Complex history of a chromosomal paralogy region: insights from amphioxus aromatic amino acid hydroxylase genes and insulin-related genes

Aromatic amino acid hydroxylase (AAAH) genes and insulin-like genes form part of an extensive paralogy region shared by human chromosomes 11 and 12, thought to have arisen by tetraploidy in early vertebrate evolution. Cloning of a complementary DNA (cDNA) for an amphioxus (Branchiostoma floridae) hydroxylase gene (AmphiPAH) allowed us to investigate the ancestry of the human chromosome 11/12 paralogy region. Molecular phylogenetic evidence reveals that AmphiPAH is orthologous to vertebrate phenylalanine (PAH) genes; the implication is that all three vertebrate AAAH genes arose early in metazoan evolution, predating vertebrates. In contrast, our phylogenetic analysis of amphioxus and vertebrate insulin-related gene sequences is consistent with duplication of these genes during early chordate ancestry. The conclusion is that two tightly linked gene families on human chromosomes 11 and 12 were not duplicated coincidentally. We rationalize this paradox by invoking gene loss in the AAAH gene family and conclude that paralogous genes shared by paralogous chromosomes need not have identical evolutionary histories.

HPA axis related genes and response to psychological therapies: genetics and epigenetics

Background Hypothalamic–pituitary–adrenal (HPA) axis functioning has been implicated in the development of stress-related psychiatric diagnoses and response to adverse life experiences. This study aimed to investigate the association between genetic and epigenetics in HPA axis and response to cognitive behavior therapy (CBT). Methods Children with anxiety disorders were recruited into the Genes for Treatment project (GxT, N = 1,152). Polymorphisms of FKBP5 and GR were analyzed for association with response to CBT. Percentage DNA methylation at the FKBP5 and GR promoter regions was measured before and after CBT in a subset (n = 98). Linear mixed effect models were used to investigate the relationship between genotype, DNA methylation, and change in primary anxiety disorder severity (treatment response). Results Treatment response was not associated with FKBP5 and GR polymorphisms, or pretreatment percentage DNA methylation. However, change in FKBP5 DNA methylation was nominally significantly associated with treatment response. Participants who demonstrated the greatest reduction in severity decreased in percentage DNA methylation during treatment, whereas those with little/no reduction in severity increased in percentage DNA methylation. This effect was driven by those with one or more FKBP5 risk alleles, with no association seen in those with no FKBP5 risk alleles. No significant association was found between GR methylation and response. Conclusions Allele-specific change in FKBP5 methylation was associated with treatment response. This is the largest study to date investigating the role of HPA axis related genes in response to a psychological therapy. Furthermore, this is the first study to demonstrate that DNA methylation changes may be associated with response to psychological therapies in a genotype-dependent manner.

Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes

Mecoprop-p [(R)-2-(4-chloro-2-methylphenoxy) propanoic acid) is widely used in agriculture and poses an environmental concern because of its susceptibility to leach from soil to water. We investigated the effect of soil depth on mecoprop-p biodegradation and its relationship with the number and diversity of tfdA related genes, which are the most widely known genes involved in degradation of the phenoxyalkanoic acid group of herbicides by bacteria. Mecoprop-p half-life (DT50) was approximately 12 days in soil sampled from <30 cm depth, and increased progressively with soil depth, reaching over 84 days at 70–80 cm. In sub-soil there was a lag period of between 23 and 34 days prior to a phase of rapid degradation. No lag phase occurred in top-soil samples prior to the onset of degradation. The maximum degradation rate was the same in top-soil and sub-soil samples. Although diverse tfdAα and tfdA genes were present prior to mecoprop-p degradation, real time PCR revealed that degradation was associated with proliferation of tfdA genes. The number of tfdA genes and the most probable number of mecoprop-p degrading organisms in soil prior to mecoprop-p addition were below the limit of quantification and detection respectively. Melting curves from the real time PCR analysis showed that prior to mecoprop-p degradation both class I and class III tfdA genes were present in top- and sub-soil samples. However at all soil depths only tfdA class III genes proliferated during degradation. Denaturing gradient gel electrophoresis confirmed that class III tfdA genes were associated with mecoprop-p degradation. Degradation was not associated with the induction of novel tfdA genes in top- or sub-soil samples, and there were no apparent differences in tfdA gene diversity with soil depth prior to or following degradation.

Cannabidivarin (CBDV) suppresses pentylenetetrazole (PTZ)-induced increases in epilepsy-related gene expression

To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV), have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o.) on acute, pentylenetetra- zole (PTZ: 95 mg/kg, i.p.)-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1) by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00) and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25) and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ≤ 3.25) and non-responders (criterion: seizure severity >3.25), PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV re- sponders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development.

Identification of domestication-related loci associated with flowering time and seed size in soybean with the RAD-seq genotyping method

Flowering time and seed size are traits related to domestication. However, identification of domestication-related loci/genes of controlling the traits in soybean is rarely reported. In this study, we identified a total of 48 domestication-related loci based on RAD-seq genotyping of a natural population comprising 286 accessions. Among these, four on chromosome 12 and additional two on chromosomes 11 and 15 were associated with flowering time, and four on chromosomes 11 and 16 were associated with seed size. Of the five genes associated with flowering time and the three genes associated with seed size, three genes Glyma11g18720, Glyma11g15480 and Glyma15g35080 were homologous to Arabidopsis genes, additional five genes were found for the first time to be associated with these two traits. Glyma11g18720 and Glyma05g28130 were co-expressed with five genes homologous to flowering time genes in Arabidopsis, and Glyma11g15480 was co-expressed with 24 genes homologous to seed development genes in Arabidopsis. This study indicates that integration of population divergence analysis, genome-wide association study and expression analysis is an efficient approach to identify candidate domestication-related genes.

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.

Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity

Background: Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. Methodology/Principal Findings: Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolatorreared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. Conclusions/Significance: Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.

Genes related to sex steroids, neural growth, and social-emotional behavior are associated with autistic traits, empathy, and Asperger syndrome

Genetic studies of autism spectrum conditions (ASC) have mostly focused on the "low functioning" severe clinical subgroup, treating it as a rare disorder. However, ASC is now thought to be relatively common ( approximately 1%), and representing one end of a quasi-normal distribution of autistic traits in the general population. Here we report a study of common genetic variation in candidate genes associated with autistic traits and Asperger syndrome (AS). We tested single nucleotide polymorphisms in 68 candidate genes in three functional groups (sex steroid synthesis/transport, neural connectivity, and social-emotional responsivity) in two experiments. These were (a) an association study of relevant behavioral traits (the Empathy Quotient (EQ), the Autism Spectrum Quotient (AQ)) in a population sample (n=349); and (b) a case-control association study on a sample of people with AS, a "high-functioning" subgroup of ASC (n=174). 27 genes showed a nominally significant association with autistic traits and/or ASC diagnosis. Of these, 19 genes showed nominally significant association with AQ/EQ. In the sex steroid group, this included ESR2 and CYP11B1. In the neural connectivity group, this included HOXA1, NTRK1, and NLGN4X. In the socio-responsivity behavior group, this included MAOB, AVPR1B, and WFS1. Fourteen genes showed nominally significant association with AS. In the sex steroid group, this included CYP17A1 and CYP19A1. In the socio-emotional behavior group, this included OXT. Six genes were nominally associated in both experiments, providing a partial replication. Eleven genes survived family wise error rate (FWER) correction using permutations across both experiments, which is greater than would be expected by chance. CYP11B1 and NTRK1 emerged as significantly associated genes in both experiments, after FWER correction (P<0.05). This is the first candidate-gene association study of AS and of autistic traits. The most promising candidate genes require independent replication and fine mapping.

Variation in genes related to hepatic lipid metabolism and changes in waist circumference and body weight.

We analysed single nucleotide polymorphisms (SNPs) tagging the genetic variability of six candidate genes (ATF6, FABP1, LPIN2, LPIN3, MLXIPL and MTTP) involved in the regulation of hepatic lipid metabolism, an important regulatory site of energy balance for associations with body mass index (BMI) and changes in weight and waist circumference. We also investigated effect modification by sex and dietary intake. Data of 6,287 individuals participating in the European prospective investigation into cancer and nutrition were included in the analyses. Data on weight and waist circumference were followed up for 6.9 ± 2.5 years. Association of 69 tagSNPs with baseline BMI and annual changes in weight as well as waist circumference were investigated using linear regression analysis. Interactions with sex, GI and intake of carbohydrates, fat as well as saturated, monounsaturated and polyunsaturated fatty acids were examined by including multiplicative SNP-covariate terms into the regression model. Neither baseline BMI nor annual weight or waist circumference changes were significantly associated with variation in the selected genes in the entire study population after correction for multiple testing. One SNP (rs1164) in LPIN2 appeared to be significantly interacting with sex (p = 0.0003) and was associated with greater annual weight gain in men (56.8 ± 23.7 g/year per allele, p = 0.02) than in women (-25.5 ± 19.8 g/year per allele, p = 0.2). With respect to gene-nutrient interaction, we could not detect any significant interactions when accounting for multiple testing. Therefore, out of our six candidate genes, LPIN2 may be considered as a candidate for further studies.

Influence of porcine genotype on the abundance of thyroid hormones and leptin in sow milk and its impact on growth, metabolism and expression of key adipose tissue genes in offspring

Neonatal mortality is greater in commercial porcine genotypes, compared with the ancient Meishan breed that rapidly lay down adipose tissue; this may be related to hormones, such as triiodothyronine (T3) or leptin. Leptin is present in maternal milk; however, the extent to which this supply provides the neonate with leptin is unknown, but may play a role in growth and development. We investigated whether thyroid hormones and leptin concentrations in maternal milk differed between genotypes; and whether this influenced piglet concentrations or expression of genes involved in adipose tissue regulation. Eight Meishan and six commercial sows were entered into the study and milk samples from the day of parturition to day 4 postpartum was taken daily. The median birth weight piglet in each litter had a daily venous blood sample taken and was euthanised on day 4. Gene expressions of IGF-I, IGF-binding protein 3 (IGFBP-3), peroxisome proliferators activated receptor (PPAR) and glucocorticoid receptor (GR) were measured in adipose tissue using real-time PCR. T3 was increased in Meishan milk, but not in piglet plasma. Milk thyroxine was similar between breeds but commercial piglet levels were significantly higher. Leptin was higher in commercial sow milk throughout the study. Milk leptin was strongly correlated to plasma leptin during the first postnatal days and also to organ and body weight in Meishan piglets that also had significantly higher expression of GR, but not IGF-I, IGFBP-3 or PPAR. In conclusion, we have found a significant disparity in the provision of thyroid hormones in Meishan and commercial sow’s milk. These changes are not always translated to plasma concentrations of hormone in the piglet. Leptin appears to have a stronger role in growth and development in the Meishan genotype compared with commercial; along with the increased GR expression, this may also represent a potential mechanism behind the rapid accumulation of adipose tissue in Meishan piglets.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.

Chromosomal mapping of ANTP class homeobox genes in amphioxus: piecing together ancestral genomes

Homeobox genes encode DNA-binding proteins, many of which are implicated in the control of embryonic development. Evolutionarily, most homeobox genes fall into two related clades: the ANTP and the PRD classes. Some genes in ANTP class, notably Hox, ParaHox, and NK genes, have an intriguing arrangement into physical clusters. To investigate the evolutionary history of these gene clusters, we examined homeobox gene chromosomal locations in the cephalochordate amphioxus, Branchiostoma floridae. We deduce that 22 amphioxus ANTP class homeobox genes localize in just three chromosomes. One contains the Hox cluster plus AmphiEn, AmphiMnx, and AmphiDll. The ParaHox cluster resides in another chromosome, whereas a third chromosome contains the NK type homeobox genes, including AmphiMsx and ArnphiTlx. By comparative analysis we infer that clustering of ANTP class homeobox genes evolved just once, during a series of extensive cis-duplication events of genes early in animal evolution. A trans-duplication event occurred later to yield the Hox and ParaHox gene clusters on different chromosomes. The results obtained have implications for understanding the origin of homeobox gene clustering, the diversification of the ANTP class of homeobox genes, and the evolution of animal genomes.

Protochordate Zic genes define primitive somite compartments and highlight molecular changes underlying neural crest evolution

The vertebrate Zic gene family encodes C2H2 zinc finger transcription factors closely related to the Gli proteins. Zic genes are expressed in multiple areas of developing vertebrate embryos, including the dorsal neural tube where they act as potent neural crest inducers. Here we describe the characterization of a Zic ortholog from the amphioxus Branchiostoma floridae and further describe the expression of a Zic ortholog from the ascidian Ciona intestinalis. Molecular phylogenetic analysis and sequence comparisons suggest the gene duplications that formed the vertebrate Zic family were specific to the vertebrate lineage. In Ciona maternal CiZic/Ci-macho1 transcripts are localized during cleavage stages by asymmetric cell division, whereas zygotic expression by neural plate cells commences during neurulation. The amphioxus Zic ortholog AmphiZic is expressed in dorsal mesoderm and ectoderm during gastrulation, before being eliminated first from midline cells and then from all neurectoderm during neurulation. After neurulation, expression is reactivated in the dorsal neural tube and dorsolateral somite. Comparison of CiZic and AmphiZic expression with vertebrate Zic expression leads to two main conclusions. First, Zic expression allows us to define homologous compartments between vertebrate and amphioxus somites, showing primitive subdivision of vertebrate segmented mesoderm. Second, we show that neural Zic expression is a chordate synapomorphy, whereas the precise pattern of neural expression has evolved differently on the different chordate lineages. Based on these observations we suggest that a change in Zic regulation, specifically the evolution of a dorsal neural expression domain in vertebrate neurulae, was an important step in the evolution of the neural crest.

Genistein affects the expression of genes involved in blood pressure regulation and angiogenesis in primary human endothelial cells

Background: Several lines of evidence suggest that the dietary isoflavone genistein (Gen) has beneficial effects with regard to cardiovascular disease and in particular on aspects related to blood pressure and angiogenesis. The biological action of Gen may be, at Least in part, attributed to its ability to affect cell signalling and response. However, so far, most of the molecular mechanisms underlying the activity of Gen in the endothelium are unknown. Methods and results: To examine the transcriptional response to 2.5 mu M Gen on primary human endothelial cells (HUVEC), we applied cDNA array technology both under baseline condition and after treatment with the pro-atherogenic stimulus, copper-oxidized LDL. The alteration of the expression patterns of individual transcripts was substantiated using either RT-PCR or Northern blotting. Gen significantly affected the expression of genes encoding for proteins centrally involved in the vascular tone such as endothelin-converting enzyme-1, endothetin-2, estrogen related receptor a and atria[ natriuretic peptide receptor A precursor. Furthermore, Gen countered the effect of oxLDL on mRNA levels encoding for vascular endothelial growth factor receptor 165, types 1 and 2. Conclusions: Our data indicate that physiologically achievable levels of Gen change the expression of mRNA encoding for proteins involved in the control of blood pressure under baseline conditions and reduce the angiogenic response to oxLDL in the endothelium. (c) 2005 Elsevier B.V. All rights reserved.