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R L Rill

Publications and source records attributed to R L Rill.

At least 37 records · Page 2Linked to original sources
PubMed1989

Mobile histone tails in nucleosomes. Assignments of mobile segments and investigations of their role in chromatin folding.

The 13C NMR spectrum of isolated nucleosome core particles contains many sharp resonances, including resonances of alpha- and beta-carbons, indicating that certain terminal segments of histones rich in basic residues are highly mobile (Hilliard, R. R., Jr., Smith, R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 5992-5998). Specific histone termini can be removed sequentially from nucleosome core particles by mild treatment with alpha-chymotrypsin or chymotrypsin plus trypsin (Rosenberg, N. L., Smith. R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 12375-12383). Comparisons of the 13C NMR spectra of native and several partially proteolyzed core particles indicated that a minimum of residues 1-20 of H3 and 1-11 and 118-128 of H2a are contained in mobile segments of native cores. H4 did not appear to contribute to the resonances from mobile histone segments, but a possible contribution of H2b residues 1-16 could not be ruled out. The 13C NMR spectra of oligonucleosomes containing and lacking lysine-rich histones (H1, H5) were similar to each other and to that of native nucleosome cores both when the oligonucleosomes were in an extended conformation at low ionic strength and when they were in a more compact conformation at higher ionic strength. This similarity suggests that histones H1 and H5 must be largely immobilized upon chromatin binding and that the segments of core histones that are mobile in isolated nucleosome cores are not strongly bound to adjacent linker regions in intact chromatin, and are not immobilized by compaction to the degree achieved in 50 mM phosphate buffer.

Animals↗
PubMed1989

Images of the DNA double helix in water.

The scanning tunneling microscope can image uncoated DNA submerged in water. The grooves of the double helix were clearly resolved in images of the 146-base pair fragment extracted from calf thymus nucleosome. In contrast to images obtained with dry DNA, the helix pitch varied only a small amount (36 +/- 5 angstroms). The path of the helix shows considerable variation. It is quite straight when the molecules are densely packed, but it curves and bends in isolated molecules.

Animals↗
PubMed1989

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I): effects of single base pair transitions on the cleavage of preferred pyrimidine-purine-pyrimidine triplets.

The cleavage of DNA restriction fragments by bis(1,10-phenanthroline)copper(I) [[(OP)2CuI]+] is sequence dependent: the trimer TAT is most strongly preferred, while the trimer TGT and tetramers TAAT, TAGT, and CAGT are strongly to moderately preferred [Veal, J. M., & R. L. (1988) Biochemistry 27, 1822-1827]. [(OP)2CuI]+ cleavage of a series of oligonucleotide duplexes of the type 5'-CCCTPyPuPyCCCC-3'/3'-GGGAPuPyPuGGGG-5' (Py = pyrimidine; Pu = purine) was examined to determine the effects of purine substituents in the central triplet on specificity. The relative cleavage rates of different PyPuPy triplets in oligomers were similar to those observed for restriction fragments. The undecamer duplex containing the trimer TAT (TTATC) was most preferentially cleaved, predominantly at the central adenosine and the adjacent 3'-thymidine. Duplexes differing from TTATC by a single A.T----G.C transition in the central triplet were cleaved at significantly reduced rates relative to TTATC, the order of preference being TAT greater than TGT greater than TAC greater than CAT. By contrast, duplexes differing from TTATC by a single A.T----I.C transition were cleaved at rates similar to those for TTATC when the transition occurred at the 5'-pyrimidine or central purine [i.e., C(.I)AT and TIT]. A duplex containing the trimer TAC(.I) was cleaved at a reduced rate similar to the duplex containing TAC(.G). The guanine 2-amino group at positions 1 and 2, but not position 3, of a 5'-PyPuPy-3' trimer is therefore implicated as a strong inhibitor of DNA binding by the copper-phenanthroline complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1989

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes.

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.

Animals↗
PubMed1989

STM and AFM images of nucleosome DNA under water.

We have imaged DNA from the calf thymus nucleosome using a scanning tunneling microscope (STM) operated in water. The fragments are deposited onto the interface between a buffer solution and an epitaxially grown gold surface using an electrochemical tecnique. Most of the fragments are fairly straight, and when individual polymers can be identified, their length is consistent with the expected 146 basepairs (approximately 500 A). The resolution is often adequate to show signs of the 36 A helical pitch. Some images show a structure which appears to have abrupt kinks of the sort predicted by Crick and Klug (Nature 255, 530-533, 1975). In order to check that this shape is not a consequence of binding to underlying structure on the gold substrate, we have also made images of kinked structures using an atomic force microscope (AFM) with the DNA bound to glass.

Animals↗
PubMed1989

7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D.

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.

Affinity Labels↗
PubMed1988

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I).

The bis(1,10-phenanthroline)copper(I) complex is a relatively simple molecule previously shown to cause DNA cleavage with a strong preference for gene control regions such as the Pribnow box. Sequence level mapping of sites of [(Phen)2CuI]+ cleavage in greater than 2000 bases in histone genes and the plasmid pUC9 showed that the specificity for control regions is related to a predominant preference for minor groove binding at TAT triplets, which were cleaved most strongly at the adenosine sugar ring. The related sequences TGT, TAAT, TAGPy, and CAGT (Py = pyrimidine) were moderately preferred, while CAT and TAC triplets, PyPuPuPu quartets, PuPuPuPy quartets, and CG-rich PyPuPuPy quartets were cleaved with low to average frequency. Polypurine and polypyrimidine sequences were cleaved with low frequency. The sequence preferences of [(Phen)2CuI]+ can be ascribed predominantly to (i) a requirement for binding in the minor groove at a pyrimidine 3'----5' step and (ii) stereoelectronic effects of the 2-amino group of guanine in the minor groove, which inhibit binding. Although the reagent appears primarily to recognize sequence features at the triplet or quartet level, lower than expected cleavage was observed for two TAT sequences adjacent to several other preferred sequences and higher than expected cleavage was observed at CAAGC sequences, suggesting that longer range sequence-dependent DNA conformational effects influence specificity in certain cases.

Animals↗
PubMed1988

Multiple liquid crystal phases of DNA at high concentrations.

DNA packaging in vivo is very tight, with volume concentrations approaching 70% w/v in sperm heads, virus capsids and bacterial nucleoids. The packaging mechanisms adopted may be related to the natural tendency of semi-rigid polymers to form liquid crystalline phases in concentrated solutions. We find that DNA forms at least three distinct liquid crystalline phases at concentrations comparable to those in vivo, with phase transitions occurring over relatively narrow ranges of DNA concentration. A weakly birefringent, dynamic, 'precholesteric' mesophase with microscopic textures intermediate between those of a nematic and a true cholesteric phase forms at the lowest concentrations required for phase separation. At slightly higher DNA concentrations, a second mesophase forms which is a strongly birefringent, well-ordered cholesteric phase with a concentration-dependent pitch varying from 2 to 10 micron. At the highest DNA concentrations, a phase forms which is two-dimensionally ordered and resembles smectic phases of thermotropic liquid crystals observed with small molecules.

Crystallization↗
PubMed1987

Atypical changes in chromatin structure during development in the sea urchin, Lytechinus variegatus.

Embryos of several sea urchin species express three distinct sets of developmental-stage-specific histone variants: cleavage stage, early and late variants. Urchins also contain sperm-specific histone variants. We have found that the sea urchin Lytechinus variegatus, common in shallow coastal waters of the southeastern United States, also contains these four distinct sets of variants. The number and electrophoretic characteristics of L. variegatus variants are similar to those of the related species, Lytechinus pictus, but changes in variant populations are delayed in relation to stage in L. variegatus relative to L. pictus and other species. The cleavage-stage-like variants of H2a and perhaps H1 in L. variegatus are much more abundant in pre-blastula and blastula embryos than cleavage-stage variants in other species; and the appearance of L. variegatus late variants is delayed, relative to the program in L. pictus. Likewise the nucleosome repeat length in L. variegatus embryos increases from 191 to 210 base pairs (bp) upon gastrulation, while major increases in repeat length in other urchins occur before or during blastulation. In addition, the nucleosome repeat length in L. variegatus sperm is anomalously low, 201 +/- 2 bp - lower than observed in L. variegatus gastrula to pluteus stage embryos (210 bp), and much lower than previously observed in sperm of other urchins (more than 233 bp). Direct comparisons showed that Strongylocentrotus purpuratus and L. variegatus sperm contained electrophoretically identical core histones, including two high-molecular-weight H2b variants characteristic of all urchin sperm, but nucleosome repeat lengths of 240 and 201 bp, respectively. Differences in nucleosome repeats of these species appear to be due to differences in H1 variants.

Animals↗
PubMed1986

The action of chymotrypsin on nucleosome cores. Histone products and conformational effects of limited digestion.

alpha-Chymotrypsin was used to probe accessible hydrophobic amino acid residues in nucleosome cores. Small amounts of chymotrypsin rapidly and selectively cleaved at leucine 20 of histone H3. Cleavage at this site caused partial unfolding of the nucleosome core at low ionic strengths indicated by a small decrease in sedimentation coefficient and increase in circular dichroism in the 265-285-nm range. Unfolding did not occur at moderate ionic strengths, probably because of more effective electrolyte screening of residual negative charge on the nucleosome core. More extensive treatment with chymotrypsin partially degraded other core histones in nucleosome cores at similar rates. The primary sites of cleavage were assigned to Leu115 of H2a, Val18 or Gln22 of H2b, and Leu10 plus Leu22 of H4. We conclude that these primary sites of chymotrypsin cleavage of the four core histones lie on or near the nucleosome core surface, while the large number of other hydrophobic histone residues located in more central sequences must be inaccessible. Extensive chymotrypsin treatment yielded a set of "limit" products approximately 80-100 residues long that were similar to the limit products of trypsin digestion. Sedimentation coefficients and circular dichroism spectra of nucleosome cores treated to near limits with chymotrypsin or chymotrypsin followed by trypsin were not consistent with significant unfolding of the proteolyzed cores at moderate ionic strength. These results indicate that the amino-terminal 20-30 residues of H2b, H3, and H4 and the amino- and carboxyl-terminal approximately 12 residues of H2a, in toto, interact weakly if at all with DNA of isolated nucleosome cores. These histone termini stabilize less than two turns and perhaps only one turn on each DNA terminus.

Amino Acids↗
PubMed1986

Natural abundance carbon-13 nuclear magnetic resonance studies of histone and DNA dynamics in nucleosome cores.

Natural abundance carbon-13 nuclear magnetic resonance spectra (67.9 MHz) were obtained for native nucleosome cores: cores dissociated in 2 M NaCl and 2 M NaCl, 6 M urea; and cores degraded with DNase I plus proteinase K. Phosphorus-31 NMR spectra of native and dissociated cores and core length DNA were also obtained at 60.7 MHz. The 31P resonance and spin-lattice relaxation time (T1) of DNA were only slightly affected by packaging in nucleosome cores, in agreement with other reports, but 13C resonances of DNA were essentially unobservable. The loss of DNA spectral intensity suggests that rapid internal motions of DNA sugar carbons in protein-free DNA previously demonstrated by 13C NMR methods are partly restricted in nucleosomes. The 13C spectrum of native cores contains many narrow intense resonances assigned to lysine side chain and alpha-carbons, glycine alpha-carbons, alanine alpha- and beta- carbons, and arginine side chain carbons. Several weaker resonances were also assigned. The narrow line widths, short T1 values, and non-minimal nuclear Overhauser enhancements of these resonances, including alpha- and beta-carbons, show that some terminal chain segments of histones in nucleosomes are as mobile as small random coil polypeptides. The mobile segments include about 9% of all histone residues and 25% of all lysines, but only 10% of all arginines. The compositions of these segments indicate that mobile regions are located in amino- or carboxyl-terminal sequences of two or more histones. In addition, high mobility was observed for side chain carbons of 45-50% of all lysines (delta and epsilon carbons) and about 25% of all arginines (zeta carbon) in histones (including those in mobile segments), suggesting that basic residues in terminal histone sequences are not strongly involved in nucleosome structure and may instead help stabilize higher order chromatin structure.

Amino Acids↗
PubMed1986

Liquid crystalline phases in concentrated aqueous solutions of Na+ DNA.

Concentrated aqueous saline solutions of short (146-base-pair) DNA fragments suddenly become turbid and iridescent when the DNA concentration is slightly increased or the temperature is decreased. Microscopic examination through crossed polarizing filters shows that turbidity and iridescence is due to formation of a liquid crystalline DNA phase similar to cholesteric liquid crystals formed by other semirigid, but nonelectrolyte, chiral polymers. Several distinct textures of the liquid crystalline phase or phases are observed depending on DNA concentration, temperature, and method of sample preparation. Textures observed include spherulites with Maltese crosses, striated and highly colored ribbons, whorls of periodic interference fringes, and colored flakes. The liquid crystalline DNA phase coexists in metastable equilibrium with the isotropic phase over a relatively narrow temperature/concentration range--approximately 175-250 mg/ml and 25-62 degrees C (limit of measurements). At higher concentrations and temperatures above approximately equal to 25 degrees C, the solutions appear fully liquid crystalline. When concentrated solutions are cooled below room temperature, crystals form due to precipitation of supporting electrolyte. A partial phase diagram is reported for the isotropic----liquid crystal----crystal transitions of solutions of DNA in buffered saline (2 M Na+). The general features of this phase diagram and the critical DNA volume fraction for formation of the anisotropic phase are consistent with the observed and theoretically predicted phase behavior of rodlike or semirigid nonelectrolyte polymers.

Animals↗
PubMed1984

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion.

A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0 degrees C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock , J.M., Jr., and Rill , R.L. (1979) Biochemistry 18, 3739-3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1-2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.

Animals↗
PubMed1984

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion.

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.

Acetates↗
PubMed1983

Spontaneous ordering of DNA. Effects of intermolecular interactions on DNA motional dynamics monitored by 13C and 31P nuclear magnetic resonance spectroscopy.

Effects of intermolecular DNA interactions on the motional dynamics of defined length (147-, 234-, and 437-nucleotide pair (np)) double-stranded DNA were examined by 13C and 31P nuclear magnetic resonance spectroscopy. At a critical high concentration varying inversely with length, DNA undergoes a spontaneous transition to an ordered, liquid crystalline-like state. Ordering is accompanied by the appearance of distinct opalescence, and an increase in solution viscosity. An apparent standard heat of fusion of -38 kcal/mol of helix (-0.13 kcal/mol of DNA phosphate) and an entropy change of -0.13 entropy units (per mol of helix) were determined from the temperature dependence of the phase transition of 147-np DNA. The average phosphodiester configuration, monitored by Raman spectroscopy, was typical of B-form DNA above and below the phase transition. NMR spectra and relaxation data show that intermolecular interactions are strong at concentrations well below the phase transition and cause stepwise uncoupling of internal motions at specific sites. Motions of the exocyclic C5' carbon, but not other sugar carbons, are frozen in approximately 50% of 147-np DNA molecules at concentrations as low as 6.5 mg/ml. Motions of backbone ring carbons (C3', C4') are frozen in a progressively larger fraction of molecules at concentrations above 46 mg/ml. Rapid C2' motions are unaffected below the critical concentration (193 mg/ml at 32 degrees C), and still occur in the ordered phase. We conclude that rapid internal motions of DNA monitored by NMR consist mostly of coupled, periodic bending deformations and partially uncoupled local motions within the sugar ring. Rapid, extensive C2' motions can occur without strong coupling to other sugar carbon motions. From a comparison of 13C and 31P NMR data, we conclude that 31P NMR at best yields an incomplete representation of DNA dynamics.

Animals↗
PubMed1983

Understanding DNA conformational dynamics: answering questions and questioning answers.

Phosphorus-31 and especially Carbon-13 NMR measurements have recently become primary input to the understanding of DNA solution dynamics. While the 31P measurements are inherently easier, the quality of 31P dynamics information is suspect and therefore 13C measurements are preferred. In fact, it is necessary to obtain several kinds of 13C data (T1s, NOE's, linewidths, integrated peak intensities) over a wide range of magnetic fields (13C NMR frequencies) in order to identify major features of DNA internal motions. Further information comes from variation of temperature and DNA fragment length and/or concentration. Most of our 13C measurements have been performed at 37.7-90.6 MHz on fully double stranded monomer size (147 base pair) DNA at concentrations in phosphate buffer of approximately less than 10 to approximately greater than 200 mg ml-1; temperatures studied range from 6 to 55 degrees C. Other measurements have been performed on monomer-size single-stranded DNA at 85 and 92 degrees. The large data set we have acquired appears to answer some important questions about the nature and extent of DNA overall and internal motional dynamics. However, the picture remains incomplete and a number of questions arise from these results: 1. Overall motion of the double stranded DNA fragments follows expected hydrodynamic behavior; 2. Restricted but rapid internal motion along the DNA structure is well represented by a spaghetti-like wobbling-in-a-cone model; 3. DNA-DNA Interactions and solvent ordering, present at relatively low DNA concentrations, partially quench the internal motion, consistent with hinge-model structural changes (and the spaghetti model above) but not as compatible with in-plane torsional motion models; 4. The deoxyribose C-2' sites undergo additional motion which is partially uncoupled from the internal wobbling motions: 5. At high DNA concentrations, a phase transition occurs, resulting in ordered structures which drastically affect DNA internal dynamics; 6. DNA interacting with ethidium does not greatly change its conformational mobility; 7. DNA interacting with Hg2+ ions shows less than anticipated change in internal DNA dynamics. The remaining challenge is to interpret our current results in terms of specific conformational processes and to understand why the conformational mobility of double stranded DNA is relatively unhindered by major structural perturbants such as intercalating ethidium and mercury ion.

DNA↗
PubMed1982

The accessibilities of histones in nucleosome cores to an arginine-specific protease.

Submaxillaris protease, reportedly specific for arginine residues, was used to probe accessible arginines of chicken erythrocyte nucleosome cores. The relative rates of digestion of histones in nucleosome cores by this protease were H3 greater than H2b greater than H4 much greater than H2a. At most, 8 of 52 total arginines among the four core histones were reasonably accessible to attack. Sites most rapidly cleaved were Arg-26 in H3, a site approximately 13 residues from the NH2 terminus of H2b, and Lys-12 or Arg-17 in H4. Five sites attacked more slowly were Arg-8, -128, or -129 and Arg-49, -52, or -53 in H3; Arg-3 and Arg-17 or -19 in H4; and a site near one terminus of H2b or H3. H2a and fragments resulting from the above cleavages were highly resistant to attack, even after prolonged incubation. Similar results were obtained upon digestion of histones in intact chromatin. H1 and H5 in whole chromatin were attacked at rates comparable to H3. Seven of the eight accessible sites lie outside of structure-forming histone sequences, i.e. sequences that are immobilized in histone complexes, indicating that these sequences are inaccessible in nucleosome cores. The single exceptional site noted, approximately 50 residues from the NH2 terminus of H3, is consistent with previous observations that Glu-51 and Glu-60 of H3 in nucleosomes are accessible to attack by S. aureus protease (Rill, R. L., and Oosterhof, D. K. (1981). J. Biol. Chem. 256, 12687-12691). The relationships of these protease accessibilities to NMR assignments of mobile histone tails in nucleosome cores are discussed.

Animals↗