Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment.

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.

[Nucleosomes of active chromatin from sea urchin embryo cells are rich in early histone variants]. / Nukleosomy aktivnogo khromatina émbrional'nykh kletok morskogo ezha obogashcheny rannimi variantami gistonov.

By means of selective micrococcal nuclease digestion chromatin from early stages of the sea urchin St. droebachiensis embryogenesis was divided into fractions differing by their transcriptional activity. The electrophoretic analysis of histones at the gastrula stage showed that the transcriptionally active chromatin fraction was enriched with early variants of histone H2A and H1. On the stage of pluteus, when primary cell differentiation is completed, the amount of total histone H1 in this fraction was significantly decreased, however it was enriched in an early alpha variant. It was shown that after mild micrococcal nuclease digestion mononucleosomes, which were mostly derived from active chromatin, were significantly enriched with in vivo labeled early histone variants.

Distribution of histone variants in the sea urchin chromatin fractions obtained by selective micrococcal nuclease digestion.

Chromatin fractions differing in their transcriptional activity were isolated by selective micrococcal nuclease digestion of nuclei from sea urchin embryos (Strongylocentrotus droebachiensis) at the gastrula and pluteus stage. The electrophoretic analysis of the chromatin proteins at the gastrula stage showed that a soluble, transcriptionally active fraction of chromatin was enriched with early variants of histones H1 and H2A. The early and late variants of histone H2A at the pluteus stage were distributed randomly between chromatin fractions. However, the content of both variants of histone H1 was essentially decreased in the soluble transcriptionally active fraction of chromatin.

Dissociation and isolation of chromatin proteins in salt solutions by an aqueous two-phase system.

An aqueous two-phase system containing 7% Dextran T 500-5% polyethylene glycol (PEG) 6000 has been adopted for rapid selective stepwise extractions of high-mobility-group proteins and histones from both isolated chromatin and intact nuclei of calf thymus. After the dissociated proteins in the PEG phase were precipitated with 20% trichloroacetic acid at 4 degrees C, proteins were recovered from this phase by solubilization of PEG with acidified acetone at room temperature. This method allows preparation of nuclei depleted of histone H1.

[Electrophoretic study of the acid soluble proteins of Saccharomyces cerevisiae yeast chromatin]. / Elektroforeticheskoe issledovanie kislotorastvorimykh belkov khromatina drozhzhei Saccharomyces cerevisiae.

Fractionation of acid-soluble proteins from yeast Saccharomyces cerevisiae chromatin by gel electrophoresis suggested the existence of four histonestone fractions--H2a, H2b, H3, H4 and histone H1-like protein. The latter was isolated according to the second method of Johns and extracted with 5% HClO4. Amino acid analysis of the histone H1-like protein showed a moderately high content of basic amino acids, but a much lower ratio of lysine: arginine (approximately 3) than that of the hisone H1 from higher eukaryotes. In addition to histone H1-like protein a protein X was also extracted with 5% HC1O4. The sequence of the electrophoretic mobilities of histone fractions from yeast coincides with those of histone fractions from plants--H4 greater than H3 greater than H2a greater than H2b greater than H1.

A study of histone-histone interactions by affinity chromatography.

Homologous whole histone from calf thymus was adsorbed on Sepharose 4B columns with covalently coupled histone fractions H2a, H2b, H3 or H4 in 0.01 M phosphate buffer, pH 6.7 - 1 M NaCl. The adsorbed histones were eluted from the columns with 5 M urea in the same buffer. Electrophoretic analysis has shown that the different columns exhibit selective affinity to the histone fractions: the H2b column to histone H2b and H2a (with only weak affinity to histones H3 and H4), the H2a column to histones H2b and H3 (moderate affinity to histone H2b), and the H4 column to histone H3, H4 and H2b (weak affinity to histone H2a). Histone H1 displayed no fixation by either of the columns tested.

Chromatin proteins from normal, vegetalized, and animalized sea urchin embryos.

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage.