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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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Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [GenBank:EU307304].
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Figure 6: Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [GenBank:EU307304].

Mentions: Given its utility as a biomarker, we further characterized the putative D. pulex MT, which represents one of the first MT sequences identified from a non-malacostracan crustacean species. A putative MT was identified by the microarray following the sequencing of probes whose expression was up-regulated by cadmium exposure (Figure 6a). A sequence similarity search against the NCBI protein database using Blastx revealed little similarity to other MTs. The closest match at the time was directed to MT from the giant keyhole limpet, Megathura crenulata (E-value = 0.036), but this is well below 1 × 10-5 E-value considered to be significant [43]. However, the translated sequence revealed a 59 amino acid protein of high cysteine content (30.5%) that contained no aromatic amino acids or histidine residues, unique features that are hallmarks of MT. The 18 cysteine residues were arrayed in characteristic Cys-xaa-yaa-Cys (1), Cys-x-Cys (6), and Cys-Cys (2) motifs, establishing it as a class 1 MT (Fig. 5; [47]). Nevertheless, the translated amino acid sequence was still quite diverged from other crustacean MTs (~30% similarity), including differences in the common pattern observed at the N terminus, P- [GD]-P-C-C-x(3,4)-C-x-C [48]. Despite this lack of similarity, the number of cysteine residues was identical to other crustacean MTs and these showed a high degree of conservation when aligned with sequences reported for other arthropods (Table 4). Based on amino-acid alignments with characterized MTs, the D. pulex MT formed two coordinative domains (α, C-terminus; β, N-terminus), hinging with the proline residue at position 20 (Fig. 6a, Dpu Mtn1). It should be noted that during the preparation of this manuscript, an MT gene transcript and translated amino acid sequence was reported for another daphniid, D. magna [19].


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)

Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [GenBank:EU307304].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [GenBank:EU307304].
Mentions: Given its utility as a biomarker, we further characterized the putative D. pulex MT, which represents one of the first MT sequences identified from a non-malacostracan crustacean species. A putative MT was identified by the microarray following the sequencing of probes whose expression was up-regulated by cadmium exposure (Figure 6a). A sequence similarity search against the NCBI protein database using Blastx revealed little similarity to other MTs. The closest match at the time was directed to MT from the giant keyhole limpet, Megathura crenulata (E-value = 0.036), but this is well below 1 × 10-5 E-value considered to be significant [43]. However, the translated sequence revealed a 59 amino acid protein of high cysteine content (30.5%) that contained no aromatic amino acids or histidine residues, unique features that are hallmarks of MT. The 18 cysteine residues were arrayed in characteristic Cys-xaa-yaa-Cys (1), Cys-x-Cys (6), and Cys-Cys (2) motifs, establishing it as a class 1 MT (Fig. 5; [47]). Nevertheless, the translated amino acid sequence was still quite diverged from other crustacean MTs (~30% similarity), including differences in the common pattern observed at the N terminus, P- [GD]-P-C-C-x(3,4)-C-x-C [48]. Despite this lack of similarity, the number of cysteine residues was identical to other crustacean MTs and these showed a high degree of conservation when aligned with sequences reported for other arthropods (Table 4). Based on amino-acid alignments with characterized MTs, the D. pulex MT formed two coordinative domains (α, C-terminus; β, N-terminus), hinging with the proline residue at position 20 (Fig. 6a, Dpu Mtn1). It should be noted that during the preparation of this manuscript, an MT gene transcript and translated amino acid sequence was reported for another daphniid, D. magna [19].

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