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J Widom

Publications and source records attributed to J Widom.

At least 55 records · Page 3Linked to original sources
PubMed1993

Twist constraints on linker DNA in the 30-nm chromatin fiber: implications for nucleosome phasing.

Previous work has shown that nucleosome repeat lengths, and hence linker DNA lengths, are preferentially quantized to a set of values differing by integral multiples of the helical twist of DNA. An explanation was proposed in which this preferential quantitation is due to twist constraints on linker DNA arising from nucleosome-nucleosome interactions in folded chromatin. Here we report the results of a study, using ethidium intercalation, designed to test whether twist constraints do indeed exist. Electron microscopy reveals that ethidium intercalation causes decondensation of dinucleosomes. Direct measurement of the free energy of intercalation by fluorescence spectroscopy reveals competition between chromatin folding and ethidium intercalation. Results from other laboratories establish that these effects of ethidium are due to ethidium-induced changes in the twist of linker DNA, and not to a variety of other possible effects. We conclude that twist constraints on linker DNA do exist. These may explain the observation of preferentially quantized linker DNA lengths. Implications of these results for mechanisms of nucleosome phasing and the mechanisms of drug action are discussed.

Animals↗
PubMed1993

Lifetime of the histone octamer studied by continuous-flow quasielastic light scattering: test of a model for nucleosome transcription.

An instrument for continuous-flow quasielastic light scattering is described that allows the translational diffusion coefficient of macromolecules to be determined as a function of time after the initiation of some time-dependent process by mixing. Control experiments are carried out using the proteins lysozyme and BSA to verify that flow of the solution does not lead to erroneous results. The instrument is used to determine the lifetime of the histone octamer. A solution of octamer that is artificially stabilized in 2.0 M NaCl is rapidly diluted to physiological ionic strength, and the Stokes diameter is determined as a function of the time, delta t, after mixing. We find that the octamers dissociate into their component H2A-H2B heterodimers and H(3)2H4 tetramers on a time scale that is faster than the earliest time point for which data were obtained, 1 s after mixing. This result argues against a simple mechanism for the progression of RNA or DNA polymerase through chromatin.

Animals↗
PubMed1992

Chromatin structure of Schizosaccharomyces pombe. A nucleosome repeat length that is shorter than the chromatosomal DNA length.

We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. We obtain a result of 156(+/- 2) bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. We note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and Aspergillus nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding.

Chromatin↗
PubMed1992

Co-operative binding of the globular domain of histone H5 to DNA.

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.

Animals↗
PubMed1992

A relationship between the helical twist of DNA and the ordered positioning of nucleosomes in all eukaryotic cells.

A large number of measurements of nucleosome repeat lengths are analyzed and are found to exhibit preferential quantization to a set of values related by integral multiples of the helical twist of DNA. This implies that the nucleosomal DNA content is preferentially quantized, which in turn implies that linker DNA lengths are preferentially quantized. This study confirms and extends previous observations in the literature that had suggested, but not firmly established, that linker lengths might be quantized. The quantization of repeat lengths applies even for very long repeat lengths. This suggests a model for the origin of the quantization, in which the quantization arises from the requirements of higher-order chromatin structure.

DNA↗
PubMed1991

Linker DNA bending induced by the core histones of chromatin.

We have previously reported that ionic conditions that stabilize the folding of long chromatin into 30-nm filaments cause linker DNA to bend, bringing the two nucleosomes of a dinucleosome into contact [Yao, J., Lowary, P. T., & Widom, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7603-7607]. Dinucleosomes are studied because they allow the unambiguous detection of linker DNA bending through measurement of their nucleosome-nucleosome distance. Because of the large resistance of DNA to bending, the observed compaction must be facilitated by the histones. We have now tested the role of histone H1 (and its variant, H5) in this process. We find that dinucleosomes from which the H1 and H5 have been removed are able to compact to the same extent as native dinucleosomes; the transition is shifted to higher salt concentrations. We conclude that histone H1 is not essential for compacting the chromatin filament. However, H1 contributes to the free energy of compaction, and so it may select a single, ordered, compact state (the 30-nm filament, in long chromatin) from a family of compact states which are possible in its absence.

Animals↗
PubMed1990

Direct detection of linker DNA bending in defined-length oligomers of chromatin.

Linker DNA, which connects between nucleosomes in chromatin, is short and, therefore, may be essentially straight and inflexible. We have carried out hydrodynamic and electron microscopic studies of dinucleosomes--fragments of chromatin containing just two nucleosomes--to test the ability of linker DNA to bend. We find that ionic conditions that stabilize the folding of long chromatin cause linker DNA in dinucleosomes to bend, bringing the two nucleosomes into contact. The results uphold a key prediction of the solenoid model of chromosome folding and suggest a mechanism by which proteins that are separated along the DNA can interact by direct contact.

Animals↗
PubMed1989

Higher-order structure of Saccharomyces cerevisiae chromatin.

We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast "killer" virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.

Animals↗
PubMed1988

Effectiveness of natural and synthetic complexes of porin and O polysaccharide as vaccines against Brucella abortus in mice.

A single vaccination of mice with a complex of porin and smooth lipopolysaccharide (porin-S-LPS) extracted from virulent Brucella abortus 2308 provided significant protection (P less than 0.01 to P less than 0.001) against challenge with the same strain, equivalent to that achieved by vaccination with living attenuated B. abortus 19. The porin-S-LPS vaccine given without adjuvant or in several adjuvants (trehalose dimycolate and muramyl dipeptide; the pluronic polymer L-121 and muramyl dipeptide; or complexed with Quil A in immunostimulating complexes) provided equivalent protection. In contrast, one vaccination with porin complexed with rough LPS (porin-R-LPS) from a rough mutant of strain 2308 provided no protection with any adjuvant tested. In one experiment, two inoculations with the porin-R-LPS resulted in a low level of protection, probably owing to priming of the animals for production of O-polysaccharide-specific antibodies. However, one vaccination with rough-strain porin covalently bound to purified O polysaccharide conferred protection equal to that obtained with natural complexes of porin-S-LPS or with living strain 19. A synthetic vaccine containing long chains of O polysaccharide was more effective than one prepared with short chains. Protective vaccines caused the formation of increased concentrations of circulating O-polysaccharide-specific antibodies, although there were individual exceptions to the quantitative association between O-polysaccharide-specific antibodies and protection. Antibodies specific for porin or R-LPS were found in negligible quantities in vaccinated mice. These results provide additional evidence that the O polysaccharide will constitute an essential component of an effective subcellular vaccine against B. abortus and that O-polysaccharide-specific antibodies play an important role in protective immunity in brucellosis.

Adjuvants, Immunologic↗
PubMed1986

Physicochemical studies of the folding of the 100 A nucleosome filament into the 300 A filament. Cation dependence.

The cation-induced refolding of the 100 A nucleosome filament into the 300 A filament has been studied over a wide range of concentrations of Na+, Mg2+, Co(NH3)3+6 and other cations. X-ray diffraction, electron microscopy and analytical ultracentrifugation have been used to determine the conditions under which the 300 A filament is formed. It is shown that cations induce chromatin refolding by acting as general DNA counterions. The concentration of any cation required to induce refolding is greatly dependent on the valence of that cation. Na+ (and, presumably, other monovalent cations) has dual effects: at high concentrations (greater than 45 to 65 mM) it stabilizes the 300 A filament state of chromatin; however, at low concentrations (less than approximately equal to 45 mM), when cations of higher valence are present and stabilizing the 300 A filament state, Na+ has the opposite effect, competing with the higher-valence cation for binding to the chromatin and destabilizing the 300 A filament state. It is shown that further addition of cations to chromatin in the 300 A filament state causes a further folding of the chromatin in which the sedimentation coefficient increases and the X-ray diffraction bands resulting from nucleosomal packing sharpen. This may reflect subtle structural changes within the 300 A filament, or it may reflect a shift in equilibrium constant for chromatin fluctuating between the 100 A and 300 A filament states. It is also shown that, with continued addition of cation, the 300 A filaments precipitate before any "endpoint" is reached in this further folding. The tendency of 300 A filaments to aggregate in vitro appears to be a built-in property, and may reflect the packing of 300 A filaments within metaphase chromosomes in vivo.

Animals↗
PubMed1985

Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.

Animals↗
PubMed1985

Structure of the 300A chromatin filament: X-ray diffraction from oriented samples.

X-ray diffraction patterns have been obtained from partially oriented samples of 300A chromatin filaments. The chromatin was prepared by methods that preserve its structure, and conditions were found in which the 300A filaments spontaneously form ordered aggregates, so that it was not necessary to pull fibers. The diffraction patterns show a meridional band at 110A, and equatorial bands at 340, 57, 37, and 27A. These patterns, together with patterns calculated from the known 7A electron density map of the nucleosome core particle, imply side-to-side packing of nucleosomes in the direction of the 300A filament, and radial packing around it. These results are consistent with the "solenoid" model of Finch and Klug, and are inconsistent with many other proposed models.

Animals↗
PubMed1983

Tests of spool models for DNA packaging in phage lambda.

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on lambda DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982). A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into lambda phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗
PubMed1981

3,4-Dihydroxyphenylacetate 2,3-dioxygenase. A manganese(II) dioxygenase from Bacillus brevis.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase, an enzyme which catalyzes the extradiol cleavage of catechols, has been purified from Bacillus brevis. Like other extradiol-cleaving dioxygenases, this enzyme has a molecular weight of 140,000 with four subunits of 36,000 each. Unlike the other enzymes, this dioxygenase is not activated by added ferrous ion, not inhibited by cyanide or diethyldithiocarbamate, and not inactivated by H2O2. X-ray fluorescence and atomic absorption analyses show the enzyme to contain approximately 2 g atoms of manganese per mol of protein. EPR spectra are consistent with a manganese(II) center in an environment of low symmetry. This is the first report of an oxygen-activating manganese enzyme.

3,4-Dihydroxyphenylacetic Acid↗