded DNA samples 120 and 160 nucleotide pairs long. Spectral linewidths are several-fold smaller than predicted for overall rigid rod rotation and increase with increasing field, indicating significant chemical shift dispersion contributions to high field linewidths. Spin-lattice relaxation times are short (e.g. T1 values for CH carbon atoms are approximately 0.5 s at 67.9 MHz), and increase with increasing field. Nuclear Overhauser effects of 0.6 to 0.8 (theoretical maximum = 2.0) were measured for the same protonated carbon atoms at 100.6 MHz. These short T1 values nd relatively large nuclear Overhauser effects show that double-stranded DNA undergoes rapid internal motions with effective correlation times of a few nanoseconds. Preliminary data indicate that DNA base carbon motions occur on essentially the same time scale as sugar motions, thus arguing against independent flexibility of the DNA backbone. Linewidths decreased 2- to 5-fold and nuclear Overhauser effects doubled, upon heat denaturation of DNA, as expected for increased motion. On the other hand, NT1 values of native and denatured DNA were nearly identical, suggesting insensitivity of spin-lattice relaxation times to motions in the nanosecond range in these relatively stiff chains.

Animals↗
PubMed1981

Circular dichroism, thermal denaturation, and deoxyribonuclease I digestion studies of nucleosomes highly enriched in high mobility group proteins HMG 1 and HMG 2.

Salt-soluble (S) nucleosomes that contain near equimolar high mobility group nonhistone chromosomal proteins HMG 1 and HMG 2 and lack histone H1 were isolated from mouse myeloma nuclei. Comparisons of the sedimentation, near-UV circular dichroism, thermal denaturation, and pattern of DNase I digestion of S nucleosomes with these properties of nucleosome cores or "typical" nucleosomes containing H1 did not detect significant differences. These results indicate that HMG 1 and 2 do not affect the conformation and stability of nucleosomes or nucleosomal DNA and are consistent with the proposal that major functions of HMG 1 and 2 are to replace H1 and maintain the (micro) solubility and accessibility of local chromatin regions. In contrast to these similarities, the initial rate of DNase I digestion of S nucleosomes was approximately 3 times that of chromatin depleted in S nucleosomes. This is consistent with a relation of S nucleosomes to transcription and suggests that subtle factors (not necessarily HMG 1 and 2) determine DNase I susceptibility.

Animals↗
PubMed1979

Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins.

Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0 degrees C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg2+, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6--8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and HMG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relationship of this soluble fraction to transcriptionally active chromatin is discussed.

Amino Acids↗
PubMed1977

Chromatin subunits from baker's yeast: isolation and partial characterization.

The organization of proteins along DNA in chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease. Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 base pairs apart. Nucleoprotein fragments were resolved and isolated by centrifugation on linear, 5-20% sucrose gradients. The predominant 11S component appears to be identical to chromatin "subunits" or "nucleosomes" isolated from higher eukaryotes, containing a 150-160 base pair length of DNA and approximately equimolar amounts of four proteins that coelectrophorese with calf histones H2A, H2B, H3, and H4, plus small amounts of three proteins that electrophorese similarly to H1 histones. Thus, the structural organization of the yeast genome is similar to that of more developed organisms, except for the smaller total repeat length. None of the yeast subunit proteins, including the possible H1 proteins, contains cysteine.

Centrifugation, Density Gradient↗
PubMed1977

Histones H2A and H2B are neighbors along DNA in chromatin: characterization of subnucleosomal particles containing H2A+H2B.

Two specific slow sedimenting nucleoprotein particles containing equimolar amounts of histones H2A and H2B and 38 or 49 base pair (bp) lengths of DNA have been isolated by centrifugation on sucrose gradients. The 3.4S particles containing 38 bp DNA and H2A+H2B thermally denature at 61 degrees, considerably higher than Proteinase K treated particles (44 degrees), but lower than 11S nucleosomes (76 degrees). Treatment with Proteinase K increases the circular dichroism of 3.4S particles at 280 nm by 63% and decreases the sedimentation coefficient to 2.1S. These results indicate that H2A and H2B are proximate along DNA in nucleosomes and alone can alter the optical activity and perhaps conformation of local regions of DNA.

Animals↗
PubMed1977

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.

Animals↗
PubMed1975

Nucleas action on chromatin: evidence for discrete, repeated nucleoprotein units along chromatin fibrils.

The time course of the fragmentation of calf thymus chromatin by DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6) has been examined by sedimentation of chromatin digests through linear (5-20%) sucrose gradients. The action of nuclease is decidedly nonrandom, ultimately producing roughly equal amounts of acid-soluble oligonucleotides and 11S nucleoprotein particles. The 11S particles contain double-stranded DNA that is approximately 400 A or 120 base-pairs long, as measured by electron microscopic examination of deproteinized samples, and is maintained in a compact conformation within the intact particles. In addition, 15S nucleoprotein particles containing predominantly 800-A lengths of DNA have been isolated from less extensively digested chromatin. Evidence is presented which indicates that the 11S particles are fundamental structural units that are arranged in tandem along certain regions of chromatin fibrils. Preliminary experiments with different nucleases and with chromatin from different mammalian species indicate that these results are a natural consequence of the arrangement of DNA and proteins in mammalian chromatin and are not peculiar to the system described in detail.

Animals↗
PubMed1975

Heterogeneity of chromatin fragments produced by micrococcal nuclease action.

Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites.

Animals↗
PubMedDate not supplied

Actinomycin D binding to unstructured, single-stranded DNA.

Actinomycin D is an anticancer antibiotic best know for inhibiting transcription by binding double-stranded DNA. Tight, sequence selective binding of actinomycin to single-stranded DNA is also known, however, and is implicated in biological activities including inhibition of (-) strand transfer by HIV reverse transcriptase. Oligonucleotide d(GTTAACCATAG) is one of the rare single-stranded DNAs that lack GC steps yet have high affinity for actinomycin. Oligonucleotide sequence and length requirements for drug binding were investigated by monitoring association of the fluorescent surrogate, 7-aminoactinomycin D, to d(GTTAACCATAG) and 31 related oligomers. The TAG-3' terminal sequence was essential for high-affinity binding, but was not sufficient. Five oligomers with TAG sequences on or near the 3'-end had high affinity [K(d) < or = 200 nM (oligomer)]. A sixth oligomer, d(GTAACCATATG), had moderately lower affinity (Kd = 370 nM), and other homologous oligomers had much lower affinity. The minimum length sequence for tight binding of 7-aminoactinomycin D was identified as only eight nucleotides, corresponding to d(AACCATAG). This octanucleotide is unstructured in the absence of actinomycin, and has the highest drug affinity of all oligomers examined (Kd = 125 nM). These studies show that high-affinity binding of 7-aminoactinomycin, and actinomycin D by extension, to single-stranded DNA does not require pre-existing secondary structure or any apparent propensity for secondary structure. It is proposed that actinomycin D binds to certain single-stranded DNA sequences by an induced-fit mechanism favored by participation of at least eight nucleotides, or the equivalent of four base pairs.

Base Sequence↗