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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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Confirmation of array results. Quantitative-real time (Q-RT)-PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subunit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biological validation, open bars, mean ± SD, n = 5).
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Figure 5: Confirmation of array results. Quantitative-real time (Q-RT)-PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subunit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biological validation, open bars, mean ± SD, n = 5).

Mentions: Following sequence analysis, expression levels of a subset of genes identified as cadmium-responsive (Table 2) were measured to validate microarray output. This included three cadmium responsive genes (i.e., cuticle protein-2, Contig 257; 2-domain haemoglobin protein subunit, Contig 262; metallothionein, Contig 221) and one gene for which expression was not altered (i.e., serine-threonine kinase, Contig 274). Expression levels were confirmed by Q-RT-PCR (Table 3) using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation) and RNA collected from repeated, independent biological exposures (biological validation). In all instances, expression levels measured by Q-RT-PCR agreed with microarray output (Figure 5) both in terms of direction (M) and relative magnitude (A) of the response.


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)

Confirmation of array results. Quantitative-real time (Q-RT)-PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subunit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biological validation, open bars, mean ± SD, n = 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Confirmation of array results. Quantitative-real time (Q-RT)-PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subunit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biological validation, open bars, mean ± SD, n = 5).
Mentions: Following sequence analysis, expression levels of a subset of genes identified as cadmium-responsive (Table 2) were measured to validate microarray output. This included three cadmium responsive genes (i.e., cuticle protein-2, Contig 257; 2-domain haemoglobin protein subunit, Contig 262; metallothionein, Contig 221) and one gene for which expression was not altered (i.e., serine-threonine kinase, Contig 274). Expression levels were confirmed by Q-RT-PCR (Table 3) using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation) and RNA collected from repeated, independent biological exposures (biological validation). In all instances, expression levels measured by Q-RT-PCR agreed with microarray output (Figure 5) both in terms of direction (M) and relative magnitude (A) of the response.

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