Can eukaryotic cells monitor the presence of unreplicated DNA?

Completion of DNA replication before mitosis is essential for genome stability and cell viability. Cellular controls called checkpoints act as surveillance mechanisms capable of detecting errors and blocking cell cycle progression to allow time for those errors to be corrected. An important question in the cell cycle field is whether eukaryotic cells possess mechanisms that monitor ongoing DNA replication and make sure that all chromosomes are fully replicated before entering mitosis, that is whether a replication-completion checkpoint exists. From recent studies with smc5–smc6 mutants it appears that yeast cells can enter anaphase without noticing that replication in the ribosomal DNA array was unfinished. smc5–smc6 mutants are proficient in all known cellular checkpoints, namely the S phase checkpoint, DNA-damage checkpoint, and spindle checkpoint, thus suggesting that none of these checkpoints can monitor the presence of unreplicated segments or the unhindered progression of forks in rDNA. Therefore, these results strongly suggest that normal yeast cells do not contain a DNA replication-completion checkpoint.

Mitochondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial DNA mutator mice

Background: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase c, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Dym). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

EndoG links Bnip3-induced mitochondrial damage and caspase-independent DNA fragmentation in ischemic cardiomyocytes

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.

Dano de frio em limas-ácidas Tahiti, colhidas em diferentes épocas e submetidas a tratamentos térmicos e bioquímicos

O estudo objetivou o estabelecimento de um método efetivo e satisfatório do controle do dano de frio em limas-ácidas. Os frutos colhidos no município de Boa Vista-RR, 140 e 150 dias após a floração, apresentaram valores médios de 7,9 e 8,2 ºBrix; 6,3 e 6,0 mL de ácido cítrico.100mL de polpa-1 e pH de 2,8 e 3,0, respectivamente, nas duas colheitas. Após cada colheita, os frutos foram levados ao laboratório de Fitotecnia/UFRR, onde foram selecionados, limpos e submetidos aos tratamentos: T1 - controle; T2, T3 e T4 - condicionamento a 35ºC, por 6, 12 e 24 horas, respectivamente; T5 - aquecimento intermitente a 20ºC, por 8 horas, após 5 e 10 dias a 1ºC; T6 - aquecimento intermitente a 20ºC, por 8 horas, após 10 e 20 dias a 1ºC; T7 - ethephon a 1.500 mg.L-1; T8 - ethephon a 3.000 mg.L-1. Os tratamentos T9 ao T16, diferenciaram-se dos tratamentos T1 a T8, apenas, na data da colheita (10 dias após a primeira). O experimento foi avaliado a cada 15 dias, durante 75 dias, a 1 ± 0,5 ºC e 92 ± 5 % de UR, quanto ao dano de frio, aspecto visual, perda de massa fresca, sólidos solúveis (SS), acidez titulável (AT), SS/AT (ratio - RT), clorofila total e ácido ascórbico. O atraso na colheita não proporcionou efeito significativo algum. Todos os tratamentos, à exceção do controle e do aquecimento intermitente aos 10 e 20 dias, foram eficientes no controle do dano de frio. No entanto, o tratamento químico e o condicionamento térmico aceleraram precocemente o metabolismo dos frutos, principalmente no que concerne à perda de massa fresca e ao aspecto visual. O maior teor de clorofila total e de ácido ascórbico, bem como o melhor aspecto visual, a não-incidência de podridões e a menor perda de massa fresca foram detectadas nos frutos submetidos ao aquecimento intermitente aos 5 e 10 dias. Os SS, AT e RT estavam dentro dos padrões de qualidade e não variaram estatisticamente entre os tratamentos.

Casein Kinase 1γ Ensures Monopolar Growth Polarity under Incomplete DNA Replication Downstream of Cds1 and Calcineurin in Fission Yeast.

Cell polarity is essential for various cellular functions during both proliferative and developmental stages, and it displays dynamic alterations in response to intracellular and extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of polarity transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1γ homolog) is a critical regulator to ensure persistent monopolar growth during S phase. Unlike the wild type, cki3 mutant cells undergo bipolar growth when S phase is blocked, a condition known to delay transition from monopolar to bipolar growth (termed NETO [new end takeoff]). Consistent with this role, Cki3 kinase activity is substantially increased, and cells lose their viability in the absence of Cki3 upon an S-phase block. Cki3 acts downstream of the checkpoint kinase Cds1/Chk2 and calcineurin, and the latter physically interacts with Cki3. Autophosphorylation in the C terminus is inhibitory toward Cki3 kinase activity, and calcineurin is responsible for its dephosphorylation. Cki3 localizes to the plasma membrane, and this localization requires the palmitoyltransferase complex Erf2-Erf4. Membrane localization is needed not only for proper NETO timing but also for Cki3 kinase activity. We propose that Cki3 acts as a critical inhibitor of cell polarity transition under S-phase arrest.

The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages

Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.

Mitochondrial DNA damage and animal longevity: insights from comparative studies

Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA). ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

Identificação de microssatélites para o mamoeiro por meio da exploração do banco de dados de DNA

Os marcadores microssatélites são ferramentas úteis em diversas análises genéticas em plantas. No caso do mamoeiro (Carica papaya L.), poucos locos de microssatélites foram descritos até o momento. Assim, o objetivo deste trabalho foi explorar a base de dados do GenBank / NCBI (National Center of Biotechnoloy Information) à procura de microssatélites de mamoeiro, visando a seu futuro uso em estudos genéticos e moleculares aplicados ao melhoramento genético. As seqüências foram obtidas no GenBank / NCBI, no formato FASTA, e analisadas para a presença de microssatélites com um mínimo de 20; 7 e 5 repetições dos motivos de mono-, di- e trinucleotídeos, respectivamente, e acima de 4 repetições para tetra- e pentanucleotídeos. Seqüências com mais de 90% de similaridade foram consideradas redundantes e, portanto, eliminadas das análises. Foram analisadas 44.591 seqüências, das quais 3.180 foram não-redundantes e apresentaram 3.947 microssatélites. Desse total, 3.587 foram classificados como microssatélites perfeitos, 8 imperfeitos, 65 interrompidos, 239 compostos-perfeitos, 8 compostos-imperfeitos e 40 compostos-interrompidos. As repetições de di- e trinucleotídeos representaram 65,7 e 14,4% do total de seqüências analisadas, respectivamente. Somente os motivos do tipo AT/TA representaram 44,1% dos microssatélites encontrados. Os motivos mais comuns de tri-, tetra- e pentanucleotídeos foram AAT, AATT e TTTAA, respectivamente. Observou-se que, nas seqüências disponíveis, o genoma do mamoeiro apresenta, em média, um microssatélite a cada 5,65 kb.

Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma.

BACKGROUND: HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma. RESULTS: We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively. CONCLUSIONS: Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Suscetibilidade a dano pelo frio em abacaxi 'pérola' tratado com 1-metilciclopropeno

Este trabalho avaliou o efeito de 1-metilciclopropeno (1-MCP) na suscetibilidade ao dano pelo frio em abacaxi 'Pérola', colhido em Santa Rita-PB, na maturidade comercial. Os frutos foram tratados com 1-MCP (0; 300; 600 e 1.200 ppb), por 12 horas, sob condição ambiente e armazenados: a) durante 42 dias a 10ºC, avaliados a cada 7 dias, e quando transferidos para o ambiente (25ºC e 65±5% U.R.), após 21; 28; 35 e 42 dias, e também foram avaliados após sete dias; b) durante 32 dias, a 7ºC, avaliados a cada 8 dias, e transferidos para o ambiente após 8; 16; 24 e 32 dias, sendo avaliados após sete dias. Em frutos mantidos a 10ºC, não se observou efeito do 1-MCP em retardar a perda de qualidade. Para frutos a 7ºC, o 1-MCP minimizou a incidência de dano pelo frio quando transferidos para a condição ambiente.

Influência do dano da abelha-irapuá em flores de mirtileiro sobre a frutificação efetiva e as frutas produzidas

A abelha-irapuá, Trigona spinipes, é considerada um inseto-praga de várias culturas, por se alimentar de folhas e principalmente de flores e frutos. Os objetivos deste trabalho foram caracterizar o dano provocado pela irapuá em flores de mirtileiro (Vaccinium ashei Read.) e avaliar a frutificação efetiva e a qualidade da fruta produzida. O experimento foi conduzido no pomar experimental de mirtileiro, da Embrapa Clima Temperado em Pelotas, RS. Foram marcadas 200 flores de mirtileiro, seleção 103, sendo 100 destas com dano feito pela irapuá e 100 sem o dano. Após a floração, foi observada a frutificação efetiva, e por ocasião da colheita, foram determinados o teor de sólidos solúveis totais (SST), o diâmetro dos frutos e o número de sementes por fruto. O delineamento experimental foi o inteiramente casualizado. Nas flores sem danos da irapuá, houve maior percentagem de frutificação efetiva, e as frutas oriundas das mesmas apresentaram maior diâmetro e maior quantidade de sementes. O teor de SST nas frutas de mirtilo, oriundas tanto das flores com dano como daquelas sem dano, foi semelhante. Esses resultados sugerem que a T. spinipes é prejudicial à cultura do mirtilo, principalmente na época de floração, pois os danos causados pelo inseto provocaram baixa frutificação, frutas de tamanho reduzido e com menor quantidade de sementes.

DnaSP Version 5. DNA Sequence Polymorphism

DnaSP, DNA Sequence Polymorphism, is a software package for the analysis of nucleotide polymorphism from aligned DNA sequence data. DnaSP can estimate several measures of DNA sequence variation within and between populations (in noncoding, synonymous or nonsynonymous sites, or in various sorts of codon positions), as well as linkage disequilibrium, recombination, gene flow and gene conversion parameters. DnaSP can also carry out several tests of neutrality: Hudson, Kreitman and Aguadé (1987), Tajima (1989), McDonald and Kreitman (1991), Fu and Li (1993), and Fu (1997) tests. Additionally, DnaSP can estimate the confidence intervals of some test-statistics by the coalescent. The results of the analyses are displayed on tabular and graphic form.

Qualidade de maçãs 'Royal gala' submetidas ao dano mecânico por impacto e aplicação de 1-metilciclopropeno em dois sistemas comerciais de armazenamento

O objetivo deste trabalho foi avaliar os efeitos do dano mecânico por impacto e da aplicação de 1-metilciclopropeno (1-MCP) sobre a qualidade de maçãs 'Royal Gala' mantidas em armazenamento refrigerado (AR) e em atmosfera controlada (AC). Os tratamentos avaliados foram dano mecânico (sem e com dano por impacto) combinado com a aplicação de 1-MCP (0 e 625 nL L-1). Os frutos foram armazenados durante quatro meses em armazenamento refrigerado (AR; 0 ºC ± 1 ºC e 92 ± 2 % de UR) (experimento 1) e durante oito meses em atmosfera controlada (AC; 1,2 kPa de O2 + 2,0 kPa de CO2; 0 ºC ± 0,1 ºC e 96 ± 2 % de UR) (experimento 2). Em AR, os frutos tratados com 1-MCP apresentaram maior firmeza de polpa, além de maior área escurecida no local danificado, na saída da câmara. Nesta condição de armazenamento, após sete dias em condição ambiente, os frutos tratados com 1-MCP apresentaram acidez titulável mais elevada, maior escurecimento da epiderme e menor profundidade de escurecimento da polpa no local danificado. Em AC, a aplicação do 1-MCP proporcionou, após a saída da câmara, frutos com menor teor de sólidos solúveis e maior escurecimento da epiderme no local danificado, sendo que, após sete dias em condição ambiente, os frutos apresentaram maior profundidade de escurecimento do tecido da polpa no local danificado. O dano por impacto ocasionou escurecimento da polpa de maçãs 'Royal Gala'. O 1-MCP não inibiu os efeitos do dano, mas preservou a qualidade dos frutos, especialmente em AR.

Complexes of Pd(II) and Pt(II) with 9-aminoacridine: reactions with DNA and study of their antiproliferative activity

Four new metal complexes {M = Pd(II) or Pt(II)} containing the ligand 9-aminoacridine (9AA) were prepared. The compounds were characterized by FT-IR and 1H, 13C, and 195Pt NMR spectroscopies. Crystal structure of the palladium complex of formulae [Pd(9AA)(μ-Cl)]2 · 2DMF was determined by X-ray diffraction. Two 9-acridine molecules in the imine form bind symmetrically to the metal ions in a bidentate fashion through the imine nitrogen atom and the C(1) atom of the aminoacridine closing a new five-membered ring. By reaction with phosphine or pyridine, the Cl bridges broke and compounds with general formulae [Pd(9AA)Cl(L)] (where L = PPh3 or py) were formed. A mononuclear complex of platinum of formulae [Pt(9AA)Cl(DMSO)] was also obtained by direct reaction of 9-aminoacridine and the complex [PtCl2(DMSO)2]. The capacity of the compounds to modify the secondary and tertiary structures of DNA was evaluated by means of circular dichroism and electrophoretic mobility. Both palladium and platinum compounds proved active in the modification of both the secondary and tertiary DNA structures. AFM images showed noticeable modifications of the morphology of the plasmid pBR322 DNA by the compounds probably due to the intercalation of the complexes between base pairs of the DNA molecule. Finally, the palladium complex was tested for antiproliferative activity against three different human tumor cell lines. The results suggest that the palladium complex of formula [Pd(9AA)(μ-Cl)]2 has significant antiproliferative activity, although it is less active than cisplatin.