Expression and localization of the aryl hydrocarbon receptors and cytochrome P450 1A during early development of Atlantic cod (Gadus morhua)

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of dioxins and dioxin-like compounds (DLCs) in vertebrates. Two clades of the Ahr family exist in teleosts (Ahr1 and Ahr2), and it has been demonstrated that Ahr2 is the main protein involved in mediating the toxicity of dioxins and DLCs in most teleost species. Recently, we characterized the Atlantic cod (Gadus morhua) Ahr1a and Ahr2a receptors. To further explore a possible subfunction partitioning of Ahr1a and Ahr2a in Atlantic cod we have mapped the expression and localization of ahr1a and ahr2a in early developmental stages. Atlantic cod embryos were continuously exposed in a passive-dosing exposure system to the Ahr agonist, benzo[a]pyrene (B[a]P), from five days post fertilization (dpf) until three days post hatching (dph). Expression of ahr1a, ahr2a, and the Ahr-target genes, cyp1a and ahrrb, was assessed in embryos (8 dpf and 10 dpf) and larvae (3 dph) with quantitative real-time PCR analyses (qPCR), while in situ hybridization was used to assess the localization of expression of ahr1a, ahr2a and cyp1a. Quantitative measurements showed an increased cyp1a expression in B[a]P-exposed samples at all sampling points, and for ahr2a at 10 dpf, confirming the activation of the Ahr-signalling pathway. Furthermore, B[a]P strongly induced ahr2a and cyp1a expression in the cardiovascular system and skin, respectively, of embryos and larvae. Induced expression of both ahr2a and cyp1a was also revealed in the liver of B[a]P-exposed larvae. Our results suggest that Ahr2a is the major subtype involved in mediating responses to B[a]P in early developmental stages of Atlantic cod, which involves transcriptional regulation of biotransformation genes, such as cyp1a. The focused expression of ahr1a in the eye of embryos and larvae, and the presence of ahr2a transcripts in the jaws and fin nodes, further indicate evolved specialized roles of the two Ahrs in ontogenesis.publishedVersio

Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1,2,3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages

Quantitative ultra performance liquid chromatography tandem mass spectrometry analysis of amphetamines in blood

.Nasjonalt folkehelseinstitutt, divisjon for rettsmedisin og rusmiddelforskning, avdeling for rusmiddelanalyser

Dialects of the DNA Uptake Sequence in <i>Neisseriaceae</i>

<div><p>In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen <i>Neisseria meningitidis</i> and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS–dependent genetic transformation have remained elusive. Bioinformatic analyses of family <i>Neisseriaceae</i> genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust <i>Neisseriaceae</i> phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5′-CTG-3′ is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in <i>N. meningitidis</i>, <i>N. elongata</i>, <i>Kingella denitrificans</i>, and <i>Eikenella corrodens</i>, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS–dependent transformation is documented in the genera <i>Eikenella</i> and <i>Kingella</i> for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation in bacteria, and define the phylogenetic relationships within the <i>Neisseriaceae</i> family.</p></div

Effects of point mutations in the DUS on quantitative transformation and DNA binding and uptake of <i>N. meningitidis</i> MC58.

<p>(A) Quantitative transformation of <i>Neisseria meningitidis</i> strain MC58 with plasmid DNA containing a panel of modified DUS sequences. Transformation rates are shown as percentage relative to the AT-DUS. Standard deviations from 7 independent experiments are indicated by bars. (B) DNA binding (gray bars) and uptake (white bars) assay with radiolabelled plasmid DNA. Average values from 4 independent experiments are shown as percentage of total DNA added. T-test results for (A) transformation rates compared to AT-DUS and (B) for DNA binding versus uptake are indicated by stars (p≤0.2 = *, p≤0.05 = **, p≤0.001 = ***). DUS sequence transversions are given as abscissa labels. The sequence logo is based on the 2742 occurrences of DUS, with a single nucleotide divergence allowed, found in the genome of <i>N. meningitidis</i> MC58.</p

Quantitative transformation of <i>N. meningitidis</i> and binding and uptake of DNA with DUS from other <i>Neisseriaceae.</i>

<p>(A) Quantitative transformation of <i>Neisseria meningitidis</i> strain MC58 with plasmid DNA containing different DUS dialects and with the <i>Haemophilus influenzae</i> USS. Transformation rates are shown as percent relative to the AT-DUS. The standard deviations from 6 independent experiments are indicated by bars. (B) DNA binding (gray bars) and uptake (white bars) assay with radiolabelled plasmid DNA. Average values from 3 independent experiments are shown as percentage of total DNA added. Abscissa labels give the DUS variant. Statistic analysis as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003458#pgen-1003458-g003" target="_blank">Figure 3</a>.</p

Alignment of eight distinct DUS dialects.

<p>Alignment of the eight different DUS dialects identified in the genomes of <i>Neisseriaceae</i> family members. Two nucleotides at the 5′-end of DUS are included and the numbering is given on the top. Relative shading indicate the level of sequence conservation.</p

Quantification of DNA binding and uptake of <i>Neisseriaceae</i> with different DUS variants.

<p>Total DNA binding shown as gray bars and benzonase resistant DNA (uptake) shown as white bars plotted as percentage of DNA added. The species tested are <i>Neisseria mucosa</i> (A), <i>Neisseria elongata</i> subsp. <i>glycolytica</i> (B), <i>Kingella oralis</i> (C) and <i>Eikenella corrodens</i> (C). Abscissa labels give the DUS variant. Represented are the results from 3 (A, D) and 5 (B, C) independent experiments. Student's t-test values are indicated (p≤0.1 = *, p≤0.05 = **, p≤0.001 = ***).</p