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G Felsenfeld

Publications and source records attributed to G Felsenfeld.

At least 127 records · Page 7Linked to original sources
PubMed1978

Chromatin.

The approximate shape of the chromatin subunit called the nucleosome is now known, but its internal architecture is not well understood. Recent studies reveal details of the organisation of DNA within the nucleosome, and show that the arginine-rich histones are essential to DNA folding. Nucleosomes or structures related to them seem to be present at points of DNA replication and transcription; interactions within and between nucleosomes are likely to play a critical part in these processes.

Animals↗
PubMed1978

DNAase I, DNAase II and staphylococcal nuclease cut at different, yet symmetrically located, sites in the nucleosome core.

We have determined the relative location of pancreatic DNAase (DNAase I), spleen acid DNAase (DNAase II) and staphylococcal nuclease cleavage sites in the nucleosome core. Each of these three enzymes cleaves the DNA of chromatin at 10.n nucleotide intervals (n integer); this specificity presumably reflects the internal structure of the nucleosome. We have already reported that DNAase I cleaves nucleosomal DNA so that nearest adjacent cuts on opposite strands are staggered by 2 nucleotides, 3' end extending (Sollner-Webb and Felsenfeld, 1977). Here we show that the nearest cuts made by DNAase II in nucleosomal DNA are staggered by 4 nucleotides, 3' end extending, while cuts made by staphylococcal nuclease have a stagger of 2 nucleotides, 5' end extending. The cutting sites of the three enzymes thus do not coincide. Each pair of staggered cuts, however, is symmetrically located about a common axis-that is, the "dyad axes" that bisect nearest pairs of cutting sites coincide for all three enzymes. This result is consistent with the presence of a true dyad axis in the nucleosome core. Our results support the conclusion that a structural feature of the nucleosome, having a 10 nucleotide periodicity, is the common recognition site for all three nucleases. The position of the cut is determined, however, by the individual characteristics of each enzyme. Sites potentially available to nuclease cleavage span a region of 4 nucleotides out of this 10 nucleotide repeat, and a large fraction of these sites are actually cut. Thus much of the nucleosomal DNA must in some sense be accessible to the environment.

Animals↗
PubMed1978

A simple model of DNA superhelices in solution.

Closed circular DNA molecules in aqueous solution take the form of interwound superhelices over a wide range of superhelix densities. We describe a very simple model of such a superhelix in which twisting and bending forces are in balance, subject both to topological constraints and to a limitation on the distance of closet approach of the interwound duplexes of the superhelix. The model is consistent with some of the observed physical properties of closed circular DNA, and suggests that there may be severe limits to the range of allowable geometries for the superhelix structure of minimum energy.

DNA, Superhelical↗
PubMed1978

An octamer of histones H3 and H4 forms a compact complex with DNA of nucleosome size.

Equimolar mixtures of histones H3 and H4 have been reconstituted onto DNA of nucleosome core size. Two distinct complexes are formed in a relative abundance that depends on the starting ratio of H3 + H4 to DNA. One of these complexes contains two H3-H4 dimers for each DNA molecule, and has a sedimentation coefficient of 7.5S. The other complex contains an octamer consisting of four H3-H4 dimers, and has a sedimentation coefficient of 10.4S. On the basis of these measurements, we conclude that the octamer complex (but not the tetramer complex) is a fully folded, compact structure resembling the nucleosome.

Animals↗
PubMed1978

A new method for the purification and identification of covalently closed circular DNA molcules.

A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.

DNA, Circular↗
PubMed1977

Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside triphosphates.

We have employed mercury-substituted UTP to study the transcription of duck reticulocyte chromatin in vitro by Escherichia coli RNA polymerase. We find that the use of this method results in large overestimates of the amount of de novo synthesis of globin-specific RNA sequences. The artefact arises because endogenous globin RNA can serve as a template for the RNA polymerase, resulting in the formation of a duplex product in which one strand is the endogenous message, and the other is the mercury-labeled complementary strand. Subsequent purification of the mercury-substituted RNA on thiol-agarose results in copurification of endogenous globin sequences. We document the details of this mechanism and describe methods which will eliminate the artefact.

Animals↗
PubMed1977

Pancreatic DNAase cleavage sites in nuclei.

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.

Animals↗
PubMed1977

Histone H3 disulfide dimers and nucleosome structure.

The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and H4. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations. The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.

Chromatin↗
PubMed1977

Supercoiling energy and nucleosome formation: the role of the arginine-rich histone kernel.

We have formed complexes of relaxed closed circular Col E1 DNA with various combinations of histones, and examined the effects of treating the complexes with nicking-closing enzyme. Germond et al (1) have shown that when a mixture of the four core histones of the nucleosome (HIA, H2B, H3 and H4) is used in such an experiment, the subsequently isolated DNA is supercoiled. We find that the arginine-rich histone pair, H3 and H4, is sufficient to induce the supercoiling observed in this experiment. Both H3 and H4 are required, and in the absence of either, no other histones are effective. H3 and and H4 are as efficient, per unit weight, as a mixture of the four histones in inducing supercoils. We also show that there is a large difference between the DNA bending energy needed to form a nucleosome and that needed to form one turn of normal superhelical DNA. These two processes are energetically quite distinct and probably separable. We estimate the free energy of interaction between DNA-bound histone pairs, and find that one or two such interactions would generate enough energy to fold the DNA into a nucleosome.

Arginine↗
PubMed1976

Chromatin structure as probed by nucleases and proteases: evidence for the central role of histones H3 and H4.

We have examined the role by each histone in forming the structure of the nu-body. When DNAase I, DNAase II, trypsin and chymotrypsin attack chromatin, characteristic discrete DNA and protein digest fragments are produced. Using this restriction of accessibility as diagnostic for chromatin structure, we have examined complexes of DNA with virtually all possible combinations of histones. The results strongly support our previous conclusion (Camerini-Otero, Sollner-Webb, and Felsenfeld, 1976) that the arginine-rich histones are unique in their ability to create, with DNA a structure with many features of native chromatin. Acting together, slightly lysine-rich histones then modify this complex into one very similar to native chromatin. An analysis of the rate constants of staphylococcal nuclease digestion also confirms that the complex of H3, H4, and DNA is crucial to the structure of the nu-body.

Animals↗
PubMed1976

The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel.

We have examined the role played by various histones in the organization of the DNA of the nucleosome, using staphylococcal nuclease as a probe of DNA conformation. When this enzyme attacks chromatin, a series of fragments evenly spaced at 10 base pair intervals is generated, reflecting the histone-DNA interactions within the nucleosome structure. To determine what contribution the various histones make to DNA organization, we have studied the staphylococcal nuclease digestion patterns of complexes of DNA with purified histones. Virtually all possible combinations of homogeneous histones were reconstituted onto DNA. Exhaustive digestion of a complex containing the four histones H2A, H2B,H3, and H4 yields a DNA fragment pattern very similar to that of whole chromatin. The only other combinations of histones capable of inducing chromatin-like DNA organization are H2A/H2B/H4 and those mixtures containing both H3 and H4. From an examination of the kinetics of digestion of H3/H4 reconstitutes, we conclude that although the other histones have a role in DNA organization within the nucleosome, the arginine-rich histone pair, H3/H4, can organize DNA segments the length of the nucleosome core in the absence of all other histones.

Animals↗
PubMed1975

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.

Animals↗
PubMed1974

Specific sites of interaction between histones and DNA in chromatin.

Staphylococcal nuclease digestion of purified chromatin from duck reticulocytes or calf thymus results in the production of a series of double-stranded DNA fragments of discrete molecular size, ranging from about 130 to 45 base pairs, which can be detected by polyacrylamide gel electrophoresis. Similar patterns of protected DNA fragments are obtained from limit digests of chromatin "reconstituted" from purified DNA and chromatin proteins. The results obtained with reconstituted material do not depend upon the origin of the DNA, which may be derived from a bacterial, viral, or homologous source. The specificity of the protective mechanism, therefore, resides in the structure of the bound histones, and probably not in any special nucleotide sequences present in the DNA. Removal of lysine-rich histones from chromatin before digestion results principally in disappearance from the digest of a DNA fragment about 130 base pairs long. Our preliminary results suggest that other elements of the digest pattern can be assigned uniquely to the remaining histone components. These results indicate that the binding of histones to DNA in chromatin involves a limited number of specific and very well defined contacts between protein and nucleic acid, which arise from structural properties of the histones.

Animals↗