RESUMO
Cytogenetic analyses of human colon cancer cells have revealed a high frequency of chromosome 1p deletions among other chromosomal abnormalities. In order to find out whether these chromosomal alterations are manifestations of loss of genetic material, we surveyed DNA of 62 primary tumors, 7 metastases, and matching peripheral blood cells with a panel of polymorphic DNA probes that detect different loci on chromosome 1p. A portion of the probes was derived from a microclone bank generated by microdissection and microcloning of 1p35----pter DNA. In 42% of the colon carcinomas allelic loss was observed with at least one probe. The deletions were of different sizes but always included a region involving band 1p35, except for two tumors in which allelic loss was detected more proximally. The frequency of 1p deletion in the metastases was higher than in the primary tumors. These data indicate that genetic information related to tumorigenesis is located within or nearby region 1p35 and that deletion of this region occurs later in tumor development. Our results add to the number of genetic changes presumably involved in colon cancerogenesis.
Assuntos
Carcinoma/genética , Cromossomos Humanos Par 1 , Neoplasias Colorretais/genética , Alelos , Carcinoma/patologia , Deleção Cromossômica , Mapeamento Cromossômico , Neoplasias Colorretais/patologia , Sondas de DNA , Heterozigoto , Humanos , Polimorfismo de Fragmento de RestriçãoRESUMO
N-Myc encodes a nuclear phosphoprotein that contains a basic region (BR), a helix-loop-helix (HLH) and a leucine zipper (Zip). These motifs are hallmarks of certain transcription factors. In pursuit of the question if N-Myc can activate transcription, we have employed an experimental model involving the yeast transcription factor Gal4. We have first generated fusion proteins containing the Gal4 DNA-binding domain joined to portions of N-Myc. Subsequently we have analysed if chimeric proteins can transactivate the transcription of a reporter under the control of Gal4 binding sites. Here we show that the amino terminal portion of N-Myc activates transcription. Activation maps to a domain highly conserved among Myc-proteins and to other non-conserved sequences, suggesting functional redundancy. Previous studies had documented in vitro association of the RB1 protein with N-Myc (Rustig et al., 1991). We here confirm this observation and identify the region encompassing the transactivation domain as responsible for RB1 binding. Analyses of N-Myc transactivation in retinoblastoma cell line WERI lacking a function RB1 protein gave results similar to those with cell lines having an intact RB1 protein, showing that RB1 protein is not required for transactivation by N-Myc. The present findings leave open the question if deregulated expression of N-Myc contributes to tumorigenesis by transcriptional activation of as yet unidentified target genes or by functionally inactivating the protein encoded by the tumor suppressor gene RB1, or by a combination of both.
Assuntos
Proteínas Fúngicas/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Transcrição Gênica , Proteínas de Ligação a DNA , Células HeLa , Humanos , Luciferases/análise , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/genética , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases alpha and delta in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina A/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase I/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Vírus Miúdo do Camundongo/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Replicação Viral , Animais , Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Humanos , Camundongos , Vírus Miúdo do Camundongo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A , Proteínas não Estruturais Virais/metabolismoRESUMO
Human neuroblastomas show a high incidence of deletions in the distal region of the short arm of chromosome 1. In pursuit of a molecular analysis of these deletions, we have generated a microclone bank from microdissected 1p35-pter chromosomal fragments. To allow a rapid localization of the microclones, we have also generated a panel of (human x mouse) hybrid cell lines through microcell-mediated chromosome transfer. The hybrid cells contained different portions of the human chromosome 1 on a murine background. A total of 20 randomly chosen single or low-copy microclones were localized by Southern analysis on DNA of the hybrid panel: All probes were derived from chromosome I. Sixteen mapped in region 1p36.1-pter, two in 1p22-p36.1, and another two in 1cen-qter. The mapping of ten of these microclones was further refined by in situ hybridization. Cells of the neuroblastoma line GI-ME-N carry two types of chromosome 1, one cytogenetically normal and another with a translocation reported to be in 1p36.2, i.e., a t(1;?) (p36.2;?) marker. Using cell hybridization, we separated the two chromosome 1 types of GI-ME-N into different hybrid cell clones. Southern hybridization of three microclones from distal Ip to DNA of the hybrid cell clones revealed that the breakpoint in the translocated chromosome I was located in 1p36.1.
Assuntos
Deleção Cromossômica , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Neuroblastoma/genética , Bacteriófago lambda , Southern Blotting , Clonagem Molecular/métodos , DNA de Neoplasias/genética , Dissecação/métodos , Vetores Genéticos , Humanos , Células Híbridas , Cariotipagem , Metáfase/genética , Vírus da Leucemia Murina de Moloney , Hibridização de Ácido NucleicoRESUMO
We have characterized two previously cloned genes, F1 and F2 (1) that code for elongation factor EF - 1 alpha of Drosophila melanogaster. Genomic Southern blot hybridization revealed that they are the only gene copies present. We isolated cDNA clones of both transcripts from embryonal and pupal stage of development that cover the entire transcription unit. The 5' ends of both genes have been determined by primer extension and for F1 also by RNA sequencing. These start sites have been shown to be used consistently during development. Comparison of cDNA and genomic sequences revealed that EF - 1 alpha,F1 consists of two and EF - 1 alpha,F2 of five exons. The two described elongation factor genes exhibit several regions of strong sequence conservation when compared to five recently cloned eucaryotic elongation factors.
Assuntos
Drosophila melanogaster/genética , Fatores de Alongamento de Peptídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica , Genes , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The complete sequence of a 40247 bp DNA segment located on the left arm of chromosome X of Saccharomyces cerevisiae has been determined and analysed. The sequence encodes the 5' coding region of the URA2 gene and 18 open reading frames of at least 100 amino acids. Ten of these correspond to known genes, whereas eight correspond to new genes. In addition, the sequence contains a tRNA-Ala gene, a tRNA-Asp gene, a Ty4 transposable element and three delta elements.
Assuntos
Cromossomos Fúngicos/genética , DNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Elementos de DNA Transponíveis , Fatores de Transcrição GATA , Genes Fúngicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
We have recently isolated a rat cDNA encoding a novel cellular protein able to interact with the major nonstructural protein NS1 of parvovirus H-1 and have termed this protein SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein. Here we report the isolation of a cDNA from human placenta encoding the human homologue, human SGT. SGT from rat and human contain 314 and 313 amino acids, respectively, and share 91% sequence identity at the protein level. The highest degree of similarity is present within the central region containing three TPR motifs in tandem array. The similarities, however, also extend beyond this region. Human SGTtranscript was found to be ubiquitously present in all human tissues tested. By fluorescence in situ hybridization analysis we have mapped the human gene to chromosome 19p13. The SGT-coding sequences are evolutionarily conserved, since we could identify genes encoding proteins of similar size and structure in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans.
Assuntos
Caenorhabditis elegans/genética , Proteínas/química , Proteínas/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Proteínas de Transporte , Mapeamento Cromossômico , Cromossomos Humanos Par 19/genética , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/isolamento & purificação , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas/isolamento & purificação , Ratos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Proteins encoded by the proto-oncogenes c-myc, L-myc, and N-myc contain at their carboxy-terminus a tripartite segment comprising a basic DNA binding region (BR), a helix-loop-helix (HLH) and a leucine zipper motif (Zip), that are believed to be involved in DNA binding and protein-protein interaction. The N-Myc oncoprotein is overexpressed in certain human tumors that share neuroectodermal features due to amplification of the N-myc gene. Using a monoclonal antibody directed against an N-terminal epitope of the N-Myc protein in immunoprecipitations performed with extracts of neuroblastoma cells, two nuclear phosphoprotein, p20/22, forming a hetero-oligomeric complex with N-Myc are identified. Both proteins are phosphorylated by casein kinase II in vitro. By partial proteolytic maps we show that p20 and p22 are structurally related to each other and that p20 is identical with Max, a recently described in vitro binding partner of myc proteins. Time course experiments show the presence of the complex in cellular extracts immunoprecipitated within a 5 min interval after the preparation of the cell extract. While the expression of N-myc is restricted, expression of both Max(p20/22) and the murine homolog Myn(p20/22) was observed in cells of diverse human and murine embryonal lineages as detected by heterologous complex formation. By introduction of expression vectors containing the wild type N-myc gene or N-myc genes with in frame deletions or point mutations into recipient cells and subsequent immunoprecipitation of the resulting N-Myc proteins we show that the HLH-Zip region is essential to the formation of the N-Myc-p20/22 complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neuroblastoma/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Caseína Quinases , Humanos , Zíper de Leucina , Camundongos , Mutagênese , Neuroblastoma/patologia , Fosforilação , Testes de Precipitina , Biossíntese de Proteínas , Proteínas Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Electrophoretic mobility shift assays performed with nuclear extracts from human fibroblasts revealed the formation of two major protein complexes with an oligonucleotide (nucleotides 78 to 107) from the palindromic region located upstream from the minute virus of mice (MVM) P4 promoter. It was shown that this oligonucleotide bound USF at the enhancer E box CACATG. The second complex contained the transcription factor NF-Y, whose association was surprising because its target sequence lacks the canonical CCAAT motif present in all mammalian NF-Y binding sites identified so far. The MVM NF-Y recognition element instead contains the CCAAC sequence. USF and NF-Y had distinct but overlapping sequence requirements for binding, suggesting that their associations with MVM DNA were mutually exclusive. Because of the palindromic nature of MVM DNA terminal sequences, NF-Y associated with the three nucleotide configurations corresponding to the hairpin structure and to the external and internal arms of the extended duplex replication form, respectively. However, owing to the imperfection of the palindrome, the binding of USF was restricted to the internal arm. Point mutations that suppressed the in vitro binding of NF-Y to the internal palindromic arm reduced the activity of the resident P4 promoter, while those preventing complex formation with USF did not, as determined by transient expression assays using the luciferase reporter gene. The data led to the identification of a novel P4 upstream regulatory region capable of interacting with two transcription factors, from which one (NF-Y) appeared to upmodulate the activity of the promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus Miúdo do Camundongo/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , DNA Viral/metabolismo , Humanos , Dados de Sequência Molecular , Fatores de Transcrição/metabolismo , Fatores Estimuladores UpstreamRESUMO
The nonstructural protein NS1 of autonomous parvoviruses is essential for viral DNA amplification and gene expression and is also the major cytopathic effector of these viruses. NS1 acts as nickase, helicase, and ATPase and upregulates P38-driven transcription of the capsid genes. We report here the identification of a novel cellular protein that interacts with NS1 from parvovirus H-1 and which we termed SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein. The cDNA encoding full-length SGT was isolated through a two-hybrid screen with, as bait, the truncated NS1dlC69 polypeptide, which lacks the C-terminal transactivation domain of NS1. Full-length NS1 and SGT interacted in the two-hybrid system and in an in vitro interaction assay. Northern blot analysis revealed one major transcript of about 2 kb that was present in all rat tissues investigated. Rat sgt cDNA coded for 314 amino acids, and the protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 34 kDa. SGT could be detected in both the nucleus and the cytoplasm of rat cells, as determined by indirect immunofluorescence analysis and Western blotting of fractionated cellular extracts with an affinity-purified antiserum raised against recombinant SGT (AC1.1). In H-1 virus-infected rat and human cells, compared to mock-infected controls, differences in the migration of SGT polypeptides were revealed after Western blot analysis of total cellular extracts. Moreover, the transient expression of NS proteins was sufficient to induce SGT modification. These results show that cellular SGT, which we have identified as an NS1-interacting protein, is modified by parvovirus infection as well as NS expression.
Assuntos
Parvovirus/metabolismo , Proteínas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Complementar , DNA Viral , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Saccharomyces cerevisiae , Homologia de Sequência de AminoácidosRESUMO
The nonstructural NS2 proteins of autonomous parvoviruses are known to act in a host cell-dependent manner and to play a role in viral DNA replication, efficient translation of viral mRNA, and/or encapsidation. Their exact function during the parvovirus life cycle remains, however, still obscure. We report here the characterization of the interaction with the NS2 proteins from the parvovirus minute virus of mice (MVM) and rat as well as mouse homologues of the human CRM1 protein, a member of the importin-beta family recently identified as an essential nuclear export factor. Using the two-hybrid system, we could detect the interaction between the carboxy-terminal region of rat CRM1 and each of the three isoforms of NS2 (P [or major], Y [or minor], and L [or rare]). NS2 proteins were further shown to interact with the full-length CRM1 by coimmunoprecipitation experiments using extracts from both mouse and rat cell lines. Our data show that CRM1 preferentially binds to the nonphosphorylated isoforms of NS2. Moreover, we observed that the treatment of MVM-infected cells with leptomycin B, a drug that specifically inhibits the CRM1-dependent nuclear export pathway, leads to a drastic accumulation of NS2 proteins in the nucleus. Both NS2 interaction with CRM1 and nuclear accumulation upon leptomycin B treatment strongly suggest that these nonstructural viral proteins are actively exported out of the nuclei of infected cells via a CRM1-mediated nuclear export pathway.
Assuntos
Proteínas de Transporte/metabolismo , Carioferinas , Vírus Miúdo do Camundongo/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular , Núcleo Celular , Clonagem Molecular , DNA Complementar , Ácidos Graxos Insaturados/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosforilação , Testes de Precipitina , Isoformas de Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/genética , Proteína Exportina 1RESUMO
The complete sequence of a 36 196 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. The sequence includes the 5' coding region of the SNF2 gene, the CPA1 leader peptide sequence and 17 open reading frames (ORFs) of at least 100 amino acids. Two of these correspond to previously known genes (CPA1, SLY41), whereas 15 correspond to new genes. The putative translation products of three ORFs show significant similarity with known proteins: one is a putative transport ATPase, another appears to be a ribosomal protein, and the third is an Snf2p homologue.
Assuntos
Cromossomos Fúngicos/genética , Genes Fúngicos/genética , Proteínas Nucleares , Fases de Leitura Aberta/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
The large nonstructural protein NS1 of the minute virus of mice and other parvoviruses is involved in essential steps of the viral life cycle, such as DNA replication and transcriptional regulation, and is a major contributor to the toxic effect on host cells. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to NS1. Homo-oligomerization is a prerequisite for a number of proteins to be fully functional. In particular, helicases generally act as homo-oligomers. Indirect evidence of NS1 self-association has been recently obtained by a nuclear cotransport assay (J. P. Nüesch and P. Tattersall, Virology 196:637-651, 1993). In order to demonstrate the oligomerizing property of NS1 in a direct way and localize the protein region(s) involved, the yeast two-hybrid system was used in combination with deletion mutagenesis across the whole NS1 molecule, followed by high-resolution mapping of the homo-oligomerization domain by a peptide enzyme-linked immunosorbent assay method. This study led to the identification of a distinct NS1 peptide that contains a bipartite domain involved in NS1 oligomerization. Furthermore, this isolated peptide was found to act as a specific competitive inhibitor and suppress NS1 helicase activity in vitro and parvovirus DNA replication in vivo, arguing for the involvement of NS1 oligomerization in these processes. Our results point to drug targeting of oligomerization motifs of viral regulatory proteins as a potentially useful antiviral strategy.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Vírus Miúdo do Camundongo/fisiologia , Fragmentos de Peptídeos/farmacologia , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Animais , Sítios de Ligação , DNA Helicases/química , Proteínas de Ligação a DNA , Proteínas Fúngicas/biossíntese , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/biossíntese , Replicação Viral/efeitos dos fármacosRESUMO
We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells.
Assuntos
Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Parvovirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Proteínas de Transporte , Linhagem Celular , Núcleo Celular/metabolismo , Replicação do DNA , Humanos , Hibridização in Situ Fluorescente , Microscopia Confocal , Microscopia Eletrônica , Chaperonas Moleculares , Infecções por Parvoviridae/virologia , Parvovirus/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas/metabolismo , RatosRESUMO
At least 70% of human neuroblastomas display cytogenetically visible aberrations in the short arm of chromosome 1. We have used a panel of probes detecting polymorphic DNA loci, most of which were derived from a library of microdissected distal 1p chromosome fragments, to compare the hybridization pattern of DNA on nine different tumors and the corresponding normal tissue. In eight of the neuroblastomas allelic loss was observed with at least two probes. The deletions were of different size. Since a consensus deletion in all eight tumors included the segment 1p36.1-2, we conclude that genetic information related to neuroblastoma tumorigenesis is located within this approximately 10 megabase segment. Previous studies have revealed the amplification of MYCN in neuroblastomas. Our study did not provide evidence for a correlation between MYCN amplification and the 1p deletion, suggesting that the two genetic alterations result from molecular mechanisms that are not directly related to each other.
Assuntos
Deleção Cromossômica , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Neuroblastoma/genética , Alelos , Sequência Consenso/genética , Sondas de DNA , DNA de Neoplasias/análise , Amplificação de Genes/genética , Marcadores Genéticos/genética , Heterozigoto , Humanos , Polimorfismo Genético/genéticaRESUMO
Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP), an enzyme which uses NAD+ as substrate. Binding of PARP to DNA single-strand or double-strand breaks leads to enzyme activation. Inhibition of poly(ADP-ribose) formation impairs the cellular recovery from DNA damage. Here we describe stable transfectants of the Chinese hamster cell line CO60 that constitutively overexpress human PARP (COCF clones). Immunofluorescence analysis of gamma-irradiation-stimulated poly(ADP-ribose) synthesis revealed consistently larger fractions of cells positive for this polymer in the COCF clones than in control clones, which failed to express human PARP. HPLC-based quantitative determination of in vivo levels of poly(ADP-ribose) confirmed this result and revealed that the basal polymer levels of undamaged cells were significantly higher in the COCF clones. The COCF clones were sensitized to the cytotoxic effects of gamma irradiation compared with control transfectants and parental cells. This effect could not be explained by depletion of cellular NAD+ or ATP pools. Together with the well-known cellular sensitization by inhibition of poly(ADP-ribosyl)ation, our data lead us to hypothesize that an optimal level of cellular poly(ADP-ribose) accumulation exists for the cellular recovery from DNA damage.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Adenosina Difosfato Ribose/efeitos da radiação , Células CHO/enzimologia , Poli(ADP-Ribose) Polimerases/biossíntese , Trifosfato de Adenosina/efeitos da radiação , Animais , Células CHO/metabolismo , Células CHO/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Cricetinae , Raios gama , Humanos , NAD/efeitos da radiação , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/efeitos da radiação , TransfecçãoRESUMO
Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.
Assuntos
Cromossomos Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Fúngico , Fases de Leitura AbertaRESUMO
The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome.
Assuntos
Cromossomos Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Centrômero , Mapeamento Cromossômico , Códon de Terminação , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta , RNA de Transferência/química , TelômeroRESUMO
In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.