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Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins.

Shaw JR, Colbourne JK, Davey JC, Glaholt SP, Hampton TH, Chen CY, Folt CL, Hamilton JW - BMC Genomics (2007)

Bottom Line: Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined.The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies.Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Dartmouth College, Hanover, New Hampshire 03755, USA. joeshaw@indiana.edu

ABSTRACT

Background: Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant.

Results: Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs.

Conclusion: The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

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Cadmium effects on gene expression: Regulated genes. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorarray elements determined to significantly different (p ≤ 0.05) using the empirical Bayes (ebayes) method to shrink gene-wise error estimate in cadmium treated vs. control animals are highlighted (up-regulated elements in pink; down-regulated elements in green).
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Figure 3: Cadmium effects on gene expression: Regulated genes. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorarray elements determined to significantly different (p ≤ 0.05) using the empirical Bayes (ebayes) method to shrink gene-wise error estimate in cadmium treated vs. control animals are highlighted (up-regulated elements in pink; down-regulated elements in green).

Mentions: Empirical Bayes statistics using a p-value cut-off of 0.05 revealed 99 elements (2.9% of the array) for which expression was increased following cadmium treatment and 30 elements (1.1% of the array) for which expression decreased (Table 2, Figure 3). Of these elements, 95 were sequenced from the 5'-end. These ESTs clustered into 42 assembled sequences. Sequence analysis and alignment comparisons of these assembled sequences using the Blastx program and an expectation-value cut-off of 1 × 10-3 and 33 matched amino acids [43] against the non-redundant protein sequence inventory at the NCBI identified 27 unique cDNAs with sequence similarities to known genes. These included 22 up and five down-regulated genes. Fifteen assembled sequences were unidentifiable, sharing no similarities to known genes. The difference between the number of sequenced elements identified as cadmium responsive (i.e., 95) and the total number of likely unique cDNAs (i.e., 42) was due to redundancy on the microarray. Although many of the elements whose ESTs were clustered into assembled sequences shared similar expression levels, in some cases their expression levels deviated by as much as two-fold (Table 2). The observed differences among clustered elements were even more pronounced when all sequenced elements belonging to the set of differentially regulated genes (EST cluster) were included in the analysis. The variation was not a function of differences in their intensity values, because deviations from the mean value of A were negligible (data not shown). Without further data, we were unable to verify whether some assembled sequences were composed of alternatively spliced transcripts and/or recently duplicated loci that differed in their responses to cadmium.


Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins.

Shaw JR, Colbourne JK, Davey JC, Glaholt SP, Hampton TH, Chen CY, Folt CL, Hamilton JW - BMC Genomics (2007)

Cadmium effects on gene expression: Regulated genes. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorarray elements determined to significantly different (p ≤ 0.05) using the empirical Bayes (ebayes) method to shrink gene-wise error estimate in cadmium treated vs. control animals are highlighted (up-regulated elements in pink; down-regulated elements in green).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2234263&req=5

Figure 3: Cadmium effects on gene expression: Regulated genes. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorarray elements determined to significantly different (p ≤ 0.05) using the empirical Bayes (ebayes) method to shrink gene-wise error estimate in cadmium treated vs. control animals are highlighted (up-regulated elements in pink; down-regulated elements in green).
Mentions: Empirical Bayes statistics using a p-value cut-off of 0.05 revealed 99 elements (2.9% of the array) for which expression was increased following cadmium treatment and 30 elements (1.1% of the array) for which expression decreased (Table 2, Figure 3). Of these elements, 95 were sequenced from the 5'-end. These ESTs clustered into 42 assembled sequences. Sequence analysis and alignment comparisons of these assembled sequences using the Blastx program and an expectation-value cut-off of 1 × 10-3 and 33 matched amino acids [43] against the non-redundant protein sequence inventory at the NCBI identified 27 unique cDNAs with sequence similarities to known genes. These included 22 up and five down-regulated genes. Fifteen assembled sequences were unidentifiable, sharing no similarities to known genes. The difference between the number of sequenced elements identified as cadmium responsive (i.e., 95) and the total number of likely unique cDNAs (i.e., 42) was due to redundancy on the microarray. Although many of the elements whose ESTs were clustered into assembled sequences shared similar expression levels, in some cases their expression levels deviated by as much as two-fold (Table 2). The observed differences among clustered elements were even more pronounced when all sequenced elements belonging to the set of differentially regulated genes (EST cluster) were included in the analysis. The variation was not a function of differences in their intensity values, because deviations from the mean value of A were negligible (data not shown). Without further data, we were unable to verify whether some assembled sequences were composed of alternatively spliced transcripts and/or recently duplicated loci that differed in their responses to cadmium.

Bottom Line: Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined.The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies.Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Dartmouth College, Hanover, New Hampshire 03755, USA. joeshaw@indiana.edu

ABSTRACT

Background: Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant.

Results: Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs.

Conclusion: The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

Show MeSH
Related in: MedlinePlus