PubMed Health⌕ Search

Biomedical subjects

A Ruiz-Carrillo

Publications and source records attributed to A Ruiz-Carrillo.

At least 19 recordsLinked to original sources
PubMed2001

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.

Animals↗
PubMed1998

Elements regulating differential activity of chicken histone H1 gene promoters.

The chicken genome contains six closely related histone H1 genes, each of which encodes a different H1 protein. The four common regulatory elements previously identified in H1 histone promoters are very similar in sequence and location in all chicken H1 genes, which gives rise to the question of how the six H1 variants are expressed at significantly different levels. Transient transfections of reporter gene transcriptional fusions indicate that approximately 200 bp of each promoter is sufficient to generate the observed spectrum of H1 promoter activity. The differences in H1 promoter-driven expression are shown to be explained by the relative activity of the previously characterized G box region and that of a novel element found between CCAAT and TATA that we have termed differential upstream sequence (Dus). Gel shift analysis indicated that the primary nuclear binding protein to the G box is one or more avian homologues of the Sp1 transcription factor. The Dus region binds multiple nuclear proteins, one of which is the recently described IBR/IBF factor. The differential affinities of the G box and Dus sequences of the H1 promoters for their respective nuclear binding factors correlate well with their relative promoter activities.

Animals↗
PubMed1997

Characterization and expression of the mouse endonuclease G gene.

Endonuclease G (Endo G) is a nuclease of prokaryotic lineage found in the mitochondria of vertebrates that has been suggested to play a role in mitochondrial DNA (mtDNA) replication. We have isolated and sequenced the entire mouse endo G gene, determined the limits of the mRNA, and mapped the promoter region. The coding sequence of the single copy gene is interrupted by two introns and analysis of the transcripts does not support a model by which more than one Endo G isoform could be produced by alternative splicing. We have also characterized a full-length human Endo G cDNA and comparison at the protein level of the human, bovine, and murine nucleases indicates a high degree of conservation except in the respective mitochondrial targeting signals. Endo G is ubiquitously expressed and the steady-state levels of its mRNA vary by a factor greater than seven between different tissues. The relationship between the mtDNA copy number and Endo G mRNA levels is not strictly proportional but tissues richer in mtDNA have higher amounts of the mRNA and vice versa.

Alternative Splicing↗
PubMed1995

Chromosomal localization of mitochondrial transcription factor A (TCF6), single-stranded DNA-binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping genes involved in mitochondrial biogenesis.

By using a PCR-based screening of a somatic cell hybrid panel and FISH, we have assigned the loci of mitochondrial single-stranded DNA-binding protein (SSBP), mitochondrial transcription factor A (TCF6), and mitochondrial endonuclease G (ENDOG) genes to human chromosomes 7q34, 10q21, and 9q34.1, respectively. The products of these three genes are involved in fundamental aspects of mitochondrial biogenesis, such as replication and transcription of the mitochondrial genome. The chromosomal localization of these genes is important to testing whether the corresponding proteins may play a role in the etiopathogenesis of human disorders associated with qualitative or quantitative abnormalities of mitochondrial DNA.

Chromosome Mapping↗
PubMed1995

Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected].

Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism.

Amino Acid Sequence↗
PubMed1993

Primers for mitochondrial DNA replication generated by endonuclease G.

Endonuclease G (Endo G) is widely distributed among animals and cleaves DNA at double-stranded (dG)n.(dC)n and at single-stranded (dC)n tracts. Endo G is synthesized as a propeptide with an amino-terminal presequence that targets the nuclease to mitochondria. Endo G can also be detected in extranucleolar chromatin. In addition to deoxyribonuclease activities, Endo G also has ribonuclease (RNase) and RNase H activities and specifically cleaves mouse mitochondrial RNA and DNA-RNA substrates containing the origin of heavy-strand DNA replication (OH). The cleavage sites match those found in vivo, indicating that Endo G is capable of generating the RNA primers required by DNA polymerase gamma to initiate replication of mitochondrial DNA.

Amino Acid Sequence↗
PubMed1993

Transcription of the histone H5 gene is regulated by three differentiation-specific enhancers.

Histone H5, an early marker of the avian erythroid lineage, is expressed at low levels in early erythroid precursors and at higher levels in more mature cells. We show that the increase in H5 expression is due to transcriptional activation of the H5 gene following differentiation of precursor CFU(E). We have found and characterized two upstream enhancers, E1 (between -2233 and -1878 from the site of transcription initiation, +1) and E3 (between -1321 and -1163), and confirmed the presence of a downstream enhancer (C. D. Trainor, S. J. Stamler, and J. D. Engel, Nature [London] 328:827-830, 1987) E7 (between +846 and +1181) which are responsible for the increase in H5 gene transcription. The enhancers had a weak effect in nondifferentiated CFU(E) but a strong effect when the cells were induced to differentiate. Cooperation among the three enhancers, however, was not required for H5 gene activity in the differentiated cells. The enhancers contain binding sites for several ubiquitous and erythroid cell-specific nuclear proteins, including GATA-1, as demonstrated with GATA-1-specific antibodies. Although the GATA sites were required for enhancer function, the concentration of GATA-1, GATA-2, and GATA-3 decreased during cell differentiation, and overexpression of these factors had little effect on H5 transcription. Hence, the differentiation-specific effect of the enhancers is not mediated by changes in relative levels of the GATA factors. Functional analysis of the H5 promoter indicated that the requirement of several elements, including a GC box necessary for transcription enhancement, did not change during the early stages of CFU(E) differentiation. However, the UPE, a positive element in proliferating CFU(E) recognized by the transcription factor H4TF2, was dispensable in the differentiated cells. These results suggest that as the cells enter the final stages of differentiation, there is a reprogramming of the regulatory factors that control H5 transcription and that the enhancers rescue and increase the activity of the promoter.

Amino Acid Sequence↗
PubMed1992

Repression of the H5 histone gene by a factor from erythrocytes that binds to the region of transcription initiation.

Expression of histone H5, like that of other erythrocyte specific proteins, declines during the latter stages of erythroid maturation because of a decrease in the rate of gene transcription. Here, we report the isolation of cIBR (chicken initiation binding repressor), a 75 kDa DNA binding glycoprotein from mature chicken erythrocytes that recognizes sequences spanning the transcription start sites of the H5 gene. cIBR was found to repress transcription from the H5 promoter in vitro and this effect could be relieved by mutations that lowered the affinity of the factor for its cognate sequence. cIBR inhibited transcription by interfering with assembly of the initiation complex, but it did not affect transcription from pre-assembled complexes. Consistent with this, binding of bacterially expressed human TFIID to the TATA element prevented subsequent binding of cIBR, although the opposite was not true. This, and the fact that cIBR had no effect when bound in a location upstream from the promoter, suggests that binding of cIBR to the start site region causes repression by direct interference with general transcription factors other than TFIID, possibly TFIIB. cIBR was found in mature and relatively late erythrocytes but not in early erythroid cells which actively transcribe the H5 gene; the transcriptionally active cells contain instead cIBF (chicken initiation binding factor). Purified cIBF is a non-glycosylated 68-70 kDa DNA binding protein(s) which also recognizes the region of transcription initiation of the H5 gene.

Animals↗
PubMed1992

Increased histone H1(0) expression in differentiating mouse erythroleukemia cells is related to decreased cell proliferation.

The amount of histone H1(0) increases relative to other H1 subtypes in terminally differentiated cells, and its expression has been associated with the onset of differentiation. We have studied the kinetics of H1(0) accumulation in mouse erythroleukemia (MEL) cells and found that the levels of H1(0) reflect the rate of cell proliferation rather than the state of differentiation. This suggests that changes in the relative amount of H1(0) during MEL cell differentiation are primarily a consequence of cell cycle arrest.

Animals↗
PubMed1990

Replacement of histone H1 by H5 in vivo does not change the nucleosome repeat length of chromatin but increases its stability.

In vivo competition between histones H1 and H5 for chromatin has been studied in rat sarcoma XC10 cells transfected with a glucocorticoid responsive MMTV-H5 gene. Activation of H5 expression results in accumulation of H5 in the nuclei where it partially replaces H1. H5 displaces H1 from its primary binding sites presumably during chromatin replication and also binds with high affinity to secondary chromatin sites normally not occupied by H1. Replacement of H1 by H5 to levels similar to those of mature chicken erythrocytes does not alter the nucleosome repeat length of chromatin. This indicates that H5 is not solely responsible for the increase in nucleosome spacing of maturing erythroid cells. Exchange of H1 by H5 in vivo or in vitro results in a higher compaction/stability of chromatin.

Animals↗
PubMed1989

Basal expression of the histone H5 gene is controlled by positive and negative cis-acting sequences.

Sequences from -3500 to +1365 of the chicken histone H5 gene have been analyzed for the presence of cis-acting elements in H5 expressing (transformed CFU-E) and non-expressing cells (fibroblasts). The region from -3500 to -115 had little effect on transcription. Proximal upstream sequences contain a negative element (UNE, -115 to -95), capable to also repress the activity of the heterologous HSV tk promoter, and two positive elements, a consensus GC-box (-83 to -74) and a proximal element (UPE, -54 to -38). The sequence of the UPE is highly related to the histone H4 subtype-specific element and it has been conserved in the duck H5 and the human and mouse H1(0) genes at equivalent positions. Although the effect of the UNE, GC-box and UPE was not tissue-specific, sequences from -38 to +77 appear to confer a degree of tissue specificity to the promoter. An activating erythroid-specific element (DE) was found downstream of the H5 gene (+1042 to +1185). The activity of the DE was modest but independent of position and orientation and required the presence of the promoter proximal elements. The DE harbors the sequence AGATAA that is recognized by a protein factor, presumably the same that binds to other erythrocyte-specific enhancers. The low activity of DE in the CFU-E may be related to the low concentration of the AGATAA-binding factor in the differentiation-blocked cells.

Animals↗
PubMed1989

Histone H5 in the control of DNA synthesis and cell proliferation.

The linker histones (H1, H5, H1 degrees) are involved in the condensation of chromatin into the 30-nanometer fiber. This supranucleosome organization correlates with the resting state of chromatin, and it is therefore possible that the linker histones play an active role in the control of chromatin activity. The effect of H5 has been directly determined by expression of an inducible transfected H5 gene in rat sarcoma cells, which do not produce H5. Transfection resulted in the reversible inhibition of DNA replication and arrest of cells in G1, at which time H5 concentrations approached that of terminally differentiated avian erythrocytes. The arrest of proliferation was accompanied by specific changes in gene expression probably related to the cell cycle block. The selectivity of these effects suggest that H5 plays an active role in the control of DNA replication and cell proliferation.

Animals↗
PubMed1989

Recognition of (dG)n.(dC)n sequences by endonuclease G. Characterization of the calf thymus nuclease.

We report the purification of endonuclease G (Ruiz-Carrillo, A., and Renaud, J. (1987) EMBO J. 6, 401-407) from calf thymus nuclei and whole tissue. The enzyme has been enriched 29,000-fold, and the activity was unambiguously identified with a 26-kDa protein after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native nuclease behaves as a 50-kDa species by gel filtration, suggesting that it is composed of two subunits, presumably identical. In terms of absolute amounts, endonuclease G (endo G) is a nuclear enzyme although it was also detected in purified mitochondria. Endo G is highly specific for (dG)n.(dC)n tracts in DNA, nicking either strand of relaxed substrates with similar kinetics. The sensitivity of the homopolymer tracts is proportional to their length (from n = 8 to 29), insofar as the flanking sequences are constant. However, the overall rate of cleavage is influenced by the composition of the flanking DNA. Minor cleavage sites contain shorter (dG)n.(dC)n clusters (n = 3-7). Endo G efficiently cleaves (dC)n but not (dG)n runs in single-stranded DNA, suggesting that it may recognize an asymmetric strand conformation of the homopolymer tracts. Endo G does not recognize other homo(co)-polymer sequences or cruciform structures in DNA.

Animals↗
PubMed1988

The globular region of histone H5 is equally accessible to antibodies in relaxed and condensed chromatin.

The accessibility of histone H5 in chromatin was examined with monoclonal antibodies recognizing several epitopes of the globular region (GH5) of the histone (Rózalski, M., Lafleur, L., and Ruiz-Carrillo, A. (1985) J. Biol. Chem. 260, 14379-14385). The stoichiometry of the chromatin-antibody complexes indicated that while 0-86% of the H5 molecules were able to react, depending on the particular epitope, the extent of antibody binding to relaxed chromatin (in 5 mM KCl) and condensed chromatin (in 100 mM KCl or 0.35 mM MgCl2) was virtually identical. This indicates that the topography of H5 does not change during the conformational transition of chromatin. The data suggest that H5 is not completely internalized in the 30-nm fiber or that the fiber is flexible enough to allow full exposure of the GH5 epitopes. Several control experiments, including monoclonal antibody binding, sedimentation analysis, DNase II digestion, and glutaraldehyde cross-linking, showed that epitope accessibility is not due to H5 exchange or to perturbation of the chromatin fiber. The accessibility of GH5 suggests ways in which inactive chromatin may be unfolded in vivo.

Animals↗
PubMed1987

Mismatch and blunt to protruding-end joining by DNA ligases.

A nuclear DNA ligase activity from immature chicken erythrocytes, and to a lesser extent T4-induced DNA ligase, can join cohesive-ends (3 and 5-nucleotides long) having one of the mismatches, A/A, T/T, C/C, G/G, at the middle position. The rate of ligation depends on the length and stability of the mispaired intermediate (G/G, T/T greater than A/A, C/C). When the non-complementary overhanging-ends are short (i.e. 1-nucleotide) both ligases catalyze the joining of the single-stranded protruding-end with a blunt-end. This reaction occurs at low but significant rates compared to blunt-end ligation. The chicken ligase has lower flush-end joining activity than T4 DNA ligase, but it is more permissive since it joins C/C or A/A mismatched-ends, whereas the prokaryotic ligase does not. Possible biological implications of the reactions are discussed. We have also found that BstEII easily cleaves at sites harboring a C/C or a G/G mismatch at the center of its recognition sequence, whereas AvaII (T/T or A/A), HinfI (G/G) and DdeI (G/G) do not.

Animals↗
PubMed1987

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.

Animals↗
PubMed1987

Regulation of histone and beta A-globin gene expression during differentiation of chicken erythroid cells.

The expression of the genes for several histones and beta A-globin was examined in the chicken erythroid cells lineage. During the transition from CFU-(E) to the mature erythrocyte, histone H5 gradually increased fourfold in nuclei with little concomitant displacement of the H1 histones. This resulted in a 70% net increase in linker histone (H1 plus H5) content. The differential accumulation of H5 reflected (i) an increase in the transcriptional activity of the H5 gene occurring at the erythroblast stage, (ii) an apparent longer half-life of H5 mRNA, and (iii) a higher stability of the protein. Although the transcriptional activity of the histone genes (except H5) decreased with cell age, it was not tightly coupled to the S phase. On the other hand, the mRNA levels for these histones were tightly regulated during the cell cycle. Use of protein and DNA synthesis inhibitors indicated that the content of H5 mRNA was regulated at the posttranscriptional level by a control mechanism(s) differing from those for the other histones. Although the transcription rates of the H5 and beta A-globin genes were comparable, differential accumulation of beta A-globin mRNA led to a 30- to 170-fold-higher copy number of the beta A-globin mRNA as the cell matured.

Animals↗