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.

Novel fibroblast growth factor 2 transcripts are expressed in mouse embryos.

We have discovered two new exons in the mouse fibroblast growth factor 2 (FGF-2 or bFGF) gene that can be alternatively spliced to the second coding exon of the gene. The newly identified exons 1b and 1c are located at, respectively, approximately 19 and 32 kb downstream of the canonical exon 1a. Using RT-PCR analysis, mRNAs containing exon 1c and canonical exons 2 and 3 were identified in embryonic limb, placenta, face, carcass and ocular tissues. A 3.7-kb transcript present in placenta and embryonic limb hybridizes with an exon 1c-derived probe in Northern blot analysis. Alternative splicing of exon 1c to exon 2 creates a transcript for which the predicted alternative FGF-2 (altFGF-2) polypeptide contains a novel N-terminal domain. Our data indicate that in mouse embryos multiple novel mRNA variants are transcribed from the FGF-2 locus using alternative splicing. These data suggest that proteins arising from these alternative transcripts may play a role in mouse embryogenesis.

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.

The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stage-specific expression of the rat beta B2-crystallin gene.

The rat beta B2-crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the -750/-123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between -123 and -77. The proximal promoter region contains a Pax-6 binding site at -65/-51. In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co-expression of Pax-6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity: the region -37/+10 sufficed for basal promoter activity. The cooperation between the -750/ -123 region and the first intron of the beta B2-crystallin gene not only determines the differentiation stage-specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non-lens cells.

The sequence of regulatory events controlling the expression of the gamma D-crystallin gene during fibroblast growth factor-mediated rat lens fiber cell differentiation.

The transcriptional activation of tissue-specific genes during terminal differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed these events for the transcriptional activation of the rat gamma D-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced) in vitro differentiation of rat lens fiber cells. In vitro, in the presence of bFGF only, the endogenous gamma D mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and gamma D mRNA starts to accumulate at Day 8. Demethylation of the gamma D promoter region, as assessed by measuring the methylation state of the ThaI site at -16, occurs much sooner, within 1 day. By genomic footprinting, the first protein interaction with the promoter region was visible at Day 8; full occupancy of the promoter region could be detected only at Day 12. The genomic footprint identified four putative regulatory regions: -141/-131, -88/-71, -55/-45, and -15/-4. Site-directed mutagenesis of the G residues at -55 and -46 resulted in a three- to fivefold decrease in promoter activity of transfected gamma D/CAT reporter genes and also abolished interaction with nuclear extract factor(s). A G-->T mutation at -43 had no effect. The -55/-45 footprint thus derives from a proximal activator. The -88/-71 footprint identifies a silencer of the gamma D promoter in late fiber cell differentiation, as a tetramer of the -85/-67 sequence silenced a tk/CAT construct when transfected into fiber cells at a late stage, but not at an early stage, of in vitro differentiation. To time the appearance of regulatory factors, the activity of a -73/+45 gamma D/CAT (containing the activator region) and of a -1100/+45 gamma D/CAT construct was measured during fiber cell differentiation. The -73/+45 construct was active between Day 5 and Day 14, with a maximum at Day 12. The additional sequence information present in the -1100/+45 construct constrained gamma D promoter activity to between Day 8 and Day 13, with a maximum at Day 10. We conclude that the phased appearance of transacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the gamma D-crystallin gene promoter.

A novel human c-sis mRNA species is transcribed from a promoter in c-sis intron 1 and contains the code for an alternative PDGF B-like protein.

The human platelet-derived growth factor (PDGF) B chain precursor is usually translated from a 3.5 kb c-sis/PDGF B gene transcript. The first exon of the c-sis/gene contains the code for the signal peptide of the PDGF B chain precursor, preceded by a 1 kb long untranslated sequence with potent translation inhibitory activity. In this paper we show that a novel 2.6 kb c-sis mRNA present in the human choriocarcinoma cell line JEG-3 initiates at an alternative exon 1, which we refer to as exon 1a. The 90 bp long exon 1a is located in the center of the first intron of the gene. It coincides with a very pronounced DNase-I-hypersensitive site and is preceded by a functional promoter. Of the three ATG codons present in exon 1a, the third one perfectly matches the criteria of a consensus start codon. It initiates an open reading frame that is continuous with the code for the PDGF B chain precursor but lacks the code for a signal peptide. We conclude that this novel 2.6 kb c-sis mRNA species lacks the strong translation inhibitory potential of the regular exon 1 and contains the code for a PDGF B-like protein that may be targeted to the cell nucleus.

In vivo footprinting and functional analysis of the human c-sis/PDGF B gene promoter provides evidence for two binding sites for transcriptional activators.

By in vivo DMS footprint and reporter gene analyses we identified two transcription factor binding sites in the human c-sis/PDGF B gene promoter. The low basal activity of the PDGF B promoter in HeLa and undifferentiated K562 cells, which express low PDGF B mRNA levels, and in PC3 cells, which express a high PDGF B mRNA level, results from binding of a weak transcriptional activator between positions -64 and -61 relative to the transcription start site. Cytotrophoblast-like JEG-3 cells, which do not express the 3.5 kb PDGF B mRNA, contain a transcriptional activator directed at the -64/-61 sequence, but DNA methylation may render the endogenous promoter inaccessible to this activator. A CCACCCAC element at position -61/-54 was identified as the in vivo binding site for a strong transcriptional activator in phorbol ester-treated megakaryocytic K562 cells, which express a high PDGF B mRNA level. Primary human fibroblasts, which do not transcribe the PDGF B gene, contain a transcriptional activator that recognizes an element between positions -60 and -45 but does not bind to the endogenous unmethylated promoter. Our results show that the complex expression pattern of the human PDGF B gene involves the cell type-specific expression of weak and strong transcriptional activators and regulation of promoter accessibility to these factors.

Signals controlling the expression of PDGF.

PDGF is an important polypeptide growth factor that plays an essential role during early vertebrate development and is associated with tissue repair and wound healing in the adult vertebrate. Moreover, PDGF is thought to play a role in a variety of pathological phenomena, such as cancer, fibrosis and atherosclerosis. PDGF is expressed as a dimer of A and/or B chains, the precursors of which are encoded by two single copy genes. Although the PDGF genes are expressed coordinately in a number of cell types, they are independently expressed in a majority of cell types. The expression of either PDGF gene can be affected by very diverse extracellular stimuli and the type of response is dependent on the cell type that is exposed to the stimulus. Expression of the PDGF chains can be modulated at every imaginable level: by regulating accessibility of the transcription start site, by varying the transcription initiation rate, by using alternative transcription start sites, by alternative splicing, by using alternative polyadenylation signals, by varying mRNA decay rates, by regulating efficiency of translation, by protein modification, and by regulating secretion. Even upon secretion, the activity of PDGF can be modulated by non-specific or specific PDGF-binding proteins. This review provides an overview of the cell types in which the PDGF genes are expressed, of the factors that are known to affect the expression of PDGF, and of the various levels at which the expression of PDGF genes can be regulated.

DNase-I-hypersensitive sites located far upstream of the human c-sis/PDGF-B gene comap with transcriptional enhancers and a silencer and are preceded by (part of) a new transcription unit.

The human c-sis gene encodes the B chain of platelet-derived growth factor (PDGF), a potent mitogen for cultured cells of mesenchymal origin. PDGF is stored in the alpha-granules of blood platelets, which are derived from bone marrow megakaryocytes and lack transcriptional machinery. Human myeloid leukemia cell line K562 can be used as a model for megakaryocytes. Phorbol-ester-mediated megakaryocytic differentiation of K562 cells is accompanied by more than 200-fold increase in the c-sis mRNA level. We have now localized transcriptional enhancers at -8.6 kb and -9.9 kb relative to the human c-sis gene transcription start site. The enhancer at -8.6 kb increases activity of the c-sis promoter by 40-60-fold specifically in K562 cells and comaps with a DNase-I-hypersensitivity (DH) site. The enhancer at -9.9 kb increases c-sis promoter activity by 5-10-fold in K562 cells and DH at that site accompanies phorbol-ester-induced megakaryocytic differentiation. In phorbol-ester-treated K562 cells the two enhancers may be negatively influenced by a silencer that comaps with DH at -10.7/-11.0 kb. Reporter gene analysis predicted that combined activity of the upstream enhancers and the c-sis promoter may result in 100-1000-fold higher promoter activity in phorbol-ester-treated K562 cells compared with untreated cells, which can fully explain the more than 200-fold increase in c-sis mRNA level. DH at -8.6 kb and -9.9 kb was also detected in human fibroblasts and in the carcinoma cell lines HeLa and PC3, which express, respectively, undetectable, low and high levels of c-sis mRNA. Although the individual DH sites displayed 4-10-fold enhancer activity in all these cells, they lost most of their biological activity when combined in a larger fragment. In addition we localized (part of) a new transcription unit at approximately 13 kb upstream of the c-sis transcription start site. The corresponding 0.45-kb sis upstream region (sur) transcript is constitutively expressed in all cell lines examined. The expression of the sur transcript is independent of the expression of c-sis mRNA and of the pattern of DH sites far upstream of the c-sis gene. Thus, at present, there is no indication that the upstream DH sites are involved in regulation of expression of the sur gene.

Localization and functional analysis of DNase-I-hypersensitive sites in the human c-sis/PDGF-B gene transcription unit and its flanking regions.

We studied the regulation of the expression of the human c-sis/PDGF-B gene in the following panel of cell lines: K562 cells, in which expression is inducible by phorbol esters; cytotrophoblast-derived cell lines JEG-3 and JAR; carcinoma-derived cell lines PC3, T24 and HeLa, which show extensive differences in c-sis mRNA content; dermal fibroblasts, which do not express the gene. We demonstrate that the wide variety of levels of c-sis mRNA in these cells is mainly determined at the transcription level. Extensive gene rearrangements or amplifications, or significant differences in the stability of the c-sis transcript could not be found. In fibroblasts and placenta cell lines, inaccessibility of the c-sis promoter, rather than the absence of transcription factors that activate it, inhibits expression of the endogenous gene. Examination of the chromatin structure of the transcription unit and immediate flanking regions revealed several cell-type-specific DNase-I-hypersensitivity (DH) sites. Functional analysis of genomic fragments harbouring one or more DH sites showed the presence of negative regulatory elements within intron 1, and of an activating element downstream of the gene. A DH site, located immediately downstream of the promoter in dermal fibroblasts, may regulate accessibility of the promoter by means of specific nucleosome phasing.

Splicing of the platelet-derived-growth-factor A-chain mRNA in human malignant mesothelioma cell lines and regulation of its expression.

Platelet-derived-growth-factor (PDGF) A-chain transcripts differing in the presence or absence of an alternative exon-derived sequence have been described. In some publications, the presence of PDGF A-chain transcripts with this exon-6-derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse-transcription polymerase-chain-reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A-chain transcript without the exon-6-derived sequence. This sequence encodes a cell-retention signal, which means that the PDGF A-chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A-chain protein might be involved in autocrine growth stimulation via PDGF alpha receptors. However, human malignant mesothelioma cell lines only possess PDGF beta receptors. If this also holds true in vivo, the PDGF A-chain protein produced and secreted by malignant mesothelial cells might have a paracrine function. In a previous paper, we described elevated expression of the PDGF A-chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern-blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A-chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A-chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein-synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A-chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run-off analysis showed that nuclear PDGF A-chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A-chain gene in human malignant mesothelioma cell lines.