Biomedical subjects
Publications and source records attributed to J Zlatanova.
1. The issue of the existence and the properties of histone H1 zero in quiescent rabbit tissues was approached by electrophoretic, chromatographic and immunochemical techniques. 2. The results demonstrated that the rabbit tissues did contain a typical histone H1 zero, its properties being comparable to those of H1 zero from all other sources studied thus far.
The belief that histone H1 interacts primarily with DNA in chromatin and much less with the protein component has led to numerous studies of artificial H1-DNA complexes. This review summarizes and discusses the data on different aspects of the interaction between the linker histone and naked DNA, including cooperativity of binding, preference for supercoiled DNA, selectivity with respect to base composition and nucleotide sequence, and effect of H1 binding on the conformation of the underlying DNA. The nature of the interaction, the structure of the complexes, and the role histone H1 exerts in chromatin are also discussed.
By using direct competition experiments, the binding of histone H1AB (a mixture of H1A and H1B) and H1(0) to superhelical and linear DNA forms was studied. Mouse liver H1 isohistones and plasmid p alpha GD containing part of the 5' flanking and part of the coding sequence of the mouse alpha-globin gene in pUC18 were used as partners in the binding reaction. The competition experiments were performed by direct mixing of the histone with labelled supercoiled DNA (at 125 mM NaCl and at a histone/DNA ratio of 1.0) and addition to the mixture of increasing amounts of cold competitor DNA, either supercoiled or linear. The radioactivity of the complex formed was determined by filter binding. The results show that both histones H1 and H1(0) posses a strong binding preference for supercoiled DNA forms. Thus, histone H1(0) resembles the regular somatic set of histone H1 and not the other differentiation-specific histone H5 studied thus far.
Histone H1 plays a role in the formation of chromatin structure, both at the level of the nucleosome particle itself and in the formation of the higher-order structures of the chromatin fibre. Histone H1 is regarded as a part of a general repressor mechanism that ensures a strong and stable repression of gene expression. In addition to serving as a general repressor for relatively large chromatin fragments, histone H1 might also be involved in controlling the transcriptional activity of individual genes.
This work is devoted to the study of the immunoreactivity of histone H1(0) and its major structural domains in mononucleosomes. Three types of antibody populations were used: (i) anti-H1(0) which reacted with antigenic determinants situated along the whole polypeptide chain; (ii) anti-GH5 which recognized epitopes located in the globular region; and (iii) anti-C-tail antibodies reacting specifically with fragment 99-193 of the protein molecule. The anti-GH5 antibodies gave a weak reaction, the C-tail-specific antibodies reacted relatively strongly and the antiserum to the intact molecule showed an intermediate level of reactivity. The relative intensities of the immunoreaction could be interpreted as reflecting the exposure of the antigenic determinants of the individual protein domains in the monosome particle.
By using affinity-purified antibodies to H10 and to H1AB the localization of these histones was studied by indirect immunofluorescence in the nuclei of proliferating (EAT and uninduced Friend cells) and of differentiating (induced Friend cells) cell populations. While with H1AB antibodies a bright fluorescence all over the chromatin was obtained, the localization of H10 varied depending on the state of the cell population. In the proliferating EAT cells it was localized strictly in the nucleoli. The Friend cell population revealed a heterogeneous picture with two types of H10 localization-nucleolar predominating in uninduced cell populations and peripheral predominating in induced cells. A comparison with literature data suggests that H10 seems to be associated with chromatin regions containing active genes.
In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt. Perchloric-acid-extracted proteins from whole cells contain only two fractions which comigrate with 'authentic' yeast high-mobility-group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross-reaction with anti-(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1. The isolation of 'authentic' yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti-HMG antiserum whereas the richer preparation also gives considerable cross-reaction with the anti-H1 antiserum. Immunoblotting analysis performed on the salt-extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti-H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.
Antibodies were elicited against chromatographically purified histone H1 subfractions or against their complexes with RNA and their specificity studied by enzyme-linked immunosorbent assay. The results show that complexing of the pure protein with RNA does not lead to any significant increase in the antibody titer and results in obtaining antibodies predominantly against the common antigenic determinants present in the H1 histone class. On the other hand, using pure histone fractions for immunization gives rise to antibody populations reacting mainly with the subfraction-specific determinants on the histone molecule. In view of these results the literature data should be interpreted with caution.
Some terminally differentiated tissues of the bivalve mollusc Anodonta cygnea contain a histone subfraction with electrophoretic and immunological characteristics similar to those of the mammalian histone H1o.
Histone H1o was found both in neuronal and oligodendrocyte rat-brain nuclei fractionated by sucrose-gradient isopycnic centrifugation. This histone was absent during the early stages of development when the brain cells were still proliferating, but it appeared in significant amounts in the terminally differentiated cells.
The lysine-rich histones of trout liver were studied in order to see whether a protein similar to mammalian histone H1(0) exists in this lower vertebrate. The biochemical methods used involved SDS and acid-urea polyacrylamide gel electrophoresis, gel exclusion and ion-exchange chromatography. Specific rabbit antibodies were elicited against chromatographically-purified mouse liver H1(0) and applied for further characterization of the presumptive trout-liver H1(0) fraction. The results from the indirect immunofluorescence on nitrocellulose-immobilized crude H1 subfractions and from the complement fixation test supplied additional evidence for the existence of an H1(0)-like protein in trout liver.
The changes in the lysine-rich histone subfraction H1(0) have been quantitatively studied in rat liver during the regeneration period after partial hepatectomy. A gradual decrease in this protein was found early after operation with a minimal value around the time of maximal mitotic activity. The reduction in the H1(0) content paralleled well the increasing number of cells in the cell cycle.
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An extraction procedure for histone H5 for chicken erythrocytes described in the literature has been applied to mouse spleen. The SDS polyacrylamide gel electrophoresis pattern of the resulting protein preparation revealed the presence of a component with the mobility of the marked chicken erythrocyte H5. Additionally the preparation has been characterized using antiserum raised against purified chicken H5. The presumptive mouse spleen H5 preparation gave visible precipitation lines with the anti-H5 chicken-antiserum. The combined electrophoretic and immunological evidence suggests the presence of histone H5 in mammalian tissue.
Histone synthesis was studied in Friend cells growth-arrested by culturing in isoleucine-deficient medium for 20-24 hours. Such cells are characterized by a very low level of DNA synthesis (5% of the controls). In contrast, the labelling of total nuclear proteins and of total histones continues at an unreduced level for many hours. At the same time there is no accumulation of histones in the cell nucleus, suggesting histone turnover. The behaviour of histone H1 differs from that of the nucleosomal histones, a fact speaking in favour of the existence of independent control mechanisms.
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Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The parameters of the electric field do not affect separation of 'normal' DNA fragments of comparable length.