Differential gene expression of Chlamydomonas reinhardtii in
response to2,4,6-trinitrotoluene (TNT) using microarray analysis

Nrupali Patel, Vinitha Cardoza and C. Neal Stewart Jr.

Trinitrotoluene (TNT) has been extensively used as an ammunition
compound in the past century. It is a well-known xenobiotic that has
entered the environment from production activities at ammunition plants,
field usage and improper disposal. The presence of this compound is
of great concern because of its toxicity, carcinogenicity and its mutagenic
effects on a wide range of organisms. As a result, there is a need for
biomonitoring and bioremediation of this compound.

In this study, we have used the unicellular algae Chlamydomonas reinhardtii
as a model organism to study the genetic response to TNT. To provide a
large-scale expression analysis in response to this common ordnance compound,
microarray experiments were conducted with 3000 clones sequenced from the
Chlamydomonas reinhardtii cDNA library. Axenic cultures of C. reinhardtii
were grown in various concentrations of TNT to determine the treatment
conditions for microarray analysis. TNT concentrations of 1mg/ml and 3mg/ml
were selected as the optimal treatment conditions. Fluorescently labeled Cy3
and Cy5 cDNA probes were generated from 10mg total RNA using direct
incorporation with reverse transcription. These probes were hybridized on the
microarray slide. After hybridization the slides were scanned and analyzed
using GenePix pro 4.1 for spot intensities. Data was normalized and 50 genes
that were unregulated or down regulated are under investigation.

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Department of Plant Sciences and Landscape Systems
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University of Tennessee
Knoxville, TN 37996-4561, USA