On the structure of cellular and viral chromatin.
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Animals↗
Biomedical subjects
V V Bakayev
Publications and source records attributed to V V Bakayev.
Chromosome of the mature virion of simian virus 40 contains H1 histone.
In addition to free SV40 minichromosomes in the compact form, complete virions were obtained from the nuclear extract of productively infected cells. Capsid proteins VP1, VP2, and VP3, as well as histones, were observed on electrophoregrams of proteins prepared from virions. In contrast to the widely accepted view, histone H1 was found in virions in stoichiometric amounts with respect to other histones. The same is true for virions isolated by a conventional method. Free minichromosomes present in infected cells contain all histones and practically no viral proteins.
Chromatin↗
On the structure of eukaryotic, prokaryotic, and viral chromatin.
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Animals↗
Nucleosomes and subnucleosomes: heterogeneity and composition.
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Cell Line↗
Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure.
We report two new findings bearing on the "supranucleo-somal" level of the structure of the Simian Virus 40 minichromosome. I) Isolated SV40 minichromosome which contains all five histones including HI/I/ exists in solution under approximately physiological ionic conditions as a compact roughly spherical particle approximately 300 A in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiological ionic strength of solution (micron approximately 0.15). Removal of HI results in a conversion of the compact minichromosomes into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contract to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nuclease-sensitive parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter-and intranucleosomal DNA in the chronatin /2,3/ readily digests the compact form of the SV40 minichromosome. Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.
Centrifugation, Density Gradient↗
Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein.
Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images
Chromosomes, Bacterial↗
Studies on chromatin. Free DNA in sheared chromatin.
Chromatin which has been hydrodynamically sheared in a low-ionic-strength buffer lacking divalent cations (I = 0.0005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1--2 mM MgCl2 or to 0.1--0.2 M NaCl results in appearance of completely free DNA molecules. A salt-induced rearrangement of DNA-bound histones but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining deoxyribonucleoprotein particles. The percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8-9% in 1 mM MgCl2 and further to approximately 25% of the total DNA in 0.15 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde-fixed deoxyribonucleoproteins in CsCl gradients but also in non-ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium.
Animals↗
Heterogeneity of chromatin subunits in vitro and location of histone H1.
Chromatin subunits ("nucleosomes") which were purified by sucrose gradient centrifugation of a staphylococcal nuclease digest of chromatin have been studied. We found that such a preparation contains nucleosomes of two discrete types which can be separated from each other by polyacrylamide gel electrophoresis. Nucleosome of the first type contains all five histones and a DNA segment of approximately 200 base pairs long, whereas nucleosome of the second type lacks histone H1 and its DNA segment is approximately 170 base pairs long, i.e., about 30 base pairs shorter than the DNA segment of the nucleosome of the first type. Purified dimer of the nucleosome also can be fractionated by gel electrophoresis into three discrete bands which correspond to dinucleosomes containing two molecules of histone H1, one and no H1. These and related findings strongly suggest that the H1 molecule is bound to a short (approximately 30 base pairs) terminal stretch of the nucleosomal DNA segment which can be removed by nuclease (possibly in the form of H1-DNA complex) without any significant disturbance of main structural features of the nucleosome.
Animals↗
Minichromosome of simian virus 40: presence of histone HI.
In contrast to conclusions of previous studies /I-3/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. In addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.
Cell Fractionation↗
Studies on chromatin. III. v-Bodies and free DNA in chromatin lacking histone H1.
Chromatin lacking histone H1 was found by electron microscopy to contain 'beaded' deoxyribonucleoprotein fibers. Adjacent beads are connected with each other by threads having a DNA-like appearance. At least some of threads are shown to be free DNA stretches. Average length and the content of free DNA stretches in histone H1-depleted chromatin depends on the ionic conditions of the medium. The appearance of individual beads is similar to that of chromatin subunits or v-bodies [1] in the original chromatin. Thus, in agreement with the X-ray data [2], histone H1 apparently is not required for maintenance of a compact state of DNA in chromatin subunits.
Animals↗
Studies on chromatin. IV. Evidence for a toroidal shape of chromatin subunits.
Electron microscopy of purified chromatin subunits (v-bodies [17] or nucleosomes [2] revealed a hole or at least a deep indentation in the globular nucleosome. A hole in the nucleosome was visualized using rotatory shadowing with platinum-palladium or more directly, by negative staining with sodium phosphotungstate. The diameter of the hole as measured from negatively stained samples is 10-25 A. The external diameter of the negatively stained nucleosome equals 75 +/- 15 A. Although most of the data are formally compatible with either a hole or a deep indentation in the nucleosome, some views of the particles in the negatively stained samples suggest a hole rather than an indentation. The possible significance of a toroidal structure of the chromatin subunit is discussed in the accompanying paper [3].
Animals↗
Studies on chromatin. II. Isolation and characterization of chromatin subunits.
Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described.
Animals↗