TP23 Environmental Hypoxia
Tuesday, 15 November 2005: 8:00 AM - 6:30 PM in Exhibit Hall

(AAA-1117-561635) Analysis of Hypoxia Responsive Genes in Grass Shrimp by DNA Microarray.

Li, Tiandao¹, Larkin, Patrick², Brouwer, Marius¹, ¹ University of Southern Mississippi, Ocean Springs, MS, USA² EcoArray, L.L.C., Alachua, FL, USA

ABSTRACT- Occurrence of hypoxia in estuarine waters is increasing, and recovery of impacted estuaries is slow. Detection of early effects of hypoxia is needed for timely remedial action to be taken. A pilot study was initiated to identify the hypoxia responsive gene expression profiles from the grass shrimp, Palaemonetes pugio by DNA microarray technology to determine if the changes in the expression levels can serve as indicator of dissolved oxygen stress. Four hundred clones from several subtracted libraries, including chronic (1.5ppm and 2.5ppm DO) and cyclic (1.5→8ppm DO) exposures were chosen for PCR amplification and digestion. Purified cDNA and control clones were spotted in duplicate arrays on UltraGAPS coated slides using the VersArray ChipWriter. Total RNA was extracted from normoxic shrimp and hypoxic shrimp (Day0, Day 3, Day 7, and Day 14) in both chronic and cyclic treatments. The DNase-treated RNA was labeled with Amino-Allyl-dUTP. Cy3 was coupled to labeled cDNA from controls and Cy5 was coupled to cDNA from experimental shrimp. Slides were hybridized with equimolar mixtures of Cy3/Cy5 cDNA from normoxic and hypoxic shrimps. Hybridized arrays were scanned with the VersArray ChipReader using TIGR Spotfinder for initial spot finding. Raw data was imported into R and analyzed for normalization. Different microarray tools were used for clustering, visualization, classification, statistical analysis and biological theme discovery. DNA microarray technology is a powerful technology that will substantially increase the speed of molecular biological research and will help us to determine if expressions of HIF and hypoxia inducible genes in response to chronic and cyclic DO can be used as bioindicators of hypoxia-related stress in the marine environment.

Key words: grass shrimp, HIF, hypoxia responsive genes, microarray