The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach’s storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7 %). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species.
Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs.
Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.
The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1-2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28 hpf and 36 hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development.
Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.
Authors: AG McArthur, N Waglechner, F Nizam, A Yan, MA Azad, AJ Baylay, K Bhullar, MJ Canova, G De Pascale, L Ejim, L Kalan, AM King, K Koteva, M Morar, MR Mulvey, JS O’Brien, AC Pawlowski, LJV Piddock, P Spanogiannopoulos, AD Sutherland, I Tang, PL Taylor, M Thaker, W Wang, M Yan, T Yu, & GD Wright
The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.
Objective: To understand the spread of drug resistance and identifying diagnostic probes among the local tuberculosis (TB) strains in order to design rational control tools for tuberculosis controls. Methods: TA cloning and sequencing were used to characterize mutation associated with RIF resistance in 69 bp region of the gene, rpoB. The analysis identified two regions of mutations but no unusual insertion and deletion. No mutation was observed in RIF sensitive strains. Results: We employed Random Amplified Polymorphic DNA (RAPD) analysis for typing strains of M. tuberculosis to determine whether new strains were present among M. tuberculosis isolates circulating in Yaounde. Three groups (I to III) of M. tuberculosis were identified among 93 isolates randomly selected. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. RAPD was used to select the new site of diagnostic by PCR. Also single nucleotide polymorphisms between M. tuberculosis and M. bovis were found, suggesting that RAPD can be a useful technique for distinguishing between species. Conclusions: Molecular typing is defined as the integration of conventional epidemiological approach to track specific strains of pathogens in order to understand the distribution of disease in populations.
Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.
Cytochrome P450 (CYP) proteins compose a highly diverse superfamily found in all domains of life. These proteins are enzymes involved in metabolism of endogenous and exogenous compounds. In vertebrates, the CYP2 family is one of the largest, most diverse and plays an important role in mammalian drug metabolism. However, there are more than 20 vertebrate CYP2 subfamilies with uncertain evolution and fairly discrete subfamily composition within vertebrate classes, hindering extrapolation of knowledge across subfamilies. To better understand CYP2 diversity, a phylogenetic analysis of 196 CYP2 protein sequences from 16 species was performed using a maximum likelihood approach and Bayesian inference. The analyses included the CYP2 compliment from human, fugu, zebrafish, stickleback, medaka, cow, and dog genomes. Additional sequences were included from rabbit, marsupial, platypus, chicken, frog, and salmonid species. Three CYP2 sequences from the tunicate Ciona intestinalis were utilized as the outgroup. Results indicate a single ancestral vertebrate CYP2 gene and monophyly of all CYP2 subfamilies. Two subfamilies (CYP2R and CYP2U) pre-date vertebrate diversification, allowing direct comparison across vertebrate classes, while all other subfamilies originated during vertebrate diversification, often within specific vertebrate lineages. Analysis of site-specific evolution indicates that some substrate recognition sites (SRS) previously proposed for CYP genes do not have elevated rates of evolution, suggesting that these regions of the protein are not necessarily important in recognition of CYP2 substrates. Type II functional divergence analysis identified multiple residues in the active site of CYP2F, CYP2A, and CYP2B proteins that have undergone radical biochemical changes and may be functionally important.
Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.