© 2016 Elsevier B.V. The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptomeassessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time.
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© The Author(s) 2016. The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queenand brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on theearliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen.
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L-DOPA is the primary pharmacological treatment for relief of the motor symptoms of Parkinson's disease (PD). With prolonged treatment (⩾5 years) the majority of patients will develop abnormal involuntary movements as a result of L-DOPA treatment, known as L-DOPA-induced dyskinesia. Understanding the underlying mechanisms of dyskinesia is a crucial step toward developing treatments for this debilitating side effect. We used the 6-hydroxydopamine (6-OHDA) rat model of PD treated with a three-week dosing regimen of L-DOPA plus the dopa decarboxylase inhibitor benserazide (4 mg/kg and 7.5 mg/kgs.c., respectively) to induce dyskinesia in 50% of individuals. We then used RNA-seq to investigate the differences in mRNA expression in the striatum of dyskinetic animals, non-dyskinetic animals, and untreated parkinsonian controls at the peak of dyskinesia expression, 60 min after L-DOPA administration. Overall, 255 genes were differentially expressed; with significant differences in mRNA expression observed between all three groups. In dyskinetic animals 129 genes were more highly expressed and 14 less highly expressed when compared with non-dyskinetic and untreated parkinsonian controls. In L-DOPA treated animals 42 genes were more highly expressed and 95 less highly expressed when compared with untreated parkinsonian controls. Gene set cluster analysis revealed an increase in expression of genes associated with the cytoskeleton and phosphoproteins in dyskinetic animals compared with non-dyskinetic animals, which is consistent with recent studiesdocumenting an increase in synapses in dyskinetic animals. These genes may be potential targets for drugs to ameliorate L-DOPA-induced dyskinesia or as an adjunct treatment to prevent their occurrence.
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The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.
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The extent of variation in DNA methylation patterns in healthy individuals is not yet well documented. Identification of inter-individual epigenetic variation is important for understanding phenotypic variation and disease susceptibility. Using neutrophils from a cohort of healthy individuals, we generated base-resolution DNA methylation maps to document inter-individual epigenetic variation. We identified 12851 autosomal inter-individual variably methylated fragments (iVMFs). Gene promoters were the least variable, whereas gene body and upstream regions showed higher variation in DNA methylation. The iVMFs were relatively enriched in repetitive elements compared to non-iVMFs, and were associated with genome regulation and chromatin function elements. Further, variably methylated genes were disproportionately associated with regulation of transcription, responsive function and signal transduction pathways. Transcriptome analysis indicates that iVMF methylation at differentially expressed exons has a positive correlation and local effect on the inclusion of that exon in the mRNA transcript.
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An in-depth proteomic study of sheep milk whey is reported and compared to the data available in the literature for the cow whey proteome. A combinatorial peptide ligand library kit (ProteoMiner) was used to normalize protein abundance in the sheep whey proteome followed by an in-gel digest of a 1D-PAGE display and an in-solution digestion followed by OFFGEL isoelectric focusing fractionation. The peptide fractions obtained were then analyzed by LC-MS/MS. This enabled identification of 669 proteins in sheep whey that, to our knowledge, is the largest inventory of sheep whey proteins identified to date. A comprehensive list of cow whey proteins currently available in the literature (783 proteins from unique genes) was assembled and compared to the sheep whey proteome data obtained in this study (606 proteins from unique genes). This comparison revealed that while the 233 proteins shared by the two species were significantly enriched for immune and inflammatory responses in gene ontology analysis, proteins only found in sheep whey in this study were identified that take part in both cellular development and immune responses, whereas proteins only found in cow whey in this study were identified to be associated with metabolism and cellular growth.
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Background: The mammary gland is a dynamic organ that undergoes dramatic physiological adaptations during the transition from late pregnancy to lactation. Investigation of the molecular basis of mammary development and function will provide fundamental insights into tissue remodelling as well as a better understanding of milk production and mammary disease. This is important to livestock production systems and human health. Here we use RNA-seq to identify differences in gene expression in the ovine mammary gland between late pregnancy and lactation. Results: Between late pregnancy (135 days of gestation ± 2.4 SD) and lactation (15 days post partum ± 1.27 SD) 13 % of genes in the sheep genome were differentially expressed in the ovine mammary gland. In late pregnancy, cell proliferation, beta-oxidation of fatty acids and translation were identified as key biological processes. During lactation, high levels of milk fat synthesis were mirrored by enrichment of genes associated with fatty acid biosynthesis, transport and lipogenesis. Protein processing in the endoplasmic reticulum was enriched during lactation, likely in support of active milk protein synthesis. Hormone and growth factor signalling and activation of signal transduction pathways, including the JAK-STAT and PPAR pathways, were also differently regulated, indicating key roles for these pathways in functional development of the ovine mammary gland. Changes in the expression of epigenetic regulators, particularly chromatin remodellers, indicate a possible role in coordinating the large-scale transcriptional changes that appear to be required to switch mammary processes from growth and development during late pregnancy to synthesis and secretion of milk during lactation. Conclusions: Coordinated transcriptional regulation of large numbers of genes is required to switch between mammary tissue establishment during late pregnancy, and activation and maintenance of milk production during lactation. Our findings indicate the remarkable plasticity of the mammary gland, and the coordinated regulation of multiple genes andpathways to begin milk production. Genes and pathways identified by the present study may be important for managing milk production and mammary development, and may inform studies of diseases affecting the mammary gland.
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Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation.
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Many insects are capable of remarkable changes in biology and form in response to their environment or diet. The most extreme example of these are polyphenisms, which are when two or more different phenotypes are produced from a single genotype in response to the environment. Polyphenisms provide a fascinating opportunity to study how the environment affects an animal’s genome, and how this produces changes in form. Here we review the current state of knowledge of the molecular basis of polyphenisms and what can be learnt from them to understand how nutrition may influence our own genomes.
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Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.
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The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.
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Epigenetic mechanisms are proposed as an important way in which the genome responds to the environment. Epigenetic marks, including DNA methylation and Histone modifications, can be triggered by environmental effects, and lead to permanent changes in gene expression, affecting the phenotype of an organism. Epigenetic mechanisms have been proposed as key in plasticity, allowing environmental exposure to shape future gene expression. While we are beginning to understand how these mechanisms have roles in human biology and disease, we have little understanding of their roles and impacts on ecology and evolution. In this review, we discuss different types of epigenetic marks, their roles in gene expression and plasticity, methods for assaying epigenetic changes, and point out the future advances we require to understand fully the impact of this field.
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The possibility that environmentally induced epigenetic marks are passed transgenerationally through the female germline has implications for our understanding of development, disease, and evolution. Although it remains to be seen how widespread and prevalent this mechanism is amongst animal taxa, the handful of studies that we do have provides enough evidence to support the existence of an evolved mechanism for the transmission of environmental information through the maternal germline to offspring.© 2014 Elsevier Inc. All rights reserved.
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The Torso receptor tyrosine kinase has two crucial roles in Drosophila melanogaster development. One is in the control of insect moulting, which is regulated by the neuropeptide hormone PTTH (protho racicotropic hormone). PTTH activates ERK signalling via Torso in the prothoracic gland to stimulate ecdysone secretion. Torso also has a role in control of one of the earliest events in embryogenesis in Drosophila; patterning of the embryonic termini. Here Torso is activated by a different, but related, peptide called Trunk. During terminal patterning another protein, Torso-like, has a key role in mediating activation of Torso by Trunk. Torso-like is also expressed in the prothoracic gland and null-mutants have defective developmental timing in Drosophila. This function, however, has been recently shown to be independent of Torso and PTTH. We refer to these proteins, Trunk, PTTH, Torso and Torso-like, as the Torso-activation module. Outside Drosophila we see that the genes encoding the Torso-activation module have a complex phylogenetic history, with different origins and multiple losses of components of this signalling pathway during arthropod evolution. This, together with expression and functional data in a range of insects, leads us to propose that the terminal patterning pathway in Drosophila and Tribolium arose through co-option of PTTH/Trunk and Torso, which has a role in developmental timing, into a new context, and that Torso-like was recruited specifically in the ovary to modulate the specificity of this pathway.© 2014 Elsevier Inc.
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Background: Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ from each other in physiology, behaviour and life span. Results: To understand how these trajectories are established we have generated a comprehensive atlas of gene expression throughout larval development. We found substantial differences in gene expression between worker and queen-destined larvae at 6 hours after hatching. Some of these early changes in gene expression are maintained throughout larval development, indicating that caste-specific developmental trajectories are established much earlier than previously thought. Within our gene expression data we identified processes that potentially underlie caste differentiation. Queen-destined larvae have higher expression of genes involved in transcription, translation and protein folding early in development with a later switch to genes involved in energy generation. Using RNA interference, we were able to demonstrate that one of these genes, hexamerin 70b, has a role in caste differentiation. Both queen and worker developmental trajectories are associated with the expression of genes that have alternative splice variants, although only a single variant of a gene tends to be differentially expressed in a given caste. Conclusions: Our data, based on the biases in gene expression early in development together with published data, supports the idea that caste development in the honeybee consists of two phases; an initial biased phase of development, where larvae can still switch to the other caste by differential feeding, followed by commitment to a particular developmental trajectory.
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Many genes are differentially regulated by caste development in the honeybee. Identifying and understanding these differences is key to discovering the mechanisms underlying this process. To identify these gene expression differences requires robust methods to measure transcript abundance. RT-qPCR is currently the gold standard to measure gene expression, but requires stable reference genes to compare gene expression changes. Such reference genes have not been established for honeybee caste development. Here, we identify and test potential reference genes that have stable expression throughout larval development between the two female castes. In this study, 15 candidate reference genes were examined to identify the most stable reference genes. Three algorithms (GeNorm, Bestkeeper and NormFinder) were used to rank the candidate reference genes based on their stability between the castes throughout larval development. Of these genes Ndufa8 (the orthologue of a component of complex one of the mitochondrial electron transport chain) and Pros54 (orthologous to a component of the 26S proteasome) were identified as being the most stable. When these two genes were used to normalise expression of two target genes (previously found to be differentially expressed between queen and worker larvae by microarray analysis) they were able to more accurately detect differential expression than two previously used reference genes (awd and RpL12). The identification of these novel reference genes will be of benefit to future studies of caste development in the honeybee.
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The pea aphid (Acyrthosiphon pisum) can reproduce either sexually or asexually (parthenogenetically), giving rise, in each case, to almost identical adults. These two modes of reproduction are accompanied by differences in ovarian morphology and the developmental environment of the offspring, with sexual forms producing eggs that are laid, whereas asexual development occurs within the mother. Here we examine the effect each mode of reproduction has on the expression of key maternal and axis patterning genes; orthodenticle (otd), hunchback (hb), caudal (cad) and nanos (nos). We show that three of these genes (Ap-hb, Ap-otd and Ap-cad) are expressed differently between the sexually and asexually produced oocytes and embryos of the pea aphid. We also show, using immunohistochemistry and cytoskeletal inhibitors, that Ap-hb RNA is localized differently between sexually and asexually produced oocytes, and that this is likely due to differences in the 3′ untranslated regions of the RNA. Furthermore, Ap-hb and Ap-otd have extensive expression domains in early sexually produced embryos, but are not expressed at equivalent stages in asexually produced embryos. These differences in expression likely correspond with substantial changes in the gene regulatory networks controlling early development in the pea aphid. These data imply that in the evolution of parthenogenesis a new program has evolved to control the development of asexually produced embryos, whilst retaining the existing, sexual, developmental program. The patterns of modification of these developmental processes mirror the changes that we see in developmental processes between species, in that early acting pathways in development are less constrained, and evolve faster, than later ones. We suggest that the evolution of the novel asexual development pathway in aphids is not a simple modification of an ancestral system, but the evolution of two very different developmental mechanisms occurring within a single species.
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Patterning of the terminal regions of the Drosophila embryo is achieved by an exquisitely regulated signal that passes between the follicle cells of the ovary, and the developing embryo. This pathway, however, is missing or modified in other insects. Here we trace the evolution of this pathway by examining the origins and expression of its components. The three core components of this pathway: trunk, torso and torso-like have different evolutionary histories and have been assembled step-wise to form the canonical terminal patterning pathway of Drosophila and Tribolium. Trunk, torso and a gene unrelated to terminal patterning, prothoraciotrophic hormone (PTTH), show an intimately linked evolutionary history, with every holometabolous insect, except the honeybee, possessing both PTTH and torso genes. Trunk is more restricted in its phylogenetic distribution, present only in the Diptera and Tribolium and, surprisingly, in the chelicerate Ixodes scapularis, raising the possibility that trunk and torso evolved earlier than previously thought. In Drosophila torso-like restricts the activation of the terminal patterning pathway to the poles of the embryo. Torso-like evolved in the pan-crustacean lineage, but based on expression of components of the canonical terminal patterning system in the hemimetabolous insect Acyrthosiphon pisum and the holometabolous insect Apis mellifera, we find that the canonical terminal-patterning system is not active in these insects. We therefore propose that the ancestral function of torso-like is unrelated to terminal patterning and that torso-like has become co-opted into terminal patterning in the lineage leading to Coleoptera and Diptera. We also show that this co-option has not resulted in changes to the molecular function of this protein. Torso-like from the pea aphid, honeybee and Drosophila, despite being expressed in different patterns, are functionally equivalent. We propose that co-option of torso-like into restricting the activity of trunk and torso facilitated the final step in the evolution of this pathway; the capture of transcriptional control of target genes such as tailless and huckebein by this complex and novel patterning pathway.
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Development of the symbiotic association in the bipartite lichen Pseudocyphellaria crocata was investigated by characterizing two regions of the thallus. Thallus organization was examined using microscopy. A HIP1-based differential display technique was modified for use on Nostoc strains, including lichenized strains. Northern hybridization and quantitative real-time polymerase chain reaction were used to confirm differential display results, and determine expression levels of key cyanobacterial genes. Photosystem II yield across the thallus was measured using pulse-amplitude modulated fluorescence. Microscopy revealed structural differences in the thallus margins compared with the centre and identified putative heterocysts in both regions. Differential display identified altered transcript levels in both Nostoc punctiforme and a lichenized Nostoc strain. Transcript abundance of cox2, atpA, and ribA was increased in the thallus margin compared with the centre. Expression of cox2 is heterocyst specific and expression of other heterocyst-specificgenes (hetR and nifK) was elevated in the margin, whereas, expression of psbB and PSII yieldwere not. Structural organization of the thallus margin differed from the centre. Both regions contained putative heterocysts but gene expression data indicated increased heterocyst differentiation in the margins where photosystem II yield was decreased. This is consistent with a zone of heterocyst differentiation within the thallus margin.
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The Drosophila Enhancer of split complex [E(spl)-C] is a remarkable complex of genes many of which are effectors or modulators of Notch signaling. The complex contains different classes of genes including four bearded genes and seven basic helix-loop-helix (bHLH) genes. We examined the evolution of this unusual complex by identifying bearded and bHLH genes in the genome sequences of Arthropods. We find that a four-gene E(spl)-C, containing three bHLH genes and one bearded gene, is an ancient component of the genomes of Crustacea and Insects. The complex is well conserved in insects but is highly modified in Drosophila, where two of the ancestral genes of the complex are missing, and the remaining two have been duplicated multiple times. Through examining the expression of E(spl)-C genes in honeybees, aphids, and Drosophila, we determined that the complex ancestrally had a role in Notch signaling. The expression patterns of genes found inserted into the complex in some insects, or that of ancestral E(spl)-C genes that have moved out of the complex, imply that the E(spl)-C is a genomic domain regulated as a whole by Notch signaling. We hypothesize that the E(spl)-C is a Notch-regulated genomic domain conserved in Arthropod genomes for around 420 million years. We discuss the consequence of this conserved domain for the recruitment of novel genes into the Notch signaling cascade.© 2010 by Cold Spring Harbor Laboratory Press.
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Aphids exhibit unique attributes, such as polyphenisms and specialized cells to house endosymbionts, that make them an interesting system for studies at the interface of ecology, evolution and development. Here we present a comprehensive characterization of the developmental genes in the pea aphid, Acyrthosiphon pisum, and compare our results to other sequenced insects. We investigated genes involved in fundamental developmental processes such as establishment of the body plan and organogenesis, focusing on transcription factors and components of signalling pathways. We found that most developmental genes were well conserved in the pea aphid, although many lineage-specific gene duplications and gene losses have occurred in several gene families. In particular, genetic components of transforming growth factor beta (TGFβ) Wnt, JAK/STAT (Janus kinase/signal transducer and activator of transcription) and EGF (Epidermal Growth Factor) pathways appear to have been significantly modified inthe pea aphid.
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Background: Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models.Results: Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes.Conclusions: The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.© 2010 Gerardo et al.; licensee BioMed Central Ltd.
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Background: Gene clusters, such as the Hox gene cluster, are known to have critical roles in development. In eukaryotes gene clusters arise primarily by tandem gene duplication and divergence. Genes within a cluster are often co-regulated, providing selective pressure to maintain the genome organisation, and this co-regulation can result in temporal or spatial co-linearity of gene expression. It has been previously noted that in Drosophila melanogaster, three of the four runt-domain (RD) containing genes are found in a relatively tight cluster on chromosome 1, raising the possibility of a putative functional RD gene cluster in D. melanogaster. Results: To investigate the possibility of such a genecluster, orthologues of the Drosophila melanogaster RD genes were identified in several endopterygotan insects, two exopterygotan insects and two non-insect arthropods. In all insect species four RD genes were identified and orthology was assigned to the Drosophila sequences by phylogenetic analyses. Although four RD genes were found in the crustacean D. pulex, orthology could not be assigned to the insect sequences, indicating independent gene duplications from a single ancestor following the split of the hexapod lineage from the crustacean lineage. In insects, two chromosomal arrangements of these genes was observed; the first a semi-dispersed cluster, such as in Drosophila, where lozenge is separated from the core cluster of three RD genes often by megabases of DNA. The second arrangement was a tight cluster of the four RD genes, such as in Apis mellifera. This genomic organisation, particularly of the three core RD genes, raises the possibility of shared regulatory elements. In situ hybridisation of embryonic expression of the four RD genes in Drosophila melanogaster and the honeybee A. mellifera shows no evidence for either spatial or temporal co-linearity of expression during embryogenesis. Conclusion: All fully sequenced insect genomes contain four RD genes and orthology can be assigned to these genes based on similarity to the D. melanogaster protein sequences. Examination of the genomic organisation of these genes provides evidence for a functional RD gene cluster. RD genes from non-insect arthropods are also clustered, however the lack of orthology between these and insect RD genes suggests this cluster is likely to have resulted from a duplication event independent from that which created the insect RD gene cluster. Analysis of embryonic RD gene expression in two endopterygotan insects, A. mellifera and D. melanogaster, did not show evidence for coordinated gene expression, therefore while the functional significance of this gene cluster remains unknown its maintenance during insect evolution implies some functional significance to the cluster.© 2008 Duncan et al; licensee BioMed Central Ltd.
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Facial eczema (FE) is a hepatogenous mycotoxicosis in sheep caused by the fungal toxin sporidesmin. Resistance to FE is a multigenic trait. To identify QTL associated with this trait, a scan of ovine chromosomes was implemented. In addition, ABCG2 was investigated as a possible positional candidate gene because of its sequence homology to the yeast PDR5 protein and its functional role as a xenobiotic transporter. The sequence of ovine ABCG2 cDNA was obtained from liver mRNA by RT-PCR and 5′ and 3′ RACE. The predicted protein sequence shares>80% identity with other mammalian ABCG2 proteins. SNPs were identified within exon 6, exon 9 and intron 4. The intron 4 SNP was used to map ABCG2 to ovine chromosome 6 (OAR6), about 2 cM distal to microsatellite marker OarAE101. Interestingly, this chromosomal region contains weak evidence for a FE QTL detected in a previous genome-scan experiment. To further investigate the association of ABCG2 with FE, allele frequencies for the three SNPs plus three neighbouring microsatellite markers were tested for differences in sheep selected for and against FE. Significant differences were detected in the allele frequencies of the intronic SNP marker among the resistant, susceptible and control lines. No difference in the levels of ABCG2 expression between the resistant and susceptible animals was detected by Northern hybridisation of liver RNA samples. However, significantly higher expression was observed in sporidesmin-dosed sheep compared with naïve animals. Our inference is that the ABCG2 gene may play a minor role in FE sensitivity in sheep, at least within these selection lines. © 2007 AgResearch.
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Sporidesmin is an epidithiodioxopiperazine mycotoxin secreted by the saprophytic fungus Pithomyces chartarum. Ingestion of sporidesmin by ruminants grazing on the saprophyte infested pasture causes severe liver and bile duct damage leading to secondary photosensitisation. Zinc supplementation is used as an effective prophylaxis against sporidesmin toxicity in ruminants, however, the mechanism by which zinc protects is unknown. This study used the human hepatoma cell line, HepG2, as a model to examine the mechanism of zinc protection against sporidesmin toxicity. Treatment of cells with various concentrations of sporidesmin (0-10μg/ml) resulted in a sigmoidal dose response curve with an LC 50 of 5 μg/ml. Cells were protected from sporidesmin toxicity by pre-treatment for 2 h or 16 h with zinc sulphate in a concentration dependent manner, with significant protection at 50 μM zinc and maximal protection at 200 μM zinc. To determine whether zinc protection required de novo gene transcription, cells were treated with the transcriptional inhibitor actinomycin D for one hour prior to and throughout the zinc pre-treatment. The presence of actinomycin D did not significantly reduce the zinc protection against sporidesmincytotoxicity (80% protection without actinomycin D versus 71% protection with actinomycin D). Therefore, de novo gene transcription does not play a major role in the mechanism of zinc protection against sporidesmin toxicity in HepG2 cells. © 2005 Elsevier Ireland Ltd. All rights reserved.
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