Mitochondrial DNA control region diversity in a population from Espirito Santo state, Brazil

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

B chromosome of the African cichlid fish Astatotilapia latifasciata: methylation patterns and transcriptional levels of DNA methylation machinery genes

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

Genetic diversity of the Brazilian Creole cattle Pe-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pe-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pe-duro and excluded maternal Zebuine introgression. In this report, the main Pe-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pe-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes

mitochondrial genomes are generally thought to be under selection for compactness, due to their small size, consistent gene content, and a lack of introns or intergenic spacers. As more animal mitochondrial genomes are fully sequenced, rearrangements and partial duplications are being identified with increasing frequency, particularly in birds (Class Ayes). In this study, we investigate the evolutionary history of mitochondrial control region states within the avian order Psittaciformes (parrots and cockatoos). To this aim, we reconstructed a comprehensive multi-locus phylogeny of parrots, used PCR of three diagnostic fragments to classify the mitochondrial control region state as single or duplicated, and mapped these states onto the phylogeny. We further sequenced 44 selected species to validate these inferences of control region state. Ancestral state reconstruction using a range of weighting schemes identified six independent origins of mitochondrial control region duplications within Psittaciformes. Analysis of sequence data showed that varying levels of mitochondrial gene and tRNA homology and degradation were present within a given clade exhibiting duplications. Levels of divergence between control regions within an individual varied from 0-10.9% with the differences occurring mainly between 51 and 225 nucleotides 3' of the goose hairpin in domain I. Further investigations into the fates of duplicated mitochondrial genes, the potential costs and benefits of having a second control region, and the complex relationship between evolutionary rates, selection, and time since duplication are needed to fully explain these patterns in the mitochondrial genome. (C) 2012 Elsevier Inc. All rights reserved.

Genetic diversity of the Brazilian Creole cattle Pé-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pé-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pé-duro and excluded maternal Zebuine introgression. In this report, the main Pé-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pé-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

The constitutive activation of the DNA damage response pathway is a novel therapeutic target in aggressive B-cell lymphoma

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. The second part of this work is the clinical, molecular and functional description of a paradigmatic case of primary refractory Burkitt lymphoma characterized by spatial intratumor heterogeneity for the TP53 mutational status, high expression levels of genomic instability and DDR activation markers, primary resistance to chemotherapy and exquisite sensitivity to DDR inhibitors.

OPA1 isoforms and protein domains in the rescue of mitochondrial dysfunctions

Mutations in OPA1 gene have been identified in the majority of patients with Dominant Optic Atrophy (DOA), a blinding disease, and the syndromic form DOA-plus. OPA1 protein is a mitochondrial GTPase involved in various mitochondrial functions, present in humans in eight isoforms, resulting from alternative splicing and proteolytic processing. In this study we have investigated the specific role of each isoform through expression in OPA-/- MEFs, by evaluating their ability to improve the defective mitochondrial phenotypes. All isoforms were able to rescue the energetic efficiency, mitochondrial DNA (mtDNA) content and cristae integrity, but only the presence of both long and short forms could recover the mitochondrial morphology. In order to identify the OPA1 protein domains crucial for its functions, we selected and modified the isoform 1, shown to be one of the most efficient in preserving mitochondrial phenotype, to express three specific OPA1 variants, namely: one with a different N-terminus portion, one unable to generate short form owing to deletion of S1 cleavage site and one with a defective GTPase domain. We demonstrated that the simultaneous presence of the N- and C-terminus of OPA1 was essential for the mtDNA maintenance; a cleavable isoform generating s-forms was necessary to completely rescue the energetic competence and the presence of the C-terminus was sufficient to partially recover the cristae ultrastructure. Lastly, several pathogenic OPA1 mutations were inserted in MEF clones and the biochemical features investigated, to correlate the defective phenotypes with the clinical severity of patients. Our results clearly indicate that this cell model reflects very well the clinical characteristics of the patients, and therefore can be proposed as an useful tool to shed light on the pathomechanism underlying DOA.

Phylogenetic relationships of German heavy draught horse breeds inferred from mitochondrial DNA D-loop variation

We analysed a 610-bp mitochondrial (mt)DNA D-loop fragment in a sample of German draught horse breeds and compared the polymorphic sites with sequences from Arabian, Hanoverian, Exmoor, Icelandic, Sorraia and Przewalski's Horses as well as with Suffolk, Shire and Belgian horses. In a total of 65 horses, 70 polymorphic sites representing 47 haplotypes were observed. The average percentage of polymorphic sites was 11.5% for the mtDNA fragment analysed. In the nine different draught horse breeds including South German, Mecklenburg, Saxon Thuringa coldblood, Rhenisch German, Schleswig Draught Horse, Black Forest Horse, Shire, Suffolk and Belgian, 61 polymorphic sites and 24 haplotypes were found. The phylogenetic analysis failed to show monophyletic groups for the draught horses. The analysis indicated that the draught horse populations investigated consist of diverse genetic groups with respect to their maternal lineage.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial -barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

Time-dependent and somatically acquired mitochondrial DNA mutagenesis and respiratory chain dysfunction in a scleroderma model of lung fibrosis.

Reactive oxygen species (ROS) have been implemented in the etiology of pulmonary fibrosis (PF) in systemic sclerosis. In the bleomycin model, we evaluated the role of acquired mutations in mitochondrial DNA (mtDNA) and respiratory chain defects as a trigger of ROS formation and fibrogenesis. Adult male Wistar rats received a single intratracheal instillation of bleomycin and their lungs were examined at different time points. Ashcroft scores, collagen and TGFβ1 levels documented a delayed onset of PF by day 14. In contrast, increased malon dialdehyde as a marker of ROS formation was detectable as early as 24 hours after bleomycin instillation and continued to increase. At day 7, lung tissue acquired significant amounts of mtDNA deletions, translating into a significant dysfunction of mtDNA-encoded, but not nucleus-encoded respiratory chain subunits. mtDNA deletions and markers of mtDNA-encoded respiratory chain dysfunction significantly correlated with pulmonary TGFβ1 concentrations and predicted PF in a multivariate model.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

The role of the conserved N -terminal domain of STAT3 in STAT3 dephosphorylation and nuclear export

Rapid redistribution of STAT subcellular localization is an essential feature of cytokine signaling. To elucidate the molecular basis of STAT3 function, which plays a critical role in controlling innate immune responses in vivo, we initiated studies to determine the mechanisms controlling STAT3 nuclear trafficking. We found that STAT3 is transported to the nucleus in the absence of cytokine treatment, as judged by indirect immunofluorescence studies in the presence of leptomycin B, an inhibitor of CRM1-dependent nuclear export, suggesting that the non-phosphorylated STAT3 protein contains a functional nuclear import signal. An isoform lacking the STAT3 N-terminal domain (Δ133STAT3) retains the ability to undergo constitutive nuclear localization, indicating that this region is not essential for cytokine-independent nuclear import. Δ133STAT3 is also transported to the nucleus following stimulation with interleukin-6 (IL-6). Interestingly, IL-6-dependent tyrosine phosphorylation of Δ133STAT3 appears to be prolonged and the nuclear export of the protein delayed in cells expressing endogenous STAT3, consistent with defective Δ133STAT3 dephosphorylation. Endogenous STAT3 does not promote the nuclear export of Δ133STAT3, although dimerization between endogenous Stat3 and Δ133STAT3 is detected readily. Thus, the STAT3 N-terminal domain is not required for dimerization with full-length STAT3, yet appears to play a role in proper export of Stat3 from the nucleus following cytokine stimulation. STAT3-deficient cells reconstituted with Δ133STAT3 show enhanced and prolonged Stat1 signaling in response to IL-6, suggesting that induction of the STAT3-dependent negative regulator SOCS3 is impaired. In fact, Δ133STAT3 fails to induce SOCS3 mRNA efficiently. These studies collectively indicate that the STAT3 N-terminal region may be important for IL-6-dependent target gene activation and nuclear dephosphorylation, while dispensable for nuclear import. STAT3 is an oncogene. STAT3 is constitutively activated in primary tumors of many types. Thus far, research in the design of STAT3 protein inhibitors has focused on the SH2 and DNA-binding domains of STAT3. Interference with these domains eliminates all signaling through STAT3. If the N-terminal domain is involved in tetramerization on a subset of target genes, inhibition of this region may lead to a more selective inhibition of some STAT3 functions while leaving others intact. ^