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George W Smith - One of the best experts on this subject based on the ideXlab platform.

  • quantitative analysis of messenger rna abundance for ribosomal protein l 15 cyclophilin a phosphoglycerokinase β glucuronidase glyceraldehyde 3 phosphate dehydrogenase β actin and Histone H2A during bovine oocyte maturation and early embryogenesis in vitro
    Molecular Reproduction and Development, 2006
    Co-Authors: Anilkumar Bettegowda, Osman V Patel, J J Ireland, George W Smith
    Abstract:

    Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and Histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and Histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.

  • quantitative analysis of messenger rna abundance for ribosomal protein l 15 cyclophilin a phosphoglycerokinase β glucuronidase glyceraldehyde 3 phosphate dehydrogenase β actin and Histone H2A during bovine oocyte maturation and early embryogenesis in vitro
    Molecular Reproduction and Development, 2006
    Co-Authors: Anilkumar Bettegowda, Osman V Patel, J J Ireland, George W Smith
    Abstract:

    Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, mea- surement of gene expression generally requires selec- tion of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic develop- ment, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have vali- dated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, b-glucuronidase, glyceralde- hyde-3-phosphate dehydrogenase, b-actin, and his- tone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed accep- table rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycer- okinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and Histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development. Mol. Reprod. Dev. 73: 267-278, 2006. 2005 Wiley-Liss, Inc.

Vesna Rapicotrin - One of the best experts on this subject based on the ideXlab platform.

  • monoubiquitinated Histone H2A destabilizes photolesion containing nucleosomes with concomitant release of uv damaged dna binding protein e3 ligase
    Journal of Biological Chemistry, 2012
    Co-Authors: Satoshi Nakajima, Maria G Kapetanaki, Ching L Hsieh, Matthew V Fagerburg, Karen Thickman, Pedro Rodriguezcollazo, Sanford H Leuba, Arthur S. Levine, Vesna Rapicotrin
    Abstract:

    How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4BDDB2. We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating Histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4BDDB2 E3 ligase specifically binds to mononucleosomes assembled with human recombinant Histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4BDDB2 from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.

  • the cullin 4b based uv damaged dna binding protein ligase binds to uv damaged chromatin and ubiquitinates Histone H2A
    Cancer Research, 2008
    Co-Authors: Jennifer Guerrerosantoro, Maria G Kapetanaki, Ching L Hsieh, Arthur S. Levine, Ilya Gorbachinsky, Vesna Rapicotrin
    Abstract:

    By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)–based ubiquitin ligase, DDB1-CUL4ADDB2. We previously showed that DDB1-CUL4ADDB2 ubiquitinates Histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4ADDB2 and DDB1-CUL4BDDB2. To investigate CUL4B9s possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4BDDB2 E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4BDDB2 is more efficient than DDB1-CUL4ADDB2 in monoubiquitinating Histone H2A in vitro. Overall, this study suggests that DDB1-CUL4BDDB2 E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER. [Cancer Res 2008;68(13):5014–22]

  • the ddb1 cul4addb2 ubiquitin ligase is deficient in xeroderma pigmentosum group e and targets Histone H2A at uv damaged dna sites
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Maria G Kapetanaki, Ching L Hsieh, Vesna Rapicotrin, Jennifer Guerrerosantoro, Dawn C Bisi, Arthur S. Levine
    Abstract:

    Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4ADDB2. The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated Histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4ADDB2 complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4ADDB2 ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets Histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated Histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that Histone modification, mediated by the XPE factor, facilitates the initiation of NER.

Anilkumar Bettegowda - One of the best experts on this subject based on the ideXlab platform.

  • quantitative analysis of messenger rna abundance for ribosomal protein l 15 cyclophilin a phosphoglycerokinase β glucuronidase glyceraldehyde 3 phosphate dehydrogenase β actin and Histone H2A during bovine oocyte maturation and early embryogenesis in vitro
    Molecular Reproduction and Development, 2006
    Co-Authors: Anilkumar Bettegowda, Osman V Patel, J J Ireland, George W Smith
    Abstract:

    Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and Histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and Histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.

  • quantitative analysis of messenger rna abundance for ribosomal protein l 15 cyclophilin a phosphoglycerokinase β glucuronidase glyceraldehyde 3 phosphate dehydrogenase β actin and Histone H2A during bovine oocyte maturation and early embryogenesis in vitro
    Molecular Reproduction and Development, 2006
    Co-Authors: Anilkumar Bettegowda, Osman V Patel, J J Ireland, George W Smith
    Abstract:

    Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, mea- surement of gene expression generally requires selec- tion of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic develop- ment, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have vali- dated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, b-glucuronidase, glyceralde- hyde-3-phosphate dehydrogenase, b-actin, and his- tone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed accep- table rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycer- okinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and Histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development. Mol. Reprod. Dev. 73: 267-278, 2006. 2005 Wiley-Liss, Inc.

Concepción J. Puerta - One of the best experts on this subject based on the ideXlab platform.

  • comparative analysis of trypanosoma rangeli Histone H2A gene intergenic region with distinct intraspecific lineage markers
    Vector-borne and Zoonotic Diseases, 2009
    Co-Authors: Concepción J. Puerta, Thais Cristine Marques Sincero, Claudia Cuervo, Patricia Hermes Stoco, Edmundo C Grisard
    Abstract:

    Abstract This study shows the characterization of the Histone H2A intergenic region sequences (H2A IR) from Trypanosoma rangeli KP1(+) and KP1(−) strains isolated from distinct hosts and geographic regions. Also, a comparative unweighted pair-group method using arithmetic averages (UPMGA) analysis with polymerase chain reaction profiles of the 24Sα rDNA and the miniexon genes was performed. Detailed H2A IR sequence analysis revealed a discrete size polymorphism among T. rangeli strains and the presence of single-nucleotide polymorphisms and minisatellite repeats, exclusively allowing an interspecific differentiation from T. cruzi strains representing the main parasite lineages. Differently from the H2A IR, UPMGA analysis of the 24Sα rDNA and the miniexon genes profiles clearly branched T. rangeli strains into KP1(−) and KP1(+) lineages, clustering separately the Brazilian and Colombian KP1(−) strains. The evolutionary implications of these findings are discussed.

  • detection of trypanosoma cruzi and trypanosoma rangeli infection in triatomine vectors by amplification of the Histone H2A sire and the sno rna c11 genes
    Revista Do Instituto De Medicina Tropical De Sao Paulo, 2007
    Co-Authors: Paula Pavia, Gustavo Adolfo Vallejo, Marleny Montilla, Ruben Santiago Nicholls, Concepción J. Puerta
    Abstract:

    Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the Histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

  • the trypanosoma rangeli Histone H2A gene sequence serves as a differential marker for kp1 strains
    Infection Genetics and Evolution, 2006
    Co-Authors: Claudia Cuervo, M.c. López, Concepción J. Puerta
    Abstract:

    Abstract Trypanosoma rangeli has recently been divided in two primary lineages denoted as KP1(+) and KP1(−) strains because of epidemiological and evolutionary interest in the molecular differentiation of these two groups. We report the molecular characterization of the genes encoding Histone H2A protein from a T. rangeli KP1(+) strain (H14), its comparison to T. rangeli KP1(−) strain (C23) Histone H2A coding genes [Puerta, C., Cuervo, P., Thomas, M.C., Lopez, M.C., 2000. Molecular characterization of the Histone H2A gene from the parasite Trypanosoma rangeli . Parasitol. Res. 86, 916–922], and its application in a low-stringency single specific primer polymerase chain reaction (LSSP-PCR) assay to differentiate these parasite groups. The results show that the locus encoding the H2A protein in the H14 strain is formed by at least 11 gene units measuring 799 nucleotides in length, organized in tandem, and located in two chromosomes of approximately 1.9 and 1.1 Mb in size. Remarkably, in KP1(−) strains these genes are on pairs of chromosomes of about 1.7 and 1.9 Mb. In addition, there is a hybridization signal in the compression region above 2.1 Mb in all T. rangeli strains. Therefore, the chromosomal location of these genes is a useful marker to distinguish between KP1(+) and KP1(−) T. rangeli strains. The alignment of the H2A nucleotide sequences from H14 and C23 strains showed an identity of 99.5% between the coding regions and an identity of 95% between the non-coding regions. The deduced amino acid sequences proved to be identical. Based on 5% of the difference between the intergenic regions, we developed a LSSP-PCR assay which can differentiate between KP1(+) and KP1(−) strains.

Jessica A Downs - One of the best experts on this subject based on the ideXlab platform.

  • Histone H2A phosphorylation in dna double strand break repair
    FEBS Journal, 2005
    Co-Authors: Elinor R Foster, Jessica A Downs
    Abstract:

    DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in Histone H2A or the H2AX Histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that Histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.

  • binding of chromatin modifying activities to phosphorylated Histone H2A at dna damage sites
    Molecular Cell, 2004
    Co-Authors: Jessica A Downs, Stephen P Jackson, Stephane Allard, Olivier Jobinrobitaille, Ali Javaheri, Andreanne Auger, Nathalie Bouchard, Stephen J Kron, Jacques Cote
    Abstract:

    Yeast Histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.

  • a role for saccharomyces cerevisiae Histone H2A in dna repair
    Nature, 2000
    Co-Authors: Jessica A Downs, Noel F Lowndes, Stephen P Jackson
    Abstract:

    Histone proteins associate with and compact eukaryotic nuclear DNA to form chromatin. The basic unit of chromatin is the nucleosome, which is made up of 146 base pairs of DNA wrapped around two of each of four core Histones1, H2A, H2B, H3 and H4. Chromatin structure and its regulation are important in transcription and DNA replication2,3,4. We therefore thought that DNA-damage signalling and repair components might also modulate chromatin structure. Here we have characterized a conserved motif in the carboxy terminus of the core Histone H2A from Saccharomyces cerevisiae that contains a consensus phosphorylation site for phosphatidylinositol-3-OH kinase related kinases (PIKKs). This motif is important for survival in the presence of agents that generate DNA double-strand breaks, and the phosphorylation of this motif in response to DNA damage is dependent on the PIKK family member Mec1. The motif is not necessary for Mec1-dependent cell-cycle or transcriptional responses to DNA damage, but is required for efficient DNA double-strand break repair by non-homologous end joining. In addition, the motif has a role in determining higher order chromatin structure. Thus, phosphorylation of a core Histone in response to DNA damage may cause an alteration of chromatin structure that facilitates DNA repair.