Nuclear DNA content variation in life history phases of the Bonnemaisoniaceae (Rhodophyta)

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4′, 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15-1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome.

Robust DNA Methylation in the Clonal Raider Ant Brain.

Social insects are promising model systems for epigenetics due to their immense morphological and behavioral plasticity. Reports that DNA methylation differs between the queen and worker castes in social insects [1-4] have implied a role for DNA methylation in regulating division of labor. To better understand the function of DNA methylation in social insects, we performed whole-genome bisulfite sequencing on brains of the clonal raider ant Cerapachys biroi, whose colonies alternate between reproductive (queen-like) and brood care (worker-like) phases [5]. Many cytosines were methylated in all replicates (on average 29.5% of the methylated cytosines in a given replicate), indicating that a large proportion of the C. biroi brain methylome is robust. Robust DNA methylation occurred preferentially in exonic CpGs of highly and stably expressed genes involved in core functions. Our analyses did not detect any differences in DNA methylation between the queen-like and worker-like phases, suggesting that DNA methylation is not associated with changes in reproduction and behavior in C. biroi. Finally, many cytosines were methylated in one sample only, due to either biological or experimental variation. By applying the statistical methods used in previous studies [1-4, 6] to our data, we show that such sample-specific DNA methylation may underlie the previous findings of queen- and worker-specific methylation. We argue that there is currently no evidence that genome-wide variation in DNA methylation is associated with the queen and worker castes in social insects, and we call for a more careful interpretation of the available data.

Differential methylation of TCF7L2 promoter in peripheral blood DNA in newly diagnosed, drug-naïve patients with type 2 diabetes

TCF7L2 is the susceptibility gene for Type 2 diabetes (T2D) with the largest effect on disease risk that has been discovered to date. However, the mechanisms by which TCF7L2 contributes to the disease remain largely elusive. In addition, epigenetic mechanisms, such as changes in DNA methylation patterns, might have a role in the pathophysiology of T2D. This study aimed to investigate the differences in terms of DNA methylation profile of TCF7L2 promoter gene between type 2 diabetic patients and age- and Body Mass Index (BMI)- matched controls. We included 93 type 2 diabetic patients that were recently diagnosed for T2D and exclusively on diet (without any pharmacological treatment). DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Type 2 diabetic patients were more insulin resistant than their matched controls (mean HOMA IR 2.6 vs 1.8 in controls, P<0.001) and had a poorer beta-cell function (mean HOMA B 75.7 vs. 113.6 in controls, P<0.001). Results showed that 59% of the CpGs analyzed in TCF7L2 promoter had significant differences between type 2 diabetic patients and matched controls. In addition, fasting glucose, HOMA-B, HOMA-IR, total cholesterol and LDL-cholesterol correlated with methylation in specific CpG sites of TCF7L2 promoter. After adjustment by age, BMI, gender, physical inactivity, waist circumference, smoking status and diabetes status uniquely fasting glucose, total cholesterol and LDL-cholesterol remained significant. Taken together, newly diagnosed, drug-naïve type 2 diabetic patients display specific epigenetic changes at the TCF7L2 promoter as compared to age- and BMI-matched controls. Methylation in TCF7L2 promoter is further correlated with fasting glucose in peripheral blood DNA, which sheds new light on the role of epigenetic regulation of TCF7L2 in T2D.

DNA methylation in Dictyostelium discoideum

DNA methyltransferases of type Dnmt2 are a highly conserved protein family with enigmatic function. The aim of this work was to characterize DnmA, the Dnmt2 methyltransferase in Dictyostelium discoideum, and further to investigate its implication in DNA methylation and transcriptional gene silencing. The genome of the social amoeba Dictyostelium encodes DnmA as the sole DNA methyltransferase. The enzyme bears all ten characteristic DNA methyltransferase motifs in its catalytic domain. The DnmA mRNA was found by RT-PCR to be expressed during vegetative growth and down regulated during development. Investigations using fluorescence microscopy showed that both DnmA-myc and DnmA-GFP fusions predominantly localised to the nucleus. The function of DnmA remained initially unclear, but later experiment revealed that the enzyme is an active DNA methyltransferase responsible for all DNA (cytosine) methylation in Dictyostelium. Neither in gel retardation assays, nor by the yeast two hybrid system, clues on the functionality of DnmA could be obtained. However, immunological detection of the methylation mark with an α - 5mC antibody gave initial evidence that the DNA of Dictyostelium was methylated. Furthermore, addition of 5-aza-cytidine as demethylating agent to the Dictyostelium medium and subsequent in vitro incubation of the DNA isolated from these cells with recombinant DnmA showed that the enzyme binds slightly better to this target DNA. In order to investigate further the function of the protein, a gene knock-out for dnmA was generated. The gene was successfully disrupted by homologous recombination, the knock-out strain, however, did not show any obvious phenotype under normal laboratory conditions. To identify specific target sequences for DNA methylation, a microarray analysis was carried out. Setting a threshold of at least 1.5 fold for differences in the strength of gene expression, several such genes in the knock-out strain were chosen for further investigation. Among the up-regulated genes were the ESTs representing the gag and the RT genes respectively of the retrotransposon skipper. In addition Northern blot analysis confirmed the up-regulation of skipper in the DnmA knock-out strain. Bisufite treatment and sequencing of specific DNA stretches from skipper revealed that DnmA is responsible for methylation of mostly asymmetric cytosines. Together with skipper, DIRS-1 retrotransposon was found later also to be methylated but was not present on the microarray. Furthermore, skipper transcription was also up-regulated in strains that had genes disrupted encoding components of the RNA interference pathway. In contrast, DIRS 1 expression was not affected by a loss of DnmA but was strongly increased in the strain that had the RNA directed RNA polymerase gene rrpC disrupted. Strains generated by propagating the usual wild type Ax2 and the DnmA knock-out cells over 16 rounds in development were analyzed for transposon activity. Northern blot analysis revealed activation for skipper expression, but not for DIRS-1. A large number of siRNAs were found to be correspondent to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA methylation. In contrast, no siRNAs corresponding to the standard skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene regulation in Dictyostelium and that different, partially overlapping mechanisms control transposon silencing for skipper and DIRS-1. To elucidate the mechanism of targeting the protein to particular genes in the Dictyostelium genome, some more genes which were up-regulated in the DnmA knock-out strain were analyzed by bisulfite sequencing. The chosen genes are involved in the multidrug response in other species, but their function in Dictyostelium is uncertain. Bisulfite data showed that two of these genes were methylated at asymmetrical C-residues in the wild type, but not in DnmA knock-out cells. This suggested that DNA methylation in Dictyostelium is involved not only in transposon regulation but also in transcriptional silencing of specific genes.

Tissue- specific DNA methylation profiles in newborns

Experimental and epidemiological studies demonstrate that fetal growth restriction and low birth weight enhance the risk of chronic diseases in adulthood. Derangements in tissue-specific epigenetic programming of fetal and placental tissues are a suggested mechanism of which DNA methylation is best understood. DNA methylation profiles in human tissue are mostly performed in DNA from white blood cells. The objective of this study was to assess DNA methylation profiles of IGF2 DMR and H19 in DNA derived from four tissues of the newborn. We obtained from 6 newborns DNA from fetal placental tissue (n = 5), umbilical cord CD34+ hematopoietic stem cells (HSC) and CD34- mononuclear cells (MNC) (n = 6), and umbilical cord Wharton jelly (n = 5). HCS were isolated using magnetic-activated cell separation. DNA methylation of the imprinted fetal growth genes IGF2 DMR and H19 was measured in all tissues using quantitative mass spectrometry. ANOVA testing showed tissue-specific differences in DNA methylation of IGF2 DMR (p value 0.002) and H19 (p value 0.001) mainly due to a higher methylation of IGF2 DMR in Wharton jelly (mean 0.65, sd 0.14) and a lower methylation of H19 in placental tissue (mean 0.25, sd 0.02) compared to other tissues. This study demonstrates the feasibility of the assessment of differential tissue specific DNA methylation. Although the results have to be confirmed in larger sample sizes, our approach gives opportunities to investigate epigenetic profiles as underlying mechanism of associations between pregnancy exposures and outcome, and disease risks in later life.

Detecting recombination in 4-taxa DNA sequence alignments with Bayesian hidden Markov models and Markov chain Monte Carlo

This article presents a statistical method for detecting recombination in DNA sequence alignments, which is based on combining two probabilistic graphical models: (1) a taxon graph (phylogenetic tree) representing the relationship between the taxa, and (2) a site graph (hidden Markov model) representing interactions between different sites in the DNA sequence alignments. We adopt a Bayesian approach and sample the parameters of the model from the posterior distribution with Markov chain Monte Carlo, using a Metropolis-Hastings and Gibbs-within-Gibbs scheme. The proposed method is tested on various synthetic and real-world DNA sequence alignments, and we compare its performance with the established detection methods RECPARS, PLATO, and TOPAL, as well as with two alternative parameter estimation schemes.

Low levels of methylmercury induce DNA damage in rats: protective effects of selenium

In this study we examined the possible antigenotoxic effect of selenium (Se) in rats chronically exposed to low levels of methylmercury (MeHg) and the association between glutathione peroxidase (GSH-Px) activity and DNA lesions (via comet assay) in the same exposed animals. Rats were divided into six groups as follows: (Group I) received water; (Group II) received MeHg (100 mu g/day); (Group III) received Se (2 mg/L drinking water); (Group IV) received Se (6 mg/L drinking water); (Group V) received MeHg (100 mu g/day) and Se (2 mg/L drinking water); (Group VI) received MeHg (100 mu g/day) and Se (6 mg/L drinking water). Total treatment time was 100 days. GSH-Px activity was determined spectrophotometrically and DNA damage was determined by comet assay. Mean GSH-Px activity in groups I, II, III, IV, V and VI were, respectively: 40.19 +/- A 17.21; 23.63 +/- A 6.04; 42.64 +/- A 5.70; 38.50 +/- A 7.15; 34.54 +/- A 6.18 and 41.39 +/- A 11.67 nmolNADPH/min/gHb. DNA damage was represented by a mean score from 0 to 300; the results for groups I, II, III, IV, V and VI were, respectively: 6.87 +/- A 3.27; 124.12 +/- A 13.74; 10.62 +/- A 3.81; 13.25 +/- A 1.76; 86.87 +/- A 11.95 and 76.25 +/- A 7.48. There was a significant inhibition of GSH-Px activity in group II compared with group I (P < 0.05). Groups V and VI did not show a difference in enzyme activity compared with groups III and IV, showing the possible protective action of Se. Comet assay presented a significant difference in DNA migration between group II and group I (P < 0.0001). Groups V and VI showed a significant reduction in MeHg-induced genotoxicity (P < 0.001) when compared with group II. A negative correlation (r = -0.559, P < 0.05) was found between GSH-Px activity and DNA lesion, showing that the greater the DNA damage, the lower the GSH-Px activity. Our findings demonstrated the oxidative and genotoxic properties of MeHg, even at low doses. Moreover, Se co-administration reestablished GSH-Px activity and reduced DNA damage.

Evaluation of curcumin and cisplatin-induced DNA damage in PC12 cells by the alkaline comet assay

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DNA damage in patients who underwent minimally invasive surgery under inhalation or intravenous anesthesia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lower levels of oxidative DNA damage and apoptosis in lymphocytes from patients undergoing surgery with propofol anesthesia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups (n= 5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (beta)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood wleukocytes sampled from diabetic rats presented higher DNA damage values (tail moment =0.57 +/- 0.05; tail length =19.92 +/- 0.41, p < 0.05) compared to control rats (tail moment =0.34 +/- 0.02; tail length= 17.42 +/- 0.33). Non-diabetic (tail moment =0.43 +/- 0.04, p > 0.05) and diabetic rats (tail moment= 0.41 +/- 0.03, p > 0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats. (c) 2007 Elsevier B.V. All rights reserved.

Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers

A protocol for DNA damage assessment by the single-cell gel (SCG)/comet assay in human urinary bladder washing cells was established. Modifications of the standard alkaline protocol included an increase to 2% of sodium sarcosinate in the lysis solution, a reduction in the glass-slide area for comet analysis, and a cutoff value for comet head diameter of at least 30 mum, to exclude contaminating leukocytes. Distinguishing cell populations is crucial, because significant differential migration was demonstrated for transitional and nontransitional cells, phenomena that may confound the results. When applying the modified protocol to urinary bladder cells from smokers without urinary bladder neoplasia, it was possible to detect a significant (P = 0.03) increase in DNA damage as depicted by the tail moment (6.39 +/- 3.23; mean 95% confidence interval; n = 18) when compared with nonsmokers (1.94 +/- 1.41; n = 12). No significant differences were observed between ex-smokers and current smokers regarding comet parameters. Inflammation was not a confounding factor, but DNA migration increased significantly with age in nonsmokers (r = 0.68; P = 0.014). Thus, age matching should be a concern when transitional cells are analyzed in the SCG assay. As it is well known, DNA damage may trigger genomic instability, a crucial step in carcinogenesis. Therefore, the present data directly support the classification of individuals with smoking history as patients at high risk for urinary bladder cancer.

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved.

Nuclear DNA content in 20 species of Siluriformes (Teleostei: Ostariophysi) from the Neotropical region

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)