Jordanka S. Zlatanova

Immunofluorescent localization of histone H10 in the nuclei of proliferating and differentiating friend cells

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.

Immunochemical approaches to the study of histone H1 and high mobility group chromatin proteins

SummaryThis review is an attempt to summarize all existing data on histone H1 and high mobility group proteins obtained with immunochemical methods. The following issues are treated consecutively: production of specific antisera to these protein groups, antigenic structure of the polypeptide chains, use of antibodies for the identification, the quantitative estimation and the study of the tissue- and species-specificity of the proteins. Special attention is devoted to the studies of the localization of the respective antigens in the cell, the nucleus, the chromosomes and the interphase chromatin. The use of specific antibodies for the elucidation of the role these proteins play in such basic cellular processes as proliferation and differentiation, replication and transcription is also discussed. It becomes clear that the use of immunochemical approaches in the study of specific chromatin proteins both at the level of the protein molecule and at the level of chromatin can be a powerful tool for the resolution of a number of specific problems. The field is very promising and will undoubtedly develop intensely in the nearest future.

DNA repair precedes replicative synthesis during early germination in maize.

DNA synthesis was studied during germination by following the rate of incorporation of radioactive thymidine into high molecular weight DNA. A peak of DNA synthesis was observed between the 8th and the 12th hour, i.e. before the beginning of the semi-conservative replication of genomic DNA, accompanied by an increase in the DNA content of the embryo. By the use of nucleoid sedimentation and nick-translation it was shown that, during the first hours of germination, extensive repair occurs of the DNA single-strand breaks present in the dry embryo. As a result, the DNA of the 16-h-germinated embryo acquires the conformation typical of that of the root meristemic cells active in transcription and replication.In addition we have shown that cytoplasmic organelle (most probably mitochondrial) DNA synthesis is very active during the prereplicative state which confirms earlier microscopic data on mitochondrial biogenesis during early germination.

Lack of coupling between DNA and histone synthesis in growth-arrested Friend erythroleukemia cells.

SummaryHistone 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.

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.

Presence of a H1o-type histone in an invertebrate: the bivalve mollusc Anodonta cygnea

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.

Antibodies specific to histone H1 inhibitin vitro transcription in isolated mammalian nuclei

The issue of whether histone H1 is present in transcriptionally active chromatin has been approached by studying the effect specific anti-H1 antibodies have onin vitro transcription in isolated nuclei. To that end, the incorporation of radioactive RNA precursors into trichloroacetic acid-precipitable material was compared for control nuclei and nuclei that had been preincubated with specific anti-H1 antibody populations (whole sera, affinity-purified immunoglobulins and monovalent Fab fragments). The anti-H1 antibodies significantly and reproducibly inhibited the transcriptional activity in isolated nuclei. Experiments were also performed to exclude the possibility that the inhibition observed was due to some long-distance effect of the binding of the antibodies to chromatin. The results are interpreted as indicating that active gene chromatin does contain histone H1.

Antigenic structure of histone H1

In order to study the antigenic structure of histone Hl° the purified protein from mouse liver was subjected to different chemical and enzymatic treatments (CNBr, acetic acid, trypsin, chymotrypsin). The resulting peptides were fractionated in SDS-containing or acid-urea polyacrylamide gels, transferred by electroblotting onto nitrocellulose paper and probed with specific rabbit anti-H1° antiserum. The C-terminal fragments 99–193 (obtained following acetic acid hydrolysis) and 107–193 (obtained by chymotrypsin digestion) also exhibited strong immunoreactivity. Fragment 1–30 (CNBr cleavage) contained antigenic determinants while the shorter fragments 1–22 and 1–28 (acetic acid hydrolysis) failed to show any detectable reactivity. It was concluded that, in contrast to histone H5 whose reactivity is mainly concentrated to the globular domain of the molecule, the antigenic determinants in histone H1° are more or less evenly distributed along the polypeptide chain with the possible exception of the short unstructured N-nose.