T2 PM 'Omic' Technologies: Current and Future Applications to Environmental Toxicology (Part 2)
Tuesday, 15 November 2005: 1:50 PM - 5:30 PM in Ballroom 2

(SHA-1117-848505) Differences in microarray gene expression profiles of Daphnia pulex exposed to metals, characterization and comparative sequence analysis of responsive genes.

Shaw, J¹, Chen, C¹, Glaholt, S¹, Folt, C¹, Davey, J², Hampton, T², Hamilton, J², Bohuski, E³, Andrews, J³, Colbourne, J³, ¹ Darmouth College, Hanover, NH, USA² Dartmouth Medical School, Hanover, NH, USA³ Indiana University, Bloomington, IN, USA

ABSTRACT- Genomic tools that discover and monitor gene function will likely become critical modern resources to understand how organisms immediately respond to, and evolve in, toxic environments. As a result, DNA technologies were developed for the freshwater crustacean Daphnia, whose genetics are tractable in the laboratory and whose populations are accessible in nature. To highlight its potential for ecological and toxicological genomic discoveries, we conducted experiments using DNA microarrays to determine gene expression profiles for D. pulex. Daphnia were acutely exposed (48-h) to incipient concentrations of two metals (arsenic, cadmium) that are known to elicit different toxic responses. These tests revealed that (i) the two metals elicit a response from different sets of genes; (ii) arsenic has a greater gene response than cadmium in both number and magnitude; and (iii) candidate genes including metallothionein (MT) were identified using the microarrays. Real-time PCR of the candidate genes was useful in more accurately quantifying expression differences and in describing biological and technical variability. Because of its known utility as a biomarker, the MT genome sequence was characterized using an arrayed cosmid library and data within the Daphnia genome database called wFleaBase. Sequence alignment and phylogenetic analyses revealed limited sequence conservation for this gene among the arthropods (except for 18 cys) and identified it as a novel crustacean MT with three putative metal response elements residing in its promoter. In addition, greater MT expression levels were observed in cadmium-acclimated populations exposed to metals compared to metal exposed naive populations. These results agree with increases in metal tolerance observed for the acclimated populations under metal stress. Our current studies are exploring differences in the genetic regulatory response to metals among arthropods and are applying Daphnia microarrays to measure variation in expression patterns to metals within natural populations.

Key words: Daphnia pulex, microarray, metal, metalothionein