Fekadu Yadetie

Plasticity of Animal Genome Architecture Unmasked by Rapid Evolution of a Pelagic Tunicate

Ocean Dweller Sequenced The Tunicates, which include the solitary free-swimming larvaceans that are a major pelagic component of our oceans, are a basal lineage of the chordates. In order to investigate the major evolutionary transition represented by these organisms, Denoeud et al. (p. 1381, published online 18 November) sequenced the genome of Oikopleura dioica, a chordate placed by phylogeny between vertebrates and amphioxus. Surprisingly, the genome showed little conservation in genome architecture when compared to the genomes of other animals. Furthermore, this highly compacted genome contained intron gains and losses, as well as species-specific gene duplications and losses that may be associated with development. Thus, contrary to popular belief, global similarities of genome architecture from sponges to humans are not essential for the preservation of ancestral morphologies. A metazoan genome departs from the organization that appears rigidly established in other animal phyla. Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.

Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4-nonylphenol

Alkylphenol ethoxylate degradation products such as nonylphenol and octylphenol are shown to have estrogenic effects. Nonylphenol induces synthesis of vitellogenin (a precursor of egg yolk proteins) and zona radiata proteins (eggshell proteins) in juvenile and/or male fish. Little is known about the molecular mechanisms of estrogenicity of environmental chemicals such as nonylphenol. To study the mechanisms of estrogenic effects of 4-nonylphenol (NP), we examined its in vivo effects on the expression of the estrogen receptor (ER), vitellogenin (Vtg) and zona radiata protein (Zrp) genes in juvenile Atlantic salmon liver. We show that the ER mRNA synthesis is induced by NP in a dose-dependent manner in juvenile Atlantic salmon liver. The induction of the ER mRNA synthesis is followed by the induction of Zrp and Vtg mRNA synthesis. The ER transcripts reach peak levels earlier than the Zrp and Vtg mRNA and proteins, which is in agreement with the physiological effects of estradiol during zonagenesis and vitellogenesis. Various studies have also shown that NP competitively inhibits the binding of 17 beta-estradiol (E2) to ER. Our results further suggest that NP directly mimics E2 in inducing the ER, Zrp and Vtg genes in salmon liver.

Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon (Salmo salar)

Alkylphenols such as 4-nonylphenol (NP) are one of the wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) mRNA and protein synthesis in the liver are widely used biomarkers for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the pituitary. To evaluate pituitary effects and develop new potential biomarkers for xenoestrogens, the influences of NP and 17beta-estradiol (E2) on the mRNA levels of pituitary gonadotropic hormone (GTH) beta subunits [leutinizing hormone beta (LH beta or GTH II beta) and follicle stimulating hormone beta (FSH beta or GTH I beta)], prolactin (PRL), growth hormone (GH) and the pituitary specific transcription factor (Pit-1) were investigated in individual male and female juvenile Atlantic salmon (Salmo salar), 3 days after a single intraperitoneal (i.p.) injection. In one experiment, fish were injected with NP (125 mg/kg body weight (BW)) or E2 (5 mg/kg BW) and a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze LH beta and FSH beta mRNA levels. In the second experiment, fish were injected with three doses of NP (10, 50, 125 mg/kg BW) or a single dose of E2 (5 mg/kg BW) and Northern blot analysis was used to quantify LH beta, FSH beta, PRL, GH and Pit-1 mRNAs. Both NP (50 and 125 mg/kg BW) and E2 significantly induced LH beta mRNA levels (P<0.01), but only in females. The highest dose of NP (125 mg/kg BW) significantly induced Pit-1 mRNA in males (P<0.01). NP did not have significant effects on any of the other pituitary transcripts. NP induced LH beta mRNA synthesis in females by up to 6-fold and the changes appeared to correlate with the increases in hepatic Vtg and Zrp mRNA levels. The results show that LH beta mRNA assay in female juvenile salmonids may be used as a marker for pituitary effects of xenoestrogens. The data also suggest that NP may have the potential to perturb the regulation of LH beta gene expression by mimicking E2.

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens

Abstract: A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17β (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.

Global transcriptome analysis of Atlantic cod (Gadus morhua) liver after in vivo methylmercury exposure suggests effects on energy metabolism pathways.

Methylmercury (MeHg) is a widely distributed contaminant polluting many aquatic environments, with health risks to humans exposed mainly through consumption of seafood. The mechanisms of toxicity of MeHg are not completely understood. In order to map the range of molecular targets and gain better insights into the mechanisms of toxicity, we prepared Atlantic cod (Gadus morhua) 135k oligonucleotide arrays and performed global analysis of transcriptional changes in the liver of fish treated with MeHg (0.5 and 2 mg/kg of body weight) for 14 days. Inferring from the observed transcriptional changes, the main pathways significantly affected by the treatment were energy metabolism, oxidative stress response, immune response and cytoskeleton remodeling. Consistent with known effects of MeHg, many transcripts for genes in oxidative stress pathways such as glutathione metabolism and Nrf2 regulation of oxidative stress response were differentially regulated. Among the differentially regulated genes, there were disproportionate numbers of genes coding for enzymes involved in metabolism of amino acids, fatty acids and glucose. In particular, many genes coding for enzymes of fatty acid beta-oxidation were up-regulated. The coordinated effects observed on many transcripts coding for enzymes of energy pathways may suggest disruption of nutrient metabolism by MeHg. Many transcripts for genes coding for enzymes in the synthetic pathways of sulphur containing amino acids were also up-regulated, suggesting adaptive responses to MeHg toxicity. By this toxicogenomics approach, we were also able to identify many potential biomarker candidate genes for monitoring environmental MeHg pollution. These results based on changes on transcript levels, however, need to be confirmed by other methods such as proteomics.

XHM: a system for detection of potential cross hybridizations in DNA microarrays.

BackgroundMicroarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results.ResultsWe have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at http://www.bioinfo.no/tools/xhm/.ConclusionsUsing XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results.

Identification of novel growth factor-responsive genes in neuroendocrine gastrointestinal tumour cells

Targeting growth-regulatory pathways is a promising approach in cancer treatment. A prerequisite to the development of such therapies is characterisation of tumour growth regulation in the particular tumour cell type of interest. In order to gain insight into molecular mechanisms underlying proliferative responses in neuroendocrine (NE) gastrointestinal (GI) tumours, we investigated gene expression in human carcinoid BON cells after exposure to gastrin, hepatocyte growth factor (HGF), pituitary adenylate cyclase-activating polypeptide or epidermal growth factor. We particularly focused on gastrin- and HGF-induced gene expression, and identified 95 gastrin- and 101 HGF-responsive genes. The majority of these genes are known mediators of processes central in tumour biology, and a number of them have been associated with poor prognosis and metastasis in cancer patients. Furthermore, we identified 12 genes that were regulated by all four factors, indicating that they may be universally regulated during NE GI tumour cell proliferation. Our findings provide useful hypotheses for further studies aimed to search for new therapeutic targets as well as tumour markers in NE GI tumours.

Conservation and divergence of chemical defense system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics.

BackgroundAnimals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in Oikopleura dioica genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo).ResultsOikopleura genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways.ConclusionsOikopleura has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.

Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

BackgroundRNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array) procedure for rapid and parallel gene expression analysis using fluorescently labeled probes.ResultsRNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples.ConclusionRNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides.