Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable

Analysis of DNA polymorphism in ancient barley herbarium material: validation of the KASP SNP genotyping platform

The availability of crop specimens archived in herbaria and old seed collections represent valuable resources for the analysis of plant genetic diversity and crop domestication. The ability to extract ancient DNA (aDNA) from such samples has recently allowed molecular genetic investigations to be undertaken in ancient materials. While analyses of aDNA initially focused on the use of markers which occur in multiple copies such as the internal transcribed spacer region (ITS) within ribosomal DNA and those requiring amplification of short DNA regions of variable length such as simple sequence repeats (SSRs), emphasis is now moving towards the genotyping of single nucleotide polymorphisms (SNPs), traditionally undertaken in aDNA by Sanger sequencing. Here, using a panel of barley aDNA samples previously surveyed by Sanger sequencing for putative causative SNPs within the flowering-time gene PPD-H1, we assess the utility of the Kompetitive Allele Specific PCR (KASP) genotyping platform for aDNA analysis. We find KASP to out-perform Sanger sequencing in the genotyping of aDNA samples (78% versus 61% success, respectively), as well as being robust to contamination. The small template size (≥46 bp) and one-step, closed-tube amplification/genotyping process make this platform ideally suited to the genotypic analysis of aDNA, a process which is often hampered by template DNA degradation and sample cross-contamination. Such attributes, as well as its flexibility of use and relatively low cost, make KASP particularly relevant to the genetic analysis of aDNA samples. Furthermore, KASP provides a common platform for the genotyping and analysis of corresponding SNPs in ancient, landrace and modern plant materials. The extended haplotype analysis of PPD-H1 undertaken here (allelic variation at which is thought to be important for the spread of domestication and local adaptation) provides further resolution to the previously identified geographic cline of flowering-time allele distribution, illustrating how KASP can be used to aid genetic analyses of aDNA from plant species. We further demonstrate the utility of KASP by genotyping ten additional genetic markers diagnostic for morphological traits in barley, shedding light on the phenotypic traits, alleles and allele combinations present in these unviable ancient specimens, as well as their geographic distributions.

Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayestan phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes Within the Albimanus Section the monophyly of the Stroder Subgroup was strongly supported and within the Myzorhynchella Section Anopheles anrunesi and An lutzu formed a strongly supported monophyletic group The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An lanet-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An goeldii were recovered as sister species Finally, there was evidence of complexes in several species, including An antunesi, An deaneorum, and An. strodei (c) 2010 Elsevier B.V. All rights reserved

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

PHYLOGENETIC AFFINITIES OF "CHANTRANSIA" STAGES IN MEMBERS OF THE BATRACHOSPERMALES AND THOREALES (RHODOPHYTA)

This study evaluated the phylogenetic relationship among samples of ""Chantransia"" stage of the Batrachospermales and Thoreales from several regions of the world based on sequences of two genes-the plastid-encoded RUBISCO LSU gene (rbcL) and the nuclear SSU ribosomal DNA gene (SSU rDNA). All sequences of ""Chantransia macrospora"" were shown to belong to Batrachospermum macrosporum based on both molecular markers, confirming evidence from previous studies. In contrast, nine species are now associated with ""Chantransia pygmaea,"" including seven species of the Batrachospermales and two of the Thoreales. Therefore, the presence of ""C. macrospora"" in a stream can be considered reliable evidence that it belongs to B. macrosporum, whereas the occurrence of ""C. pygmaea"" does not allow the recognition of any particular species, since it is associated with at least nine species. Affinities of ""Chantransia"" stages to particular taxa were congruent for 70.5% of the samples comparing the rbcL and SSU analyses, which were associated with the same or closely related species for both markers. Sequence divergences have been reported in the ""Chantransia"" stage in comparison to the respective gametophyte, and this matter deserves further attention.

Generic relationships and dating of lineages in Winteraceae based on nuclear (ITS) and plastid (rpS16 and psbA-trnH) sequence data

Phylogenetic analyses of representative species from the five genera of Winteraceae (Drimys, Pseudowintera, Takhtajania, Tasmannia, and Zygogynum s.l.) were performed using ITS nuclear sequences and a combined data-set of ITS + psbA-trnH + rpS16 sequences (sampling of 30 and 15 species, respectively). Indel informativity using simple gap coding or gaps as a fifth character was examined in both data-sets. Parsimony and Bayesian analyses support the monophyly of Drimys, Tasmannia, and Zygogynum s.l., but do not support the monophyly of Belliolum, Zygogynum s.s., and Bubbia. Within Drimys, the combined data-set recovers two subclades. Divergence time estimates suggest that the splitting between Drimys and its sister clade (Pseudowintera + Zygogynum s.l.) occurred around the end of the Cretaceous; in contrast, the divergence between the two subclades within Drimys is more recent (15.5-18.5 MY) and coincides in time with the Andean uplift. Estimates suggest that the earliest divergences within Winteraceae could have predated the first events of Gondwana fragmentation. (C) 2009 Elsevier Inc. All rights reserved.

Phylogenetic Analyses of Eriotheca and Related Genera (Bombacoideae, Malvaceae)

Molecular and morphological data have shown that Bombacoideae and Malvoideae together form a well-supported Malvatheca clade. Phylogenetic relationships in Bombacoideae have been studied, but some genera in Bombax s. I. have not been adequately sampled for sufficiently variable molecular markers. The relationships of Eriotheca, for example, have yet to be resolved. Here, nuclear (ITS) and chloroplast (trnL-Fand matK) sequence data from 50 exemplars of Bombacoideae and seven additional taxa from other genera of Malvatheca were used to test monophyly of Eriotheca and its relationships with related genera of Bombax s. I. Parsimony and Bayesian analyses of individual and combined sequence data suggest that Eriotheca is not monophyletic as currently circumscribed but forms a paraphyletic grade containing Pachira s. 1. The newly discovered Eriotheca + Pachira clade has a probable synapomorphy of striate seeds. In addition, two other moderately supported clades emerged within the core Bombacoideae: Pseudobombax + Ceiba s. 1. and Bombax + Spirotheca + Pachira quinata. These three clades, and the African Rhodognaphalon together constitute the major clade of core Bombacoideae, whereas Adansonia appears to be more closely related to Catostemma, Scleronema, and Cavanillesia. The phylogenetic results imply three independent migrations from the New to Old World and homoplasy in staminal morphology.

Mitochondrial DNA Diversity of Orchid Bee Euglossa fimbriata (Hymenoptera: Apidae) Populations Assessed by PCR-RFLP

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085-0.289) and nucleotide diversity (0.000-0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (I center dot(st) = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (I center dot(st) = 0.161-0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.

Multiple origins of vagrant Subantarctic fur seals: A long journey to the Brazilian coast detected by molecular markers

In this study, we present the first data about putative source populations of the vagrant Subantarctic fur seal, Arctocephalus tropicalis, found on the Brazilian coast, through the comparison of their mitochondrial DNA control sequences to exclusive haplotypes from the main breeding colonies of the species. The results indicated that, despite the majority of the vagrant individuals are from Gough Island (the closest breeding site to the Brazilian coast), they also come from other reproductive colonies, such as Crozet Island, a distance around 16,500 km from the Brazilian coast. Furthermore, the molecular data identified three possible management units: (1) Gough, (2) Amsterdam, and (3) Marion, Macquarie and Crozet. This significant genetic subdivision must be taken into account in any future management plan for the species conservation, including rehabilitation and even reintroduction of vagrant fur seals.

Na+-glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: Involvement of hepatocyte nuclear factor-1 alpha expression and activity

Mutations in Na+-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1 alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1 alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1 alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1 alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1 alpha mRNA expression (similar to 50%) and binding of nuclear proteins to a HNF-1 consensus motif (similar to 100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1 alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1 alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1 alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions. (C) 2007 Elsevier B.V. All rights reserved.

Phosphoproteomics Profiling Suggests a Role for Nuclear beta IPKC in Transcription Processes of Undifferentiated Murine Embryonic Stem Cells

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal and differentiation However, the function of specific PKC Isoenzymes have yet to be determined Of the PKCs expressed in undifferentiated ESCs, beta IPKC was the only isoenzyme abundantly expressed in the nuclei To investigate the role of beta IPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one beta IPKC-specific inhibitor peptide We identified 13 nuclear proteins that are direct or indirect beta IPKC substrates in undifferentiated ESCs These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation Inhibiting beta IPKC had no effect on DNA synthesis in undifferentiated ESCs However, upon differentiation many cells seized to express beta IPKC and beta IPKC was frequently found in the cytoplasm Taken together, our results suggest that beta IPKC takes part in the processes that maintain ESCs in their undifferentiated state

Electronic Structure and Spectro-Structural Correlations of Fe(III)Zn(II) Biomimetics for Purple Acid Phosphatases: Relevance to DNA Cleavage and Cytotoxic Activity

Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a dinuclear Fe(II)M(II) center (M(II) = Fe, Mn, Zn) in the active site and are able to catalyze the hydrolysis of a variety of phosphoric acid esters. The dinuclear complex [(H(2)O)Fe(III)(mu-OH)Zn(II)(L-H)](CIO(4))(2) (2) with the ligand 2-[N-bis(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H(2)L-H) has recently been prepared and is found to closely mimic the coordination environment of the Fe(III)Zn(II) active site found in red kidney bean PAP (Neves et al. J. Am. Chem. Soc. 2007, 129, 7486). The biomimetic shows significant catalytic activity in hydrolytic reactions. By using a variety of structural, spectroscopic, and computational techniques the electronic structure of the Fe(III) center of this biomimetic complex was determined. In the solid state the electronic ground state reflects the rhombically distorted Fe(III)N(2)O(4) octahedron with a dominant tetragonal compression align ad along the mu-OH-Fe-O(phenolate) direction. To probe the role of the Fe-O(phenolate) bond, the phenolate moiety was modified to contain electron-donating or -withdrawing groups (-CH(3), -H, -Br, -NO(2)) in the 5-position. Tie effects of the substituents on the electronic properties of the biomimetic complexes were studied with a range of experimental and computational techniques. This study establishes benchmarks against accurate crystallographic struck ral information using spectroscopic techniques that are not restricted to single crystals. Kinetic studies on the hydrolysis reaction revealed that the phosphodiesterase activity increases in the order -NO(2)<- Br <- H <- CH(3) when 2,4-bis(dinitrophenyl)phosphate (2,4-bdnpp) was used as substrate, and a linear free energy relationship is found when log(k(cat)/k(0)) is plotted against the Hammett parameter a. However, nuclease activity measurements in the cleavage of double stranded DNA showed that the complexes containing the electron-withdrawing -NO(2) and electron-donating CH3 groups are the most active while the cytotoxic activity of the biomimetics on leukemia and lung tumoral cells is highest for complexes with electron-donating groups.