Identification of robust hypoxia biomarker candidates from fin of medaka (Oryzias latipes).

Aquatic hypoxia caused by organic pollution and eutrophication is a pressing worldwide water pollution problem. Better methods for monitoring oxygen levels are needed to assist efforts to maintain and protect the health of natural aquatic environments. In this project, we used a Japanese ricefish (medaka, Oryzias latipes) 8K oligonucleotide array as a platform to identify potential hypoxic biomarkers in different organs (fin, gill, liver and brain) upon exposure to hypoxia. The microarray results were validated by qRT-PCR employing a subset of candidate biomarkers. Interestingly, the largest number and most significant of hypoxia responding array features were detected in hypoxia exposed fin tissues. We identified 173 array features that exhibited a significant response (over 2 fold change in expression) upon exposure to hypoxic conditions and validated a subset of these by quantitative RT-PCR. These gene targets were subjected to annotation and gene ontology mining. Positively identifiable gene targets that may be useful for development of a rapid and accurate biomarker test using fin clips are discussed in relation to previous reports on hypoxia responsive genes.

Comparison of gene expression responses to hypoxia in viviparous (Xiphophorus) and oviparous (Oryzias) fishes using a medaka microarray.

Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka (Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species (Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant (p<0.05)+/-3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 x 10(-5)) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).

Genomic approaches in the identification of hypoxia biomarkers in model fish species.

Eutrophication leading to hypoxic water conditions has become a major problem in aquatic systems worldwide. Monitoring the levels and biological effects of lowered oxygen levels in aquatic systems may provide data useful in management of natural aquatic environments. Fishes represent an economically important resource that is subject to hypoxia exposure effects. Due to the extreme diversity of fish species and their habitats, fishes in general have evolved unique capabilities to modulate gene expression patterns in response to hypoxic stress. Recent studies have attempted to document quantitative changes in gene expression patterns induced in various fish species in response to reduced dissolved oxygen levels. From a management perspective, the goal of these studies is to provide a more complete characterization of hypoxia responsive genes in fish, as molecular indicators (biomarkers) of ecosystem hypoxic stress.The molecular genetic response to hypoxia is highly complex and overlaps with other stress responses making it difficult to identify hypoxia specific responses using traditional single gene or low throughput approaches. Therefore, recent approaches have been aimed at developing functional genomic (e.g. high density microarray and real-time PCR) and proteomic (two-dimensional fluorescence difference in gel electrophoresis coupled with mass spectrometry based peptide identification) technologies that employ fish species. Many of the fish species utilized in these studies do not have the advantages of underlying genome resources (i.e., genome or transcriptome sequences). Efforts have attempted to establish correlations between discreet molecular responses elicited by fish in response to hypoxia and changes in the genetic profiles of stressed organs or tissues. Notable progress in these areas has been made using several different versions of either cDNA or oligonucleotide based microarrays to profile changes in gene expression patterns in response to hypoxic stress.Due to these efforts, hundreds of hypoxia responsive genes have been identified both from laboratory reared aquaria fish and from feral fish derived from both fresh and saltwater habitats. Herein, we review these reports and the emergence of hypoxia biomarker development in aquatic species. We also include some of our own recent results using the medaka (Oryzias latipes) as a model to define genetic profiles of hypoxia exposure.

Perturbation of DNA repair gene expression due to interspecies hybridization.

The effect of interspecies hybridization on gene regulation was examined using real-time polymerase chain reaction (RT-PCR) to measure the expression of five base-excision repair genes in brain, eye, gill, liver, and tailfin tissues from Xiphophorus parental species and F(1) hybrids. Relative mRNA levels of uracil N-glycosylase (Ung), Apurinic/apyrimidinic endonuclease (Ape1), polymerase-beta (Polb), flap endonuclease (Fen1), and DNA ligase (Lig1) were measured in three parental Xiphophorus species (X. maculatus Jp 163 B, X. helleri Sarabia, and X. andersi andC) and in two interspecies F(1) hybrids, the Sp-helleri hybrid (X. maculatus Jp 163 BxX. helleri Sarabia) and the Sp-andersi hybrid (X. maculatus Jp 163 BxX. andersi) to identify genes that undergo changes in expression levels upon interspecies hybridization. Significant differences in gene expression were observed between parental animals and their respective F(1) hybrids in both interspecies crosses. Generally, marked increases in DNA repair gene mRNA levels were observed across all tissues in F(1) hybrid animals from the Sp-helleri cross compared to either X. maculatus or X. helleri parents. In contrast, the Sp-andersi F(1) hybrid animals generally exhibited decreased base-excision repair gene expression, although this trend was more specific to individual tissues than observed for Sp-helleri hybrids.

Multiple tissue gene expression analyses in Japanese medaka (Oryzias latipes) exposed to hypoxia.

Due in part to human population growth watersheds and coastal estuaries have been receiving increasing run-off of nutrients and genotoxins. As a consequence, the occurrences of nutrient-driven hypoxia in coastal waters appear to be increasing. Thus, understanding the molecular genetic response to hypoxia by model aquatic organisms is of interest both from environmental and physiological viewpoints. The major objectives of this study are to determine genome-wide gene expression profiles and to better understand how hypoxia influences global gene expression in medaka (Oryzias latipes), a well utilized aquatic model species. Herein we detail our development of a microarray containing 8046 medaka unigenes and describe our experimental results for measuring gene expression changes in the brain, gill, and liver of hypoxia exposed fish. Using conservative selection criteria, we determined that 501 genes in the brain, 442 in the gill, and 715 in the liver were differentially expressed in medaka exposed to hypoxia. These differentially expressed genes fell into a number of biological gene ontology groups related to general metabolism, catabolism, RNA and protein metabolism, etc. Two biological pathways, ubiquitin-proteasome and phosphatidylinositol signaling, were significantly dysregulated in medaka upon hypoxia exposure. Comparative genomics between medaka and human identified several human orthologies associated with known diseases.

DNA microarray technology in toxicogenomics of aquatic models: methods and applications.

Toxicogenomics represents the merging of toxicology with genomics and bioinformatics to investigate biological functions of genome in response to environmental contaminants. Aquatic species have traditionally been used as models in toxicology to characterize the actions of environmental stresses. Recent completion of the DNA sequencing for several fish species has spurred the development of DNA microarrays allowing investigators access to toxicogenomic approaches. However, since microarray technology is thus far limited to only a few aquatic species and derivation of biological meaning from microarray data is highly dependent on statistical arguments, the full potential of microarray in aquatic species research has yet to be realized. Herein we review some of the issues related to construction, probe design, statistical and bioinformatical data analyses, and current applications of DNA microarrays. As a model a recently developed medaka (Oryzias latipes) oligonucleotide microarray was described to highlight some of the issues related to array technology and its application in aquatic species exposed to hypoxia. Although there are known non-biological variations present in microarray data, it remains unquestionable that array technology will have a great impact on aquatic toxicology. Microarray applications in aquatic toxicogenomics will range from the discovery of diagnostic biomarkers, to establishment of stress-specific signatures and molecular pathways hallmarking the adaptation to new environmental conditions.

An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus.

Teleost fish genome projects involving model species are resulting in a rapid accumulation of genomic and expressed DNA sequences in public databases. The expressed sequence tags (ESTs) collected in the databases can be mined for the analysis of both structural and functional genomics. In this study, we in silico analyzed 49,430 unigenes representing a total of 692,654 ESTs from four model fish for their potential use in developing simple sequence repeats (SSRs), or microsatellites. After bioinformatical mining, a total of 3,018 EST derived SSRs (EST-SSRs) were identified for 2,335 SSR containing ESTs (SSR-ESTs). The frequency of identified SSR-ESTs ranged from 1.5% for Xiphophorus to 7.3% for zebrafish. The dinucleotide repeat motif is the most abundant SSR, accounting for 47%, 52%, 64%, and 78% for medaka, Fundulus, zebrafish, and Xiphophorus, respectively. Simulation analysis suggests that a majority of these EST-SSRs have sufficient flanking sequences for polymerase chain reaction (PCR) primer design. Comparative DNA sequence analyses of SSR-ESTs identified several cross-species SSRs and sequences that may be used as cross-reference genes in comparative studies. For example, the flanking sequences of one SSR (CTG)n within the pituitary tumor-transforming gene (PTTG) 1 interacting protein (PTTGIP), showed conservation spanning the medaka, Fundulus, human, and mouse genomes. This study provides a large body of information on EST-SSRs that can be useful for the development of polymorphic markers, gene mapping, and comparative genome analysis. Functional analysis of these SSR-ESTs may reveal their role in metabolism and gene evolution of these model species.

Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus.

Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase beta (Polbeta) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.