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A Ruiz-Carrillo

Publications and source records attributed to A Ruiz-Carrillo.

At least 37 records · Page 2Linked to original sources
PubMed1986

Transcription unit of the chicken histone H5 gene and mapping of H5 pre-mRNA sequences.

We have analyzed the transcription unit of the gene coding for the erythrocyte-specific histone H5. RNA transcripts elongated in vitro by permeabilized immature cells hybridized to the template strand of the structural gene as well as to 3'-flanking sequences. Approximately 90% of the engaged RNA polymerase II molecules terminate transcription within a region of about 500 base pairs immediately downstream of the polyadenylation site. S1 nuclease protection experiments indicated that the downstream sequences are also transcribed in vivo, their relative amounts reflecting the distribution of RNA polymerases observed in vitro. RNA molecules extending up to 1.14 kilobase pairs downstream of the polyadenylation site were detected, but no unique site of termination was found. The sequence of the transcription termination region shows no obvious homology to those of other RNA polymerase II termination regions. The possible involvement of altered DNA and/or chromatin structures in the transcription termination process is discussed.

Animals↗
PubMed1986

Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation.

We have analyzed the chromatin structure of a region that encompasses 14.4 X 10(3) base-pairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation. Seven of eight major lineage-specific DNase I-hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene. The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene. DNase I, micrococcal nuclease, and S1 nuclease recognize the same regions, which differ from those cleaved by S1 on supercoiled plasmid DNA. This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation. The DNA sequence of some of the DNase I target sites contains repeated motifs, (T-C-C-C)2, (T-C-C)2, (T-G-G-G-G)2, which are found in the hypersensitive sites of other genes. Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined. Notably, the first one-third of the gene is more sensitive than the rest. The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant.

Animals↗
PubMed1985

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin.

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.

Amino Acid Sequence↗
PubMed1984

The histone H5 gene is flanked by S1 hypersensitive structures.

The potential of the cloned histone H5 gene to form altered DNA structures has been examined by S1 nuclease digestion of supercoiled recombinant plasmids containing up to 8.8 kbp of chicken DNa. The three main nicking sites map at the upstream and downstream sequences flanking the structural gene. The cleavage sites share sequence homology, strand specificity, and do not seem to be single-stranded. The sequence of the S1-sensitive sites does not suggest that the fragments can adopt any of the known DNA secondary structures.

Animals↗
PubMed1983

Genomic organization of the genes coding for the six main histones of the chicken: complete sequence of the H5 gene.

The organization of the genes coding for histones in the chicken has been examined, with special reference to that coding for the tissue-specific, developmentally regulated histone H5. Two recombinant phages containing sequences complementary to cloned H5 cDNA have been isolated from a genomic chicken library. The clones have been characterized by heteroduplex formation, restriction nuclease analysis, hybridization to cloned homologous histone gene probes, and DNA sequencing. Hybridization to genomic DNA has shown that there is only one copy of the H5 gene per haploid genome, whereas there are six to 11 copies of the genes for the other histones. Examination of 29 X 10(3) base-pairs of DNA sequences flanking the H5 gene has revealed the absence of any other histone genes which, although not tandemly reiterated, for the most part appear to reside in loosely organized clusters. The complete DNA sequence of the H5 gene and flanking regions, as well as the mapping of the 5'-end of its messenger RNA by primer extension with AMV reverse transcriptase, has shown that the gene has no introns and little homology to other histone genes, including those for H1.

Animals↗
PubMed1983

Isolation of a 167 basepair chromatosome containing a partially digested histone H5.

A test has been made of the proposal that protection of the 167 basepair DNA length in the 'chromatosome' is due only to the central globular domain of the lysine-rich histones. Chicken erythrocyte chromatin was treated with trypsin to leave only the limit peptide from histones H1 and H5. Nucleosome monomers were then isolated on sucrose gradients following micrococcal nuclease digestion and were found to contain the 167 basepair DNA band as in intact chromatin. The presence of the limit peptide from H5 on the monomers was confirmed using an antibody to H5. It is concluded that the trypsin-susceptible domains of the lysine-rich histones are not involved in the protection of the 2-turn 167 basepair length of DNA in the nucleosome.

Animals↗
PubMed1982

Nucleosomes containing histones H1 or H5 are closely interspersed in chromatin.

The distribution of histones H1 and H5 along chromatin fibers has been examined in the nucleated hen erythrocyte. Nucleosome oligomers, produced by micrococcal nuclease digestion of nuclei, were sequentially reacted with affinity-chromatography purified rabbit anti-H5 and sheep anti-rabbit antibodies. Quantitation of the relative amounts of H1 and H5 in the precipitated and supernatant fractions as a function of the oligomer number was consistent with a close interspersion of both types of histones, probably a random one. This conclusion was supported by the immunoprecipitation of longer chromatin fibers. This pattern of distribution appears to apply both to bulk chromatin and to chromatin inactivated during the maturation of the erythrocyte.

Animals↗
PubMed1982

Effect of histone composition on the stability of chromatin structure.

The mode of fragmentation of chromatin by micrococcal nuclease has been studied in nuclei from different sources at physiological ionic strength and low temperature. During digestion, the size of chromatin was reduced until an average S value of 95-100 (hen erythrocyte) or 60-65 (rat liver) was attained. The accumulation of these structures correlated with the period of maximum solubility (80%), indicating that the bulk of chromatin behaved in this manner. Further digestion did not result in a corresponding decrease in S value but in a bimodal sedimentation pattern. As opposed to this behavior, chromatin containing actively acetylated core histones showed a continuous variation in size during the digestion. Indirect immunoprecipitation of chromatin by anti-H5 antibody and sheep anti-rabbit antibody revealed that the acetylated chromatin is partially depleted of H5.

Acetates↗
PubMed1982

Enrichment of histone H5-synthesizing polysomes by indirect immunoadsorption. Purification of H5 mRNA.

A method of purifying H5 mRNA from immature hen erythrocytes is described. Polysomes from red blood cells of anemic animals were allowed to react with affinity-chromatography-purified rabbit anti-H5 antibodies and immunoadsorbed to cellulose-insolubilized sheep and anti-rabbit antibodies. This method produced a 10- to 15-fold enrichment of the H5 mRNA, which was further purified by sucrose gradient centrifugation, poly(U)-Sepharose chromatography, and size fractionation in denaturing polyacrylamide gels. The H5 mRNA activity was essentially pure, as shown by translation in a reticulocyte cell-free system and immunoprecipitation with H5-antibodies. The proportion of H5 mRNA has been estimated to lie in the range 0.4-0.6% of all cellular mRNAs. These values are in agreement with the estimated relative synthesis of H5 in the immature erythrocyte.

Animals↗
PubMed1980

Stability and reversibility of higher ordered structure of interphase chromatin: continuity of deoxyribonucleic acid is not required for maintenance of folded structure.

The organization of the higher order structure of chromatin has been examined in chicken erythrocyte. Chromatin solubilized during the time course of a gentle micrococcal nuclease digestion of nuclei shows a continuous variation in the distribution of molecular weights. Electron microscopy studies of large chromatin fragments solubilized at physiological ionic strength (0.14 M NaCl or KCl) suggest that the polynucleosome chain is folded in continuous compact structures of an average diameter of 23 nm in which the individual nucleosomes are difficult to distinguish. This compact structure is destabilized even at intermediate ionic strengths (e.g., 40 mM NaCl), resulting in looser fibers of similar diameter. At 5 mM NaCl the fiber is unraveled into a continuous filament of 10-nm diameter. These conformational changes are reversible as determined by hydrodynamic and biochemical parameters. The 10-nm leads to 23-nm transition of chromatin appears to be a cooperative process requiring the full complement of histones H1 and H5. Micrococcal nuclease cleaves the DNA in the compact chromatin structure to an apparent limit of digestion corresponding to an average of eight to nine nucleosomes with little effect on the size of the fiber. Thus, the continuity of the DNA is not required for the stability of the folded chromatin fiber. Histones H1 and H5 exhibit a binding preference to larger chromatin fragments regardless of the length of the DNA. This behavior is not observed with relaxed chromatin, suggesting that multiple stabilizing interactions involving H1 (H5) are possible only in the compact configuration.

Animals↗
PubMed1980

Histone H5 messenger RNA is polyadenylated.

In most known systems, histone mRNA lacks the poly(A) sequence at the 3' end of the molecule typical of most mRNAs. Furthermore, the synthesis of histones, unlike that of most proteins, is tightly coupled to DNA synthesis. Nevertheless, histone synthesis occurs in amphibian oocytes in the absence of DNA synthesis. Moreover, it has recently been found that in amphibian oocytes most of the histone mRNA is polyadenylated, and the polyadenylate is probably removed during maturation of the oocyte. Histone H5, an H1-like tissue-specific histone occurring only in nucleated erythrocytes, is also atypical in that it is synthesised in the absence of DNA synthesis during maturation of the red blood cells. We report here that H5 mRNA is polyadenylated.

Animals↗
PubMed1979

An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.

The association of histones H2A, H2B, H3, and H4 in solution has been studied. In 2 M NaCl and at neutral pH they can assemble in a complex in which each histone is present in equimolar amounts. The complex has a weight average molecular weight of 98,000 (+/- 3700) and a sedimentation coefficient (so20,w) of 4.8. The value of the weight average molecular weight and the histone stoichiometry indicate that the complex is an octamer. The pairs of histones H2A,H2B and H3,H4 studied separately under identical conditions only associated as equimolar complexes consistent with dimeric and tetrameric structures, respectively. The stability of the core histone octamer is a function of the ionic strength, pH, and concentration of protein. The octamer dissociates by losing dimers of H2A,H2B until the main complexes existing in solution are the H3.H4 tetramer and the H2A.H2B dimer. This process is reversible upon reestablishing the original conditions.

Alkylation↗
PubMed1979

H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle.

The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.

Animals↗
PubMed1979

In vitro core particle and nucleosome assembly at physiological ionic strength.

Nucleosome core particles have been efficiently assembled in vitro by direct interaction of histones and DNA at physiological ionic strength, as assayed by digestion with DNases, supercoiling of relaxed circular DNA, and electron microscopy. Reconstitution was achieved either by the simultaneous addition of all core histones, or by the sequential binding of H3 . H4 tetramer and H2A . H2B dimer to DNA. Micrococcal nuclease digestion and electron microscopy studies indicated that there is heterogeneity in the spacings at which core particles are assembly on the DNA. Length measurements of oligomeric DNA produced during the course of the digestion suggest that the core histone octamer can organize 167 (+/- 4) rather than 145 base pairs of DNA, the extra 20 base pairs being quickly digested. Binding of histone H1 to core particles resulted in the protection of about 165 base pairs of DNA from nuclease attack. Because the core histone octamer is fully dissociated into H3 . H4 tetramer and H2A . H2B dimer at physiological ionic strength, our results would suggest that in vivo core particle assembly may also occur by interaction of these two complexes on the nascent DNA.

Animals↗