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V L Makarov

Publications and source records attributed to V L Makarov.

At least 19 recordsLinked to original sources
PubMed2004

Applications of combined DNA microarray and chromosome sorting technologies.

The sequencing of the human genome has led to the availability of an extensive mapped clone resource that is ideal for the construction of DNA microarrays. These genomic clone microarrays have largely been used for comparative genomic hybridisation studies of tumours to enable accurate measurement of copy number changes (array-CGH) at increased resolution. We have utilised these microarrays as the target for chromosome painting and reverse chromosome painting to provide a similar improvement in analysis resolution for these studies in a process we have termed array painting. In array painting, chromosomes are flow sorted, fluorescently labelled and hybridised to the microarray. The complete composition and the breakpoints of aberrant chromosomes can be analysed at high resolution in this way with a considerable reduction in time, effort and cytogenetic expertise required for conventional analysis using fluorescence in situ hybridisation. In a similar way, the resolution of cross-species chromosome painting can be improved and we present preliminary observations of the organisation of homologous DNA blocks between the white cheeked gibbon chromosome 14 and human chromosomes 2 and 17.

Cell Line↗
PubMed1999

Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266 nm laser.

We developed appropriate conditions to use a laser with 60 femtosecond pulses, a frequency of 1 KHz and a wavelength of 266 nm to efficiently crosslink proteins to DNA in human nuclei for the purpose of using immunoprecipitation to study the binding of specific proteins to specific sequences of DNA under native conditions. Irradiation of nuclei for 30 min with 1-3 GW/cm(2)pulses crosslinked 10-12% of total protein to DNA. The efficiency of crosslinking was dose and protein specific. Histones H1 and H3 were crosslinked by 15 min of irradiation with 20-25% efficiency, at least 10 times more strongly than the other histones, consistent with experiments using conventional UV light. Irradiation for 15 min did not damage proteins, as assayed by SDS-PAGE of Ku-70 and histones. Although the same level of irradiation did not cause double-strand breaks, it did make the DNA partially insensitive to Eco RI restriction enzyme, probably through formation of thymidine dimers. Immuno-analysis of crosslinked nucleoprotein showed that Ku crosslinking to nuclear DNA is detectable only in the presence of breaks in the DNA, and that nucleosomes are bound to a significant fraction of the telomeric repeat (TTAGGG) (n).

Antigens, Nuclear↗
PubMed1997

Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening.

The chromosomes of lower eukaryotes have short telomeric 3' extensions. Using a primer-extension/nick-translation technique and nondenaturing hybridization, we find long 3' G-rich tails at human chromosome ends in mortal primary fibroblasts, umbilical vein endothelial cells, and leukocytes, as well as in immortalized fibroblasts. For all cells tested, >80% of the telomeres have long G-rich overhangs, averaging 130-210 bases in length, in disagreement with the conventional model for incomplete lagging-strand replication, which predicts overhangs on 50% of the chromosome ends. The observed G tails must exist during most of the cell cycle and probably result from degradation of both chromosome ends. The average lengths of the G tails are quantitatively consistent with the observed rates of human chromosome shortening.

Alkalies↗
PubMed1996

Condensation of rat telomere-specific nucleosomal arrays containing unusually short DNA repeats and histone H1.

Vertebrate telomeres contain arrays of nucleosomes with unusually short and regular repeat lengths (Makarov, V. L., Lejnine, S., Bedoyan, J., and Langmore, J. P.(1993) Cell 73, 775-787; Lejnine, S., Makarov, V., and Langmore, J. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2393-2397). In order to better define the specific structural features of telomere chromatin, we examined the condensation and H1 content of telomere nucleoproteins from rat liver. Velocity sedimentation analysis shows that telomeric nucleosome arrays condense with increasing ionic strength and molecular weight in a manner comparable with that of bulk chromatin despite the very short repeat length. However, these condensed structures do not exhibit the approximately 100-base pair deoxyribonuclease II repeat characteristic of condensed bulk chromatin. Frictional coefficient calculations suggest that telomere-specific higher order structure is more compact than bulk chromatin. Nucleoprotein gel electrophoresis shows that telomeric dinucleosomes from soluble chromatin contain H1. Finally, direct isolation and analysis of telomere nucleoproteins from formaldehyde-cross-linked nuclei indicate the presence of core histone proteins and H1. These results are consistent with the view that a major fraction of the long telomeres of rat are organized as specialized nucleosome arrays with features similar but not identical to those of bulk chromatin.

Animals↗
PubMed1995

Conserved nucleoprotein structure at the ends of vertebrate and invertebrate chromosomes.

Eukaryotic chromosomes terminate with telomeres, nucleoprotein structures that are essential for chromosome stability. Vertebrate telomeres consist of terminal DNA tracts of sequence (TTAGGG)n, which in rat are predominantly organized into nucleosomes regularly spaced by 157 bp. To test the hypothesis that telomeres of other animals have nucleosomes, we compared telomeres from eight vertebrate tissues and cell cultures, as well as two tissues from an invertebrate. All telomeres have substantial tracts of (TTAGGG)n comprising 0.01-0.2% of the genome. All telomeres are long (20-100 kb), except for those of sea urchin, human, and some chicken chromosomes, which are 3-10 kb in length. All of the animal telomeres contained nucleosome arrays, consistent with the original hypothesis. The telomere repeat lengths vary from 151 to 205 bp, seemingly uncorrelated with telomere size, regularity of nucleosome spacing, species, or state of differentiation but surprisingly correlated with the repeat of bulk chromatin within the same cells. The telomere nucleosomes were consistently approximately 40 bp smaller than bulk nucleosomes. Thus, animal telomeres have highly conserved sequences and unusually short nucleosomes with cell-specific structure.

Animals↗
PubMed1995

Increased Na+,K(+)-pump activity in erythrocytes of rabbits fed cholesterol.

Na+,L(+)-pump activity, intracellular sodium, potassium and magnesium concentrations and membrane cholesterol content were studied in erythrocytes of rabbits fed cholesterol. The average activity of the Na+,K(+)-pump in erythrocytes of rabbits with high plasma cholesterol was twice that in erythrocytes of control animals. Analysis showed a positive correlation between the pump activity and plasma cholesterol. The sodium content in erythrocytes correlated negatively with plasma cholesterol, as well as with the Na+,K(+)-pump activity. No significant differences in potassium and magnesium concentrations or in the membrane cholesterol content were observed between the two groups. The results indicate that modulation of the pump activity by cholesterol is not necessarily mediated by changes in the membrane viscosity.

Animals↗
PubMed1995

[Modulation of Na+,K+-pump activity and Na+,Li+-exchange in rabbit erythrocytes at various stages of cholesterol feeding].

The activities of the Na+,K(+)-pump and the Na+/Li(+)-exchange and the intracellular concentration of Na+ in erythrocytes of rabbits with experimental hypercholesterolemia have been studied at different stages of cholesterol (Ch) feeding as well as at different intervals after stopping of cholesterol-rich diets. After sufficiently long periods of feeding the animals with Ch (1.5-2 months), a significant activation of the Na+,K(+)-pump, reduction of the Na+/Li(+)-exchange rate and lowering of the intracellular sodium content in erythrocytes of rabbits from experimental groups were observed. Several months after stopping Ch-rich diets there was a partial normalization of the parameters under study. However, at the initial stage of the experiment (20 days after the beginning of Ch feeding) as well as two weeks after Ch feeding had been stopped, there occurred no changes in the activities of the ion transport systems or in [Na+]i despite substantial changes in the plasma Ch level. It is suggested that the observed effect of dietary Ch on the activity of ion carriers may be mediated by affecting the expression of the corresponding genes.

Animals↗
PubMed1994

[Transport of monovalent cations into erythrocytes of rabbits with experimental hypercholesterolemia: correlation with plasma cholesterol].

The activities of the Na+, K(+)-pump, Na+, K+, 2Cl- and K+, Cl(-)-cotransports and Na+, Li+ exchange as well as intracellular concentrations of Na+, K+, Mg2+ and cholesterol content in erythrocyte membranes of rabbits with experimental hypercholesterolemia have been studied. The activity of the Na+, K(+)-pump recorded as the ouabain-inhibited component of 86Rb influx is, on the average, by 100% higher than that in control erythrocytes of rabbits fed on a cholesterol-rich diet for 2 months and correlates significantly with the concentration of cholesterol (Ch) and low density lipoproteins (LDL) in the plasma as well as with Na+ concentration in erythrocytes. The activity of the Na+, K+, 2Cl- and K+, Cl(-)-cotransports recorded, correspondingly, as the bumetanide- and furosemide-inhibited component of 86Rb influx, is unobserved in rabbit erythrocytes irrespective of the Ch level in the plasma. The activity of the Na+, Li+ exchange is markedly reduced in erythrocytes of rabbits with hypercholesterolemia and correlates with Ch and LDL levels in the plasma. The K+ and Mg2+ concentrations in erythrocytes do not depend on Ch plasma levels. There was a negative correlation between the intracellular Na+ content with plasma Ch and LDL levels. The Ch content in erythrocyte ghosts is, on the average, identical for both groups of experimental animals.

Animals↗
PubMed1994

[Na+/H+- and Na+/Na+-countertransport in human, rabbit, and rat erythrocytes: evidence for the existence of two independent ion-transporting systems].

The activity and regulatory features of the Na+/H(+)- and Na+/Na(+)-exchange were studied in human, rabbit and rat red blood cells. No basal activity of the Na+/H(+)-exchange (the amyloride-inhibited component of the 22Na+ influx) in erythrocytes of these species was observed. The rate of 22Na+ influx increased rapidly when the experiments were carried out on acid-loaded cells in an alkaline (pH0 = 8.0) incubation medium (delta mu H(+)-induced Na+/H(+)-exchange). The ratio of delta mu H(+)-induced Na+/H(+)-exchange activities in human, rabbit and rat red blood cells was 1.0 : 1.1 : 2.3, respectively, whereas that of the Na+/Na(+)-exchange activities (the phloretin-inhibited component of the 22Na+ influx) in erythrocytes of these species was 1.0 : 4.6 : 0.2. The osmotic shrinkage of rat and rabbit erythrocytes led to the stimulation of the Na+/H(+)- (but not Na+/Na+) exchange. Amyloride (1 mM) inhibited the shrinkage-induced 22Na+ entry as well as the delta mu H(+)-induced 22Na+ entry--by 95 and 10-20%, respectively. Heat treatment (10 min, 49-51 degrees C), disturbing the membrane cytoskeleton suppressed both the shrinkage-induced activation and the delta mu H(+)-induced activation of the Na+/H(+)-exchange. The data obtained indicate that the both transport systems are mediated by two distinct transport carriers. It may be suggested that the delta mu H(+)-induced Na+/H(+)-exchange, on the one hand, and the shrinkage-induced Na+/H(+)-exchange, on the other, are mediated by two different Na+/H(+)-exchanger subtypes.

Amiloride↗
PubMed1993

Nucleosomal organization of telomere-specific chromatin in rat.

Rat liver interphase chromosomes have telomeres 20-100 kb in length. Micrococcal nuclease digestion of nuclei cleaves telomeres with a uniform 157 bp periodicity, producing soluble particles that sediment in sucrose gradients exactly like oligonucleosomes. The monomeric telomere particles comigrate with nucleosome core particles on nucleoprotein and DNA gels but do not bind H1. DNAase I cleaves telomere nucleoprotein into a series of bands spaced by about 10.4 bp and with the same intensity distribution as bands from bulk nucleosomes. Removal of H1 from chromatin alters the sedimentation properties of telomeres in parallel with bulk chromatin. Thus, telomeres of mammals are constructed of closely spaced nucleosomes, in contrast with the telomeres of lower eukaryotes, which show no evidence of nucleosomal structure.

Animals↗
PubMed1992

[The effect of physical training loads on the body function of sailors on a long cruise].

The article studies the influence of physical exercises at sports simulators upon the state of some bioenergetic systems of seaman's organism in the conditions of a 4-month cruise. The article discloses the dynamics of maximum oxygen consumption, pH value, the quantity of buffered bases and standard bicarbonate in blood; activity of lactic dehydrogenase and its isoenzymes in serum; activity of lactic dehydrogenase, alpha-glycerophosphate dehydrogenase, etc. in lymphocytes of peripheral blood. The authors discuss the reasons of changes that take place in aerobic and anaerobic processes of cells and the whole organism.

Adult↗
PubMed1991

The structure and dynamics of H1-depleted chromatin.

The size of DNA involved in the interaction with a histone octamer in H1-depleted chromatin was re-examined. We compared the thermal untwisting of chromatin DNA and naked DNA using CD and electrophoretic topoisomer analysis, and found that DNA of 175 +/- 10 base pairs (bp) in length interacted with the histone core under physiological conditions. The decrease of ionic strength below 20 mM NaCl reduced this length down to 145 bp: apparently, an extra 30 bp DNA dissociated from the histone core to yield well-known 145-bp core particle. Histone cores partly dissociate within the temperature range of 25 to 40 degrees C. Quantitative analysis of histone thermal dissociation from DNA shows that the size of DNA protected against thermal untwisting would be significantly overestimated if this effect is neglected. The results presented in this paper also suggest that the dimers (H2A, H2B) act as a lock, which prevents transmission of conformational alterations from a linker to nucleosome core DNA. The histone core dissociation as well as (H2A, H2B) dimer displacement are discussed in the light of their possible participation in the eukaryotic genome activation.

Animals↗
PubMed1990

The B-A transition in superhelical DNA.

Relaxation of a DNA superhelical stress due to the B to A transition induced by trifluoroethanol has been studied by assessing the change of DNA orientation in a flow gradient. Using DNAs of different superhelical densities, a decrease in the winding angle during the B----A shift of DNA was found to be 1.5 degrees per base pair in solution. Accepting the winding angle for B-DNA in solution to be 34.1 degrees, that for A-DNA must have a value of 32.6 degrees which agrees with the X-ray data for A-DNA in the condensed state. The date obtained within the B-A transition interval make it possible to conclude that there is an increase in winding at each B/A junction, which is about 5 degrees per one junction.

DNA Topoisomerases, Type I↗
PubMed1990

The chromatin fiber: structure and conformational transitions as revealed by optical anisotropy studies.

Structure and conformational transitions of the chromatin fiber as revealed by optical anisotropy studies are reviewed. The data in the literature do not allow a definite interpretation; in fact some of them are contradictory. The major findings are reported here and an attempt is made to analyse them with respect to the internal dynamics and the various models suggested for the organization of the chromatin fiber.

Cations↗
PubMed1988

NaCl-induced chromatin condensation. Application of static light scattering at 90 degrees and stopped flow technique.

We have studied the NaCl-induced condensation of calf thymus chromatin by static light scattering of 90 degrees and shown that the increase in NaCl concentration up to 120-170 mM results in a large increase in scattering intensity of the total chromatin. Histones H1-depleted and trypsinized chromatin preparations do not reveal such a large increase in scattering intensity. The increase in the scattering intensity reflects the folding of the chromatin filaments, but not their aggregation. We have used this effect to monitor the kinetics of the chromatin condensation in response to a jump to higher NaCl concentrations by means of a stopped-flow technique. The results show that the condensation is a fast complex process consisting of at least two steps. The first step is only partially resolved by the stopped-flow apparatus. The second step has a time constant in the range of 20-50 ms, which does not depend on chromatin concentration.

Chromatin↗
PubMed1988

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies.

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.

Chromatin↗
PubMed1988

Polyamine-DNA interactions. Condensation of chromatin and naked DNA.

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.

Cations↗