Symmetric expression of ohnologs encoding conserved antiviral responses in tetraploid common carp suggest absence of subgenome dominance after whole genome duplication.

Allopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp.

Differential gene expression in narrow- and broad-headed European glass eels (Anguilla anguilla) points to a transcriptomic link of head shape dimorphism with growth rate and chemotaxis.

One of the major challenges in evolutionary biology is to understand the mechanisms underlying morphological dimorphism and plasticity, including the genomic basis of traits and links to ecology. At the yellow eel stage of the European eel (Anguilla anguilla), two morphotypes are found: broad- and narrow-heads. This dimorphism has been linked to dietary differences, with broad-heads feeding on harder, larger prey than narrow-heads. However, recent research showed that both morphotypes could be distinguished at the glass eel stage, the nonfeeding predecessor of the yellow eel stage, implying that nondietary factors play a role in the development of this head shape dimorphism. Here, we used transcriptome profiling (RNAseq) to identify differentially expressed genes between broad- and narrow-headed glass eels. We found 260 significantly differentially expressed genes between the morphotypes, of which most were related to defence and immune responses. Interestingly, two genes involved in growth (soma and igf2) were significantly upregulated in narrow-heads, while nine genes involved in chemotaxis showed significant differential expression. Thus, we found support for the observation that head shape is associated with somatic growth, with fast-growing eels developing a narrower head. Additionally, observations in the wild have shown that slow-growers prefer freshwater, while fast-growers prefer brackish water. The differential expression of genes involved in chemotaxis seems to indicate that glass eel growth rate and habitat choice are linked. We hypothesize that two levels of segregation could take place in the European eel: first according to habitat choice and second according to feeding preference.

A full-body transcriptome and proteome resource for the European common carp.

BACKGROUND: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. RESULTS: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. CONCLUSIONS: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

In- and outdoor reproduction of first generation common sole Solea solea under a natural photothermal regime: Temporal progression of sexual maturation assessed by monitoring plasma steroids and gonadotropin mRNA expression.

Reproduction of many temperate fishes is seasonal and maturation and spawning of gametes are under photothermal control. Reproductive success of first generation (G1) common sole Solea solea in captivity has been low. In this study, the sexual maturation status has been assessed during the prespawning months in G1 sole that were housed (a) outdoor under the natural photoperiod and temperature, or (b) indoor under artificial photothermal induction. Maturation was assessed in male and female G1 broodstock in November as controls, after which the remaining population was divided over two outdoor flow-through tanks placed in a pond and two indoor recirculating aquaculture system (RAS) tanks. Subsequently, maturation status (gonadosomatic index GSI and plasma levels of testosterone T and 17ß-estradiol E2) was assessed in one tank for each condition in January, February and during spawning in early April, while fish in the other tank were not disturbed in achieving reproductive success. Quantitative real-time PCR was performed to determine species-specific gonadotropin mRNA expression in females. Successful G1 spawning and egg fertilisation occurred in all experimental tanks. Gonadal development was similar under both conditions. Higher E2 and T levels were found in indoor housed females. Gonadotropin expression revealed similar profiles between outdoor and indoor housed females. G1 sole could be reproduced in the outdoor tanks under the natural photoperiod and in the indoor tanks under artificial simulation of this regime that includes a potentially crucial chilling period of 2-3 months at 5-7 °C.

Quantitative bioassays for measuring biologically functional gonadotropins based on eel gonadotropic receptors.

Significant declines in eel stocks have been noted in many parts of the world. Because eel aquaculture is dependent on wild-caught juveniles, there is a need to achieve artificial reproduction. Adult eel maturation is currently induced by repeated injections of purified gonadotropin (human chorionic gonadotropin [hCG]) or pituitary extract. Thus the determination of the biological efficacy and quantification of internal levels of gonadotropic hormones is important for optimizing artificial reproduction protocols. To quantify the plasma levels of biologically functional gonadotropic hormones, we developed a bioassay for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) based on the stable expression of receptors in HEK293 cells of the Japanese eel Anguilla japonica LH (ajLHR) and the European eel Anguilla anguilla FSH (aaFSHR), respectively. Such cells also contain a firefly luciferase reporter gene driven by a cAMP-responsive element (CRE-Luc). We found that the obtained stable cells, with ajLHR, responded linearly to a more than 100,000-fold concentration range of hCG diluted in saline. The cells with aaFSHR showed a linear response to a 1000-fold concentration range of salmon pituitary extract mixed with saline. The biological functionality of the LH and FSH bioassays was validated using hCG, human FSH, and pituitary extracts from salmon, carp and eel. Since the toxins in eel plasma damaged the HEK293 cells, the protocol was adapted to selectively inactivate the toxins by heating at 37°C for 24h. This process successfully enabled the monitoring of hormone levels in blood plasma sampled from hCG-injected eels. In this paper, we describe the development of gonadotropin bioassays that will be useful for improving reproduction protocols in eel aquaculture.

Models for the study of tendinopathy.

Tendinopathy refers to the clinical presentation of activity-related pain, focal tendon tenderness, and intratendinous imaging changes. The underlying pathology was once thought to be due to inflammation ('tendinitis'), but is now considered to predominantly result from degeneration ('tendinosis'). While some progress has been made in understanding tendinosis, the condition remains poorly understood and a need exists for suitable exploratory preclinical models. It is unlikely that one suitable model exists because of the complexity of the underlying pathology and myriad of possible causes. This paper provides an overview of current models utilized in tendinopathy research. It progresses hierarchically from in vitro and ex vivo models to in vivo models. For each model, rationale for use, pertinent findings, and advantages and disadvantages are discussed. By improving on these models, new methods for the prevention and treatment of tendinopathy may be explored with the ultimate outcome being a reduction in the occurrence and effects of the condition in humans.

Is single-dose NVP relevant in the era of more efficacious PMTCT regimens? Lessons from Zambia.

For almost a decade, single-dose nevirapine (sdNVP) has been proven to be a safe and effective drug for the prevention of mother-to-child transmission (PMTCT) of HIV. With the advent of the use of more efficacious combination therapy strategy in reducing mother-to-child transmission, sdNVP has been relegated as a lower tier intervention. Availability of infrastructural capacity coupled with the practical reality that very few women attend an antenatal clinic more than once makes universal implementation of combination therapy a challenge. This retrospective review examined PMTCT programmatic indicators following the introduction of sdNVP at first contact in selected sites. Data from 79 PMTCT sites was reviewed from April 2006 to March 2007 (when sdNVP was offered only after 32 weeks) and compared to the period of April 2007-March 2008. In the pre-intervention period (April 2006-March 2007), the monthly average of pregnant women who received sdNVP per site was 5.02. Post-intervention (April 2007-March 2008), the monthly average increased by 59% to 7.97 (p-value<0.05). In pre-intervention period when sdNVP was dispensed at 32 weeks, the average proportion of pregnant women who received antiretroviral prophylaxis was 59%. This increased to 82% after the intervention. Current systems for dispensing sdNVP may be used as a foundation for implementation of more efficacious PMTCT regimens. The sdNVP administered at first contact should be a safety net for women who are unable to receive more efficacious regimen.

Mutational analysis of fibrillarin and its mobility in living human cells.

Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant approximately 0.02 microm(2) s(-1)), and has a significantly larger mobile fraction in CBs than in nucleoli.

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR.

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

Identification of regulatory sequences in the promoter of the PDGF B-chain gene in malignant mesothelioma cell lines.

Platelet-derived growth factor (PDGF) B-chain mRNA is readily detectable in malignant mesothelioma (MM) cell lines, but not in normal mesothelial (NM) cell lines. The high affinity receptor for PDGF B-chain dimers, the PDGF beta-receptor, is expressed in MM cell lines. NM cell lines predominantly express the PDGF alpha-receptor. Coexpression of the PDGF beta-receptor and its ligand may lead to an autocrine growth stimulating loop in the malignant cell type. In nuclear run off experiments, PDGF B-chain mRNA was detectable in MM cells only, indicating an increased level of transcription in this cell type. The proximal promoter of the PDGF B-chain gene contains DNaseI hypersensitive (DH) sites and mediates reporter gene activation in both normal and malignant cells. Nuclear proteins, extracted from both cell types, interact with DNA sequences within the proximal promoter around bp-64 to -61 relative to the transcription start site. Electrophoretic mobility shift assays (EMSAs) indicate that these factors are more abundantly present in the malignant than in the normal cell type. A DH site around -9.9 kb was found in both cell types. When tested in CAT assays, this region exerted a stimulatory effect on transcription in malignant cells. The elevated level of transcription of the PDGF B-chain gene in malignant cells may well be the result of interaction of regulatory sites in the proximal promoter and an enhancing element located at -9.9 kb from the transcription start site.

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.

Nucleotide sequence and expression of a beta-tubulin gene from Plasmodium falciparum, a malarial parasite of man.

Genomic and cDNA clones, containing a Plasmodium falciparum beta-tubulin coding sequence (pf-bTub), were isolated and characterized. Comparison of the genomic sequence with the cDNA sequence showed that the malarial bTub-coding region is interrupted by two introns, the positions of which are not found in any beta-tubulin gene (btub) from other species. The gene appears to be present as a single-copy gene in the P. falciparum genome and is expressed as a 2.3-kb transcript both in the asexual blood stages and in the sexual stages (gametes/zygotes) of the parasite. The deduced polypeptide product of the pf-btub gene is a protein of 445 amino acids (aa) (Mr 49,517). Comparison of the aa sequence of pf-bTub with that of bTubs from other species revealed that the malarial protein shows a high degree of similarity to mammalian bTubs. Upon examination of the colchicine-binding sites of pf-bTub we predict that this tubulin probably has an altered sensitivity to this inhibitor.

Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus.

Fluorescence in situ hybridization and immunocytochemical techniques have contributed significantly to our current understanding of how transcription, RNA processing, and RNA transport are spatially and temporally organized in the cell nucleus. New technologies enabling the visualization of nuclear components in living cells specifically advanced our knowledge of the dynamic aspects of these nuclear processes. The picture that emerges from the work reviewed here shows that the positioning of genes within the three-dimensional nuclear space is of crucial importance, not only for its expression, but also for the efficient processing of its transcripts. Splicing factors are recruited from speckles to sites of active transcription, which can be present within, at the periphery, or at a relatively large distance from speckles. Furthermore, results are discussed showing that transcripts are exported by means of random diffusion.

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

Differential expression in blood stages of the gene coding for the 21-kilodalton surface protein of ookinetes of Plasmodium berghei as detected by RNA in situ hybridisation.

The developmentally regulated transcription of the gene encoding the ookinete surface protein, Pbs21, has been investigated in the rodent malaria parasite, Plasmodium berghei, by RNA in situ hybridisation using fluorescently labelled DNA probes. We used a procedure that will allow the visualisation of cytoplasmic mRNA in the parasite and of high copy DNA repeats in the nucleus. Specific hybridisation to Pbs21 mRNA occurred in the cytoplasm of female gametocytes, zygotes and ookinetes, while asexual blood stages, male gametocytes and gametes showed no fluorescence. Analysis of the transcription of the Pbs21 gene during blood stage development in two tightly synchronised parasite clones using the same methodology revealed that transcription is restricted to sexual stages and is initiated in immature gametocytes at 19 h post invasion (hpi). At this point in development it is not yet possible to discriminate between the morphology of asexual trophozoites and immature gametocytes. At 24 hpi approximately 50% of the gametocytes transcribed the Pbs21 gene and the morphology of these gametocytes was identical and female. The distribution of the mRNA encoding Pbs21 confirmed that post-transcriptional control of expression occurred in the cytoplasm by repression of translation and not through delayed transport of the message to the cytoplasm. The transcription of the Pbs21 gene is the earliest demonstrated event in gametocytogenesis in rodent malaria species to date.

Expression of the egg-laying hormone genes in peripheral neurons and exocrine cells in the reproductive tract of the mollusc Lymnaea stagnalis.

The neuroendocrine caudodorsal cells play an important role in the control of reproduction in Lymnaea stagnalis. These neurons produce at least nine neuropeptides which are encoded by caudodorsal cell hormone-I and -II genes. The role of some of these peptides in the control of reproduction has been established. The present study demonstrates that the transcription and translation of the caudodorsal cell hormone genes also proceeds abundantly in the reproductive tract of this hermaphroditic animal. In the female part of the reproductive tract neurons were found to express gene I. These neurons are most likely involved in the control of transport of the eggs and egg-masses and in the regulation of secretory activity from the female accessory sex glands. In the male part of the reproductive tract exocrine secretory cells express gene I or gene II. The gene products are secreted into the male duct and transferred to the female copulant during copulation. Furthermore, putative sensory neurons in the skin were found to express gene I. The results indicate that in L. stagnalis the complex process of reproduction is regulated--at least in part--by a set of neuropeptides which are encoded by a small multigene family, viz. the caudodorsal cell gene family.

Staining of the midbody by an anti-digoxin-specific antibody.

Using RNA in situ hybridization to reveal cytoplasmic localization patterns of mRNAs in cultured cells, we noted unexpected staining of a cytoplasmic component in telophase cells. Control experiments revealed that the anti-digoxin-specific antibody was responsible for this staining. Because the staining was observed only at a position where both daughter cells are still connected, we identified the stained component as the midbody. This was confirmed by double staining of cells with anti-digoxin and anti-alpha-tubulin antibodies. We concluded that anti-digoxin-specific antibody shows crossreactivity with a component present in the midbody.

RNA polymerase II localizes at sites of human cytomegalovirus immediate-early RNA synthesis and processing.

Pre-mRNA synthesis in eukaryotic cells is preceded by the formation of a transcription initiation complex and binding of unphosphorylated RNA polymerase II (Pol II) at the promoter region of a gene. Transcription initiation and elongation are accompanied by the hyperphosphorylation of the carboxy-terminal domain (CTD) of Pol II large subunit. Recent biochemical studies provided evidence that RNA processing factors, including those required for splicing, associate with hyperphosphorylated CTDs forming "transcription factories." To directly visualize the existence of such factories, we simultaneously detected human cytomegalovirus immediate-early (IE) DNA and RNA with splicing factors and Pol II in rat 9G cells inducible for IE gene expression. Combined in situ hybridization and immunocytochemistry revealed that, after induction, both splicing factors and Pol II are present at the sites of IE mRNA synthesis and of IE mRNA processing that extend from the transcribing gene. Noninduced cells revealed no such associations. When IE mRNA-synthesizing cells were treated with a transcription inhibitor, these associations disappeared within 30 min. Our results show that the association of Pol II and splicing factors with IE DNA is dependent on its transcriptional activity and furthermore suggest that splicing factors are still associated with Pol II during active splicing.

Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.