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J Zlatanova

Publications and source records attributed to J Zlatanova.

At least 55 records · Page 3Linked to original sources
PubMed1994

Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy.

Unfixed chicken erythrocyte chromatin fibers in very low salt have been imaged with a scanning force microscope operating in the tapping mode in air at ambient humidity. These images reveal a three-dimensional organization of the fibers. The planar "zig-zag" conformation is rare, and extended "beads-on-a-string" fibers are seen only in chromatin depleted of histones H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies, suggest that chromatin fibers may exist as irregular three-dimensional arrays of nucleosomes even at low ionic strength.

Animals↗
PubMed1994

Competition between linker histones and HMG1 for binding to four-way junction DNA: implications for transcription.

Both lysine-rich histones and high mobility group proteins 1 and 2 bind to linker DNA in chromatin. While the members of the histone H1 class are considered general repressors of transcription, HMG1/2 are viewed as activators. Using stable four-way junction DNA as a model for cross-overs of linker DNA at the entry and exit to nucleosomes, we show that HMG1 can compete efficiently with H1 for binding to four-way junctions. In contrast, the erythrocyte-specific histone H5 seems to be refractory to displacement by HMG1. The results suggest that replacement of histone H1 by HMG1 may be part of the transcriptional activation by HMG1.

Animals↗
PubMed1994

Linker DNA accessibility in chromatin fibers of different conformations: a reevaluation.

New studies on chromatin fiber morphology, using the technique of scanning force microscopy (SFM), have caused us to reexamine recent analysis of nuclease digestion of chromatin. Chicken erythrocyte chromatin fibers, glutaraldehyde-fixed at 0, 10, and 80 mM NaCl, were imaged with the help of SFM. The chromatin fibers possessed a loose three-dimensional 30-nm structure even in the absence of added salt. This structure slightly condensed upon addition of 10 mM NaCl, and highly compacted, irregularly segmented fibers were observed at 80 mM NaCl. This sheds new light upon our previously reported analysis of the kinetics of digestion by soluble and membrane-immobilized micrococcal nuclease [Leuba, S. H., Zlatanova, J. & van Holde, K. (1994) J. Mol. Biol. 235, 871-880]. While the low-ionic-strength fibers were readily digested, the highly compacted structure formed at 80 mM NaCl was refractory to nuclease attack, implying that the linkers were fully accessible in the low-ionic-strength conformation but not in the condensed fibers. We now find that cleavage of the linker DNA by a small molecule, methidiumpropyl-EDTA-Fe(II), proceeds for all types of conformations at similar rates. Thus, steric hindrance is responsible for the lack of accessibility to micrococcal nuclease in the condensed fiber. Taken in total the data suggest that reexamination of existing models of chromatin conformation is warranted.

Animals↗
PubMed1994

Binding of histones H1 and H5 and their globular domains to four-way junction DNA.

We have compared chicken erythrocyte linker histones H1 and H5 binding to a synthetic four-way DNA junction. Each histone binds to form a single complex, with an affinity which permits competition against a large excess of linear duplex DNA. The affinity of H5 is higher than that of H1. The globular domain from either protein will also bind strongly, but in this case multiple binding occurs. Binding of intact H1 is inhibited by cations: Mg2+ and spermidine are very effective, Na+ much less so. This inhibition is not likely to be a general ion-competition effect, for Mg2+ is much less effective in inhibiting the binding of H1 to linear DNA. Instead, the inhibition of binding may be due to ion-dependent changes in the conformation of the four-way junction, which are known to occur under similar conditions. These results strongly suggest that the angle formed between the arms of the DNA junction could be a major determinant in the interaction of H1 with DNA crossovers.

Animals↗
PubMed1994

On the location of linker DNA in the chromatin fiber. Studies with immobilized and soluble micrococcal nuclease.

The structure of chicken erythrocyte chromatin fibers has been probed using micrococcal nuclease, both membrane-immobilized and free in solution. Under the extremely mild digestion conditions used, the linker DNA is almost completely protected against digestion with either immobilized or free enzyme in the 30 nm fibers, whereas it is readily accessible in the more extended structures. Control experiments with glutaraldehyde-fixed chromatin fibers gave essentially the same results. Experiments with fibers of intermediate degree of condensation revealed a direct relationship between the degree of compaction and the resistance of linker DNA to digestion. Our results favor models in which access to the linkers is limited by local steric hindrance due to the high compaction, rather than by internalization in the center of the fibers.

Animals↗
PubMed1994

Unusual DNA structures, chromatin and transcription.

Extensive studies of DNA secondary structure during the past decade have shown that DNA is a dynamic molecule, whose structure depends on the underlying nucleotide sequence and is influenced by the environment and the overall DNA topology. Three major non-B-DNA structures have been described (Z-DNA, triplex DNA and cruciform DNA) which are stabilized by unconstrained negative supercoiling and can be formed under physiological conditions. In this essay we summarize the DNA primary structure features that are pertinent to the formation of these conformers and present data concerning the occurrence of these sequences in the eukaryotic genome. The evidence in favor of the existence of these unusual DNA structures in vivo is discussed. The effect of alternative non-B-DNA structures on the way DNA is organized in chromatin is considered, and this is followed by evaluation of the data relating these structures to eukaryotic transcription. Some possible mechanisms by which the effect of non-B structures on transcription might be exerted are proposed.

Animals↗
PubMed1994

Histone H1 zero: a major player in cell differentiation?

Histone H1 zero was initially described as a member of the lysine-rich histone class, typically present in nondividing mammalian cells. Since then it has been found in almost every animal or plant species studied. The protein accumulates in terminally differentiated cells that have stopped dividing. It has also been implicated in changes in chromatin structure and function accompanying malignant transformation. Despite its involvement in these fundamental cellular processes, its precise role remains elusive, as do the molecular mechanisms via which it acts. This review is an attempt to summarize and critically discuss the huge relevant literature, trying to highlight the problems that still await answers.

Animals↗
PubMed1994

A spot test for protein detection and semiquantitative estimation in small samples.

Modern biological experiments, conducted on mini- and micro-scales require methods for protein detection in small samples. The method proposed here is based on visual inspection of protein dots immobilized on nitrocellulose filters and stained with the common protein dye amido black. It allows detection and semiquantitative determination of protein concentrations in a wide range.

Amido Black↗
PubMed1993

Preferential binding of histone H1 to four-way helical junction DNA.

Histone H1 is a major chromatin protein, which stabilizes the nucleosome, has an essential role in organizing nucleosomes into higher order structures, and may have a role as a repressor of transcription (van Holde, K. E. (1989) Chromatin, Springer Publishing Co., New York). Here we show that H1 forms a defined complex with a synthetic four-way junction of DNA strands even in the presence of an excess of linear nonspecific competitor DNA. The four-way junction also competes efficiently against two duplex DNA molecules, which together have the same sequence information as the four-way junction molecule. Another major chromatin protein, high mobility group protein 1, also binds four-way junction structures specifically (Bianchi, M. E., Beltrame, M., and Paonessa, G. (1989) Science 243, 1056-1059), and this similar behavior may indicate a related function of these proteins. Our finding may suggest that four-way DNA junction is a structural equivalent to the main H1 binding site in the nucleosome: a crossover of double helical DNA at the point where the DNA enters and exits the nucleosome (Allan, J., Hartman, P. G., Crane-Robinson, C., and Aviles, F. X. (1980) Nature 288, 675-679).

DNA↗
PubMed1993

Histones H1 and H5 interact preferentially with crossovers of double-helical DNA.

The interaction of the linker histones H1 and H5 from chicken erythrocyte chromatin with pBR322 was studied as a function of the number of superhelical turns in circular plasmid molecules. Supercoiled plasmid DNA was relaxed with topoisomerase I so that a population with a narrow distribution of topoisomers, containing from zero to five superhelical turns, was obtained. None of the topoisomers contained alternative non-B-DNA structures. Histone-DNA complexes formed at either 25 or 100 mM NaCl final concentration and at histone-DNA molar ratios ranging from 10 to 150 were analyzed by agarose gel electrophoresis. The patterns of disappearance of individual topoisomer bands from the gel were interpreted as an indication of preference of the linker histones for crossovers of double-helical DNA. This preference was observed at both salt concentrations, being more pronounced under conditions of low ionic strength. Isolated H5 globular domain also caused selective disappearance of topoisomers from the gel, but it did so only at very high peptide-DNA molar ratios. The observed preference of the linker histones for crossovers of double-helical DNA is viewed as a part of the mechanism involved in the sealing of the two turns of DNA around the histone octamer.

Animals↗
PubMed1993

Histone H1 deposition and histone-DNA interactions in replicating chromatin.

An immunochemical method for analyzing protein interactions with BrdUrd-substituted DNA was used to study binding of histones to nascent DNA in nuclei. The results indicate that in Ehrlich ascites tumor (EAT) cells, histone H1 deposits on newly replicated DNA simultaneously with or immediately after core histone deposition so that in chromatin replicated for 3 min, the stoichiometry of the histones is the same as in bulk chromatin. All histones, and especially histone H1, interact with nascent DNA more weakly than with bulk chromatin, although the efficiency of interaction via the globular domains seems to be the same for both types of chromatin.

Animals↗
PubMed1993

On the location of histones H1 and H5 in the chromatin fiber. Studies with immobilized trypsin and chymotrypsin.

The location of linker histones H1 and H5 in chicken erythrocyte chromatin was studied as a function of the fiber structure by the use of proteolytic enzymes immobilized onto Immobilon membranes. The immobilization of trypsin and chymotrypsin creates proteolytic probes, specific respectively to the terminal portions of the molecules or to the phenylalanine in the globular domain, that are incapable of penetrating into the interior of the condensed fiber. The chromatin fiber was studied in three different conformations: open zig-zag (in Tris buffer), closed zig-zag (upon addition of 10 mM-NaCl), or 30 nm fiber (upon addition of 0.35 mM-MgCl2). The results from digestion experiments performed on linker histones either in chicken erythrocyte chromatin, or free in solution or bound in mononucleosomes revealed several features relevant to linker histone location: (1) histone H5 is more protected than histone H1 in the fiber; (2) the N and C-terminal portions of histone H1 do not change their accessibility, and hence their location, upon compaction of the fiber; this behavior of H1 is in contrast to that of histone H5, whose tails become significantly internalized in the 30 nm fiber; (3) phenylalanine in the globular domain of both H1 and H5 is inaccessible (buried) both in the fiber and in the mononucleosomal particle. Sedimentation velocity measurements performed during the course of trypsin digestion demonstrate that the conformation of the fiber is highly sensitive to even a few cuts in some of the linker histone molecules; hence, the linker histones are an important factor in the organization of the fiber in all its different condensation states.

Animals↗
PubMed1992

An indirect-labeling procedure for the study of specific interactions of proteins with DNA.

A semiquantitative and reproducible indirect-labeling procedure for the study of specific protein/DNA interactions using nitrocellulose-filter-immobilized proteins and linear or superhelical DNA molecules is reported. Proteins were immobilized on nitrocellulose filters either by direct dotting or by electrotransfer from polyacrylamide electrophoretic gels. After incubation with the respective DNA (linear restriction fragments or closed circular recombinant DNA plasmids) the paper strips were washed, and specifically bound DNA was denatured by alkali and detected by hybridization with 32P-nick-translated DNA. The quantitation of the reaction was performed by scanning of the autoradiograms of the radioactive spots and determination of the area under the respective peaks. The intensity of the radioactive spots was proportional to the amount of protein present in the dot. The sensitivity of the assay depends primarily on the affinity of the respective DNA to the protein and in the case of mouse liver histone H1AB/mouse alpha-globin gene approximately 50 ng of protein per dot was enough for determination.

Animals↗
PubMed1992

Histone H1 interacts specifically with certain regions of the mouse alpha-globin gene.

We used fragments of a cloned mouse alpha-globin gene to determine if histone H1 interacts selectively with defined regions of a eukaryotic gene. The use of intact plasmids instead of isolated fragments permitted study of relevant sequences in their superhelical form. Several independent experimental approaches (filter binding, precipitation, binding to protein immobilized on nitrocellulose membranes, and agarose gel electrophoresis of the protein-DNA complexes) were used and the histone-DNA interaction was investigated under both noncompetitive and competitive conditions. Binding to subclones encompassing the 5' end of the gene and the first half of the coding sequence is preferred over binding to other subclones. The expression of the sequence-specific selectivity depends on the ionic strength of the binding reaction; the selectivity is mainly expressed under conditions of non-cooperative binding of the histone to DNA. No correlation is observed between AT content and relative affinity of binding to H1. Evidently, other features of DNA structure are involved in the specific H1 binding.

Animals↗
PubMed1991

Purification of histone H10 and its subfractions under non-denaturing conditions.

A quick and convenient method for a non-denaturing purification of histone H10 and its subfractions from different mammalian sources is presented. It is based on cation-exchange chromatography on a CM-Sephadex C-25 column developed by a linear gradient of NaCl. The procedure allows a satisfactory fractionation of H10 subfractions in a form suitable for further studies in assays requiring the native state of the protein.

Animals↗
PubMed1991

DNA sequence specific interactions of histone H1.

Histone H1 is know to play a role in the formation and maintenance of higher-order chromatin structure. It has been recently suggested that the linker histone might be also involved in the regulation of the activity of individual genes. If H1 is a regulatory factor in eukaryotic gene transcription, it should possess specificity of binding to defined DNA sequences. This review is an attempt to summarize and discuss the existing literature data on DNA sequence-specific interactions of histone H1.

Animals↗