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G Felsenfeld

Publications and source records attributed to G Felsenfeld.

At least 109 records · Page 6Linked to original sources
PubMed1982

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II.

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.

Animals↗
PubMed1982

The high mobility group proteins HMG 14 and 17, do not prevent the formation of chromatin higher order structure.

The high mobility group proteins, HMG 14 and 17, have been associated with the chromatin of active genes (refs 1-8), although how they function is not known. We use sedimentation and electric dichroism to investigate the effect of HMG 14 and 17 on the condensation of chicken erythrocyte chromatin into higher order structure. We find no evidence that excess HMG 14 and 17 induce an extended configuration, either in bulk chromatin or in the chromatin of the chicken beta-globulin gene.

Animals↗
PubMed1982

Effect of the B--Z transition in poly(dG-m5dC) . poly(dG-m5dC) on nucleosome formation.

We have studied the properties of complexes formed between histones and the methylated synthetic polydeoxynucleotide poly(dG-m5dC). poly(dG-m5dC). This polymer undergoes the transition from B DNA to left-handed Z DNA at moderate ionic strength. When the polymer is in the Z form it will bind histones, but nucleosomes are not detected. When the polymer in the B form is combined with equimolar quantities of the four core histones and digested with micrococcal nuclease, particles are formed which behave in all respects as normal nucleosome cores. When these core particles are placed in solvents that would result in conversion of the protein-free polymer to the Z form, no transition is observed. The formation of a nucleosome core particle thus stabilizes the B form, whereas the presence of the Z form prevents nucleosome formation. The results suggest that if Z DNA is present in eukaryotic nuclei, it will serve to disrupt the normal chromatin structure.

DNA↗
PubMed1981

Repeated sequence organization and RNA transcription map of the chicken adult beta-globin gene region.

We have used adaptations of the contact hybridization method to determine the repeated sequence map of a cloned fragment of chicken genomic DNA and to establish the map of RNA transcription in vivo from this same fragment. The cloned 6.2-kilobase pair (kbp) Eco RI fragment contains the adult beta-globin gene and a portion of the embryonic epsilon-globin gene. The technique of contact hybridization allows the rapid determination of DNA reiteration frequency or RNA abundance in a single experiment. The DNA sequence analysis reveals repeated sequence spanning about 1.5 kbp, located about 1 kbp to the 3'-side of the adult gene and immediately 5' of the embryonic gene. Examination of the in vivo RNA transcript by these methods reveals the presence of RNA homologous to both the adult beta-globin gene and to the repeated sequence region. In reticulocyte cytoplasm, these RNAs were of about equal abundance; in reticulocyte nuclei, adult globin RNA was found but the repeated sequence RNA was not detected. In erythrocyte cytoplasm, the abundance of the repeated sequence RNA was much greater than that of the globin RNA.

Animals↗
PubMed1981

A 200 base pair region at the 5' end of the chicken adult beta-globin gene is accessible to nuclease digestion.

A DNA segment close to the 5' end of the chicken adult beta-globin gene contains a hypersensitive site for nuclease action. Using a variety of endonucleases, we show that this hypersensitive site is in fact an accessible region, which extends from approximately 60 to approximately 260 base pairs 5' from the start of mRNA transcription. The presence of this hypersensitive region is correlated with gene activity; it is observed in nuclei isolated from the definitive erythrocytes of 14-day chicken embryos, but is not observed in nuclei isolated from the primitive erythrocytes of 5-day embryos (which do not express this gene) or in brain nuclei or purified DNA. The close spacing of accessible digestion sites on this DNA segment is not consistent with the presence of a normal nucleosome. Furthermore, a 115 base pair DNA fragment contained within this nuclease-sensitive region can be excised by Msp I digestion and released from nuclei, in at least 50% yield. Approximately one third of the released fragments behave as protein-free DNA.

Animals↗
PubMed1981

Effects of methylation on a synthetic polynucleotide: the B--Z transition in poly(dG-m5dC).poly(dG-m5dC).

We have compared the behavior in solution of the synthetic polynucleotide poly(dG-m5dC).poly(dG-m5dC) with that of the unmethylated polynucleotide poly(dG-dC).poly(dG-dC). In solutions containing high concentrations of salt, poly(dG-dC).poly(dG-dC) is known to exhibit altered circular dichroic and absorption spectra correlated with formation of a left-handed Z DNA structure. Poly(dG-m5dC).poly(dG-m5dC) behaves similarly, but the spectral transition from the B to the Z form occurs at much lower salt concentrations, close to usual physiological conditions. Divalent and polyvalent ions are particularly effective: The B--Z transition of poly(dG-m5dC).(dG-m5dC) can be induced at a Mg2+ concentration three orders of magnitude lower than that required for the unmethylated polymer. We have also studied mixed copolymers containing both dC and m5dC. Our results suggest that the sequence m5dC-dG, which occurs in eukaryotic DNA, can have a disproportionately large effect on the B--Z transition.

Cations↗
PubMed1980

The interaction of high mobility proteins HMG14 and 17 with nucleosomes.

The interaction of the high mobility group proteins, HMG14 and HMG17, with nucleosome core particles has been studied. The results show that two molecules of HMG14/17 can be bound tightly but reversibly to each core particle and that their affinity for core particles is greater than their affinity for histone-free DNA of core size. Thermal denaturation and nuclease digestion studies suggest that major sites of interaction are located near the ends of the nucleosome core DNA. When nucleosome preparations from chicken erythrocyte nuclei stripped of HMG proteins are partially titrated with HMG14/17, the nucleosome-HMG complex fraction is enriched in beta-globin gene sequences.

Animals↗
PubMed1980

The number of charge-charge interactions stabilizing the ends of nucleosome DNA.

It has been shown by others that the melting of DNA in the nucleosome core particle is biphasic (ref. 1) and that the initial denaturation phase is due to melting of the DNA termini (refs. 1 & 2). We analyze the salt dependence of the melting temperature of this first transition and estimate that only 15% of the phosphates of the DNA termini are involved in intimate charge-charge interactions with histones. (The simplest model yields approximately 9%, whereas a calculated overestimate yields approximately 21% neutralization.) This is a surprisingly small number of interactions but we suggest that it may nonetheless be representative of all the core particle DNA.

Animals↗
PubMed1980

Orientation of the nucleosome within the higher order structure of chromatin.

We have used electric dichroism to investigate chromatin fragments isolated from chicken erythrocytes. Both within the extended "10 nm filament" present at low salt and the condensed "30 nm solenoid" induced by the addition of divalent cations, the data give a quantitative description of the DNA conformation in the higher order structure of chromatin. In the 10 nm filament both the spacer DNA and the flat faces of the core particle discs must be oriented within 20 degrees of the fiber axis. Within the Mg++-induced 30 nm solenoid the flat faces of the core particles must also be oriented close to parallel to the solenoid axis, the estimated angle depending upon the disposition of the spacer DNA. We suggest a model for the 30 nm solenoid.

Animals↗
PubMed1980

Nucleosome structure and conformational changes.

We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests (as did earlier probe experiments) that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5-0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface.

Animals↗
PubMed1979

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.

Animals↗
PubMed1979

Reaction of nucleosome DNA with dimethyl sulfate.

We have measured the effect of the histones in the nucleosome core particle on methylation of purines in nucleosome DNA by dimethyl sulfate. By using 32P terminally labeled nucleosome cores, we have examined the pattern of strand cleavage at methylated sites in the nucleosome DNA and compared it to the pattern observed in histone-free DNA. We are unable to detect any significant difference between the reactivity of N7 of guanines in nucleosome DNA and of that in naked DNA, with the exception of a single site of enhanced reactivity at approximately nucleotide 62 from the 5' end of the nucleosome. Contrary to our expectation, there is no detectable periodic modulation of reactivity corresponding to the twist of the DNA on the nucleosome surface. We are able to place a low upper limit on the extent to which the histones of the nucleosome can protect N7 of guanine in the large groove. With somewhat less precision, we also conclude that the N3 of adenine in the small groove is largely unprotected. These results indicate that in nucleosome DNA the bases are nearly as accessible to solvent as they are in DNA free of protein.

Adenine↗
PubMed1978

Transcription of histone-covered T7 DNA by Escherichia coli RNA polymerase.

Purified core histones (H2A, H2B, H3, and H4) and bacteriophage T7 DNA have been reconstituted to form a nucleoprotein complex, and the properties of this complex as a template for transcription by Escherichia coli RNA polymerase have been studied. At low ionic strength, RNA chain elongation rates are slow, and the chains produced even after long incubation are short. At higher salt concentrations, chain-elongation rates approach those on naked DNA. Since the salt concentrations used are not in themselves sufficient to dissociate the histones from the DNA, some mechanism must exist that permits passage of the polymerase through histone-covered regions.

Chromatin↗