Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)

Linkage of loci associated with two pigment mutations on mouse chromosome 13.

Progeny from one intra- and two inter-specific backcrosses between divergent strains of mice were typed to map multiple markers in relation to two pigment mutations on mouse chromosome 13, beige (bg) and pearl (pe). Both recessive mutants on a C57BL/6J background were crossed separately with laboratory strain PAC (M. domesticus) and the partially inbred M. musculus stock PWK. The intra- and inter-specific F1 hybrids were backcrossed to the C57BL/6J parental strain and DNA was prepared from progeny. Restriction fragment length polymorphisms were used to follow the segregation of alleles in the backcross offspring at loci identified with molecular probes. The linkage analysis defines the association between the bg and pe loci and the loci for the T-cell receptor gamma-chain gene (Tcrg), the spermatocyte specific histone gene (Hist1), the prolactin gene (Prl), the Friend murine leukaemia virus integration site 1 (Fim-1), the murine Hanukuh Factor gene (Muhf/Ctla-3) and the dihydrofolate reductase gene (Dhfr). This data confirms results of prior chromosomal mapping studies utilizing bg as an anchor locus, and provides previously unreported information defining the localization of the prolactin gene on mouse chromosome 13. The relationship of multiple loci in relation to pe is similarly defined. These results may help facilitate localization of the genes responsible for two human syndromes homologous with bg and pe, Chediak-Higashi syndrome and Hermansky-Pudlak syndrome.

Analysis of Chlamydomonas reinhardtii histones and chromatin.

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.

The histones of the endosymbiont alga of Peridinium balticum (Dinophyceae).

The histones of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium balticum were characterized by amino acid analysis and peptide mapping, and compared to calf thymus histones. Using these and various other criteria we have identified two H1-like histones as well as the highly conserved histones H3 and H4. A 13,000 dalton component in sodium dodecyl sulphate (SDS) gels can be separated into two components in Triton-containing gels. We suggest that these histones (HPb1 and HPb2) correspond to the vertebrate histones H2A and H2B, respectively.

The murine gamma-chain of the T cell receptor is closely linked to a spermatocyte specific histone gene and the beige coat color locus on chromosome 13.

With the use of standard genetic techniques, we have mapped the T cell gamma-chain gene locus (Tcrg) to the proximal end of mouse chromosome 13. Close genetic linkage was demonstrated between a restriction fragment length polymorphism in the Tcrg locus and a locus (HlS) encoding a spermatocyte-specific histone electrophoretic variant (HlS) by using recombinant inbred strains. Because HlS is closely linked to the dominant visible marker extra toes (Xt), Tcrg must be within 5 cM of the beige (bg) locus. Mice homozygous for the recessive mutation beige (bg/bg) show diminished pigmentation, enlarged lysosomes in granulocytes, and a selective deficiency of natural killer cell function. The bg mutation in mice is an animal model for humans with Chediak-Higashi syndrome; we speculate that the human gamma-chain may be closely linked to the human mutation and may be a useful diagnostic tool. Seven restriction enzymes identify five haplotypes of the highly polymorphic gamma-chain in mice.

Regulation of nucleosomal core histone variant levels in differentiating murine erythroleukemia cells.

During hexamethylenebis(acetamide)-induced terminal differentiation of murine erythroleukemia (MEL) cells in vitro, the histone variant proportions undergo changes similar to those observed in vivo in terminally differentiating cells of the young mouse. Thus, there is a rapid increase in the relative amounts of the variants H2A.1 and H2B.2 in parallel with the increase in the number of hemoglobin-producing cells and the sharp decrease in the growth rate. We show that the changes in variant proportions are not associated with slower growth per se but are most likely due to differential changes in the rates of variant synthesis as a result of commitment to terminal differentiation. In addition, we observed an inducer-specific increase in the rate of synthesis and the relative amount of the minor H2A variant 4, well before hemoglobin accumulation. We also present evidence that H2A and H2B histones are synthesized and incorporated into chromatin at a significant rate even when DNA synthesis is inhibited, suggesting turnover of these histones. H2A and H2B turnover can be detected directly even in exponentially growing cells. H2A.1 and H2B.2 have higher turnover rates than H2A.2 and H2B.1, respectively, in exponentially growing cells, a difference which is even more pronounced in induced cells. The magnitude of the differential turnover is not sufficient to account for the changes in the histone variant proportions in the short life of induced MEL cells but could explain the slow accumulation of H2A.2, H2B.1, and H3.3 in nondividing adult tissues of the mouse.

Changes in nucleosomal core histone variants during chicken development and maturation.

The nucleosomal core histones H2A, H2B, and H3 of the chicken can be resolved by polyacrylamide gel electrophoresis in the presence of nonionic detergents into two primary structure variants each, which occur in different relative amounts in various adult tissues. Quantitative analysis of the histone components throughout embryonic development and posthatching maturation of the chicken revealed that the proportions of the three pairs of variants change independently. Thus, the two H2A variants occur in similar proportions throughout embryonic development and in all adult tissues. In contrast, only one variant each of H2B and H3 is detectable at the earliest stages (primitive streak). The second variant of these histones becomes detectable and increases gradually during somite formation (2-12 days of incubation) to reach a plateau at a level of about 3 and 10% of total H2B and H3 histones, respectively. After hatching, the relative amounts of the minor H2B and H3 variants remain at embryonic levels in those tissues which maintain a high mitotic activity such as blood-forming tissues, but increase with different kinetics in tissues which essentially stop cell division in adults (e.g., liver, kidney, etc.). However, while H2B.2 remains a very minor component in all tissues, H3.3 increases at a relatively high rate for more than a year to become the predominant H3 variant in the liver and kidney of older chickens. The changes in chicken core histone variant proportions appear to be related to changes in growth rate rather than cell differentiation. The extensive change of H3 variant proportions in nondividing adult tissues is most likely due to replication-independent incorporation of H3.3 into nucleosomes.

Localization of the amino acid substitution site in a new variant of human serum albumin, albumin Mexico-2.

Using an electrophoretic screening procedure, we have discovered that two species of human serum albumin Mexico occur that are indistinguishable by conventional electrophoretic methods. We suggest that these species be referred to as albumins Mexico-1 and Mexico-2. Isolation and determination of the partial sequence of the cyanogen bromide fragment of albumin Mexico-2 that differs from the corresponding fragment of the common albumin A revealed this variant to arise from at least a glycine/aspartic acid substitution at position 550. This region of the albumin molecule is involved in the binding of the fatty acid, palmitate.

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.

Physical properties of DNA and chromatin isolated from G1- and S-phase HeLa S-3 cells. Effects of histone H1 phosphorylation and stage-specific nonhistone chromosomal proteins on the molar ellipticity of native and reconstituted nucleoproteins during thermal denaturation.

To help delineate how changes in chromatin organization are related to DNA replication and transcription during the HeLa S-3 cell cycle, we have extended previous studies of the composition and structure of chromatin in synchronized G1- and S-HASE CELLS. By analyzing changes in molar ellipticity at 276 nm ([theta 276]) during thermal denaturation, it was found that double-helical DNA molecules in native chromatin have different optical activities and thermal stabilities at these two stages of the cell cycle. Furthermore, profiles of d[theta 276]/dT vs. T indicate that native G1- and S-phase chromatins contain different families of DNA superstructures. To help determine the causes and functional significance of these chromatin reorganizations during the cell cycle, we compared the optical activities and thermal stabilities of DNA in native chromatin with protein-free DNA and DNA in nucleoproteins reconstituted in vitro by NaCl-urea gradient dialysis. In addition, we examined levels of histone phosphorylation, histone acetylation, and types of histone and nonhistone chromosomal proteins (NHCP) found in G1- and S-phase cells and in purified hydroxylapatite, (HAP) fractions of these nuclear proteins which were used for in vitro reconstitution. The results of the present studies indicate that changes in H1-DNA-NHCP interactions occur in vivo, are associated with the phosphorylation of histone 1 molecules, and appear to be responsible for the relaxation of compact G1-phase chromatin superstructures into more open S-phase configurations during the HeLa S-3 cell cycle.

Species-specific suppression of histone H1 and H2B production in human/mouse hybrids.

Ten human/mouse hybrid cell lines that segregate either human or mouse chromosomes were examined for the expression of human- and mouse-specific histones H1 and H2B. Results of this study indicate that the human and mouse chromosomes in hybrid cells that segregate human chromosomes (M greater than H hybrids) contain only mouse histone H1 and H2B. Chromosomes in hybrid cells that segregate mouse chromosomes (H greater than M hybrids) contain only human H1 and H2B histones. Loss of the ability to produce either human or mouse histones does not seem to be due to the loss of specific human or mouse chromosomes because M greater than H hybrids retaining at least one copy of each human chromosome contain only mouse H1 and H2B and H greater than M hybrids retaining at least one copy of each mouse chromosome contain only human H1 and H2B histones. These results, together with those concerning histone H4 acetylation levels and ratios of variants of histones H3 and H2A that are like those in the dominant parent cell type, indicate that the control mechanisms affecting H1 and H2B expression in H greater than M and in M greater than H hybrid cells affect expression of histones H2A, H3, and H4 genes as well. The present data thus support the hypothesis that none of the histone genes that are active in the recessive parent cell type is expressed in hybrid lines that segregate recessive cell chromosomes.

Biochemical and immunological characterization of two distinct variants of histone H2A in Friend leukemia.

Changes in the relative amount of two histone H2A subfractions have been observed in cells at different proliferative stages of Friend leukemia. Biochemical analyses of the purified H2A subfractions reveal them to be different in primary structure, and not the result of postsynthetic modifications of the same parent protein. Antibodies raised against the purified H2A.2 subfraction cross react with H2A.1 and H2A.2, but show high specificity for the immunizing subfraction at higher sera dilutions. Only H2A.2 contains a methionine which appears critical to an antigenic difference that immunologically distinguishes H2A.2 from H2A.1. The observed change in the relative amounts of two nonallelic variants of a histone coincident with changes in the physiologic states of the cell may indicate a correlation between genome structure and function.

Studies of human histone messenger RNA. II. The resolution of fractions containing individual human histone messenger RNA species.

Polyribosomal 4 to 18 S RNA from S phase HeLa S-3 cells has been fractionated by chromatography on oligo(dT)-cellulose and resolved into multiple discrete components by continuous elution preparation electrophoresis. The human histone messenger RNA (mRNA) species associated with various polyadenylated [poly(A(+))] and nonpolyadenylated [poly(A(-))] components of 4 to 18 S RNA were determined by translation of these RNA fractions in vitro using a Krebs II ascites cell-free system followed by resolution of histones synthesized in vitro on polyacrylamide gels containing Triton X-100. The results of these studies indicate that poly(A(-)) 4 to 18 S RNA from S phase HeLa polyribosomes contains: (a) large quantities of discrete 7.4 and 8 S RNA species which are not functional histone mRNA; (b) a discrete 8.6 S RNA fraction which contains the templates of human histone H4; (c) 9.2 to 10.7 S RNA which contains mixtures of incompletely resolved histone H2B, H2A, and H3 mRNA (These mRNA species do not closely correspond to discrete RNA subfractions resolvable by our techniques.); (d) discrete 12 and 13 S RNA fractions which contain templates of human histone H1 polypeptides. The present studies also indicate that the mRNA templates of histone variants H3.2 and H3.3 have a slightly lower electrophoretic mobility than H3.1 mRNA and that H2A.2 mRNA has a slightly lower electrophoretic mobility than H2A.1 mRNA. In addition, appreciable quantities of H3.2, H3.3, and H2A.2 mRNA are bound to oligo(dT)-cellulose in 0.5 M KCl. These results indicate that mRNA species of the same histone class differ slightly in primary structure and are consistent with the hypothesis that some histone mRNA species contain short tracts of poly(A).

Stage-specific switches in histone synthesis during embryogenesis of the sea urchin.

Histones H2A and H2B of the sea urchin embryo have been resolved by new methods into components that are synthesized at different stages of development. One form of H2A and one form of H2B are synthesized only during the period from fertilization to the blastula stage. Subsequently, two other types of H2A and H2B molecules are synthesized. In addition, a histonelike protein was detected which is synthesized only from fertilization until the 16-cell stage when the synthesis of still another H2A-like protein begins. None of the late-appearing forms are derived from histone polypeptide chains synthesized earlier in development. Since the early components do not disappear after their synthesis stops, these modulations of histone synthesis lead to an increase in histone multiplicity, concomitant with the beginning of cell diversification and a decrease in cell division rate.

Further evidence of transcriptional and translational control of histone messenger RNA during the HeLa S3 cycle.

Using a translational assay and new analytical procedures we have found that: -Histone mRNA can be detected both associated with polyribosomes and in the postribosomal supernatant of S phase HeLa S3 cells.-Inhibition of DNA replication by cytosine arabinoside treatment causes histone mRNA to completely disappear from polyribosomes, and little histone mRNA can be detected in the postribosomal supernatant of inhibited cells. These data indicate that histone mRNA does not accumulate in the cytoplasm after the inhibition of DNA replication. -Histone mRNA species cannot be detected in the postribosomal supernatant of G1 cells synchronized by selective detachment. This observation, together with the previous finding that histone mRNA is not present on G1 polyribosomes, is consistent with the idea of a transcriptional block in histone mRNA production and transport to the cytoplasm during G1.