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In this “global” approach, the techniques most frequently used recently are differen-
tial display (Liang and Pardee 1992) and suppression subtractive hybridization (SSH)
(Diatchenko et al. 1996; Gurskaya et al. 1996). Differential display is a relatively rapid and
not very expensive technique, but showed a major inconvenience: the detection of a large
number of false positives (Martin and Pardee 2000). So, it must be accompanied by neces-
sary steps of verification. Using this technique successfully, Bultelle et al. (2002) detected
differentially expressed genes in the freshwater zebra mussel
Dreissena polymorpha
exposed
to contaminants. In the case of the fish
Fundulus heteroclitus
exposed to a polyaromatic
hydrocarbon (anthracene), Peterson and Bain (2004) revealed 26 DNA fragments displaying
differential expression; among these sequences, 8 showing homologies with data available
in international databanks were identified, the other 18 sequences being unknown. The 26
differential inductions were verified with macroarray technology. This technique consists
of leaving PCR products of genes whose expression might be disturbed on a solid support
(membrane). Membranes are then placed in contact with complementary DNAs (cDNAs)
under hybridization conditions, cDNA reflecting the transcription of exposed and unex-
posed animals. By hybridization processes, fixed fragments of DNA keep the homologous
cDNA. The comparison of the membranes obtained with the two transcriptomic extracts
allows the determination of different levels of expression between contaminated and con-
trol organisms. With this technical approach, Peterson and Bain (2004) identified three
sequences as markers of contamination by anthracene. A parallel study conducted on nat-
ural populations submitted to complex contaminations confirmed the previous inductions
but revealed other overexpressions; these results underline the difficulties of extrapolating
observations from controlled environments to natural environments.
The aim of SSH is to obtain a picture of the induction (positive regulation) or the repres-
sion (negative regulation) of several genes, the quantification of the differences between
the expression levels in two experimental or environmental conditions not being possi-
ble. The SSH technique considers the study of two banks of cDNA to identify separately
the two kinds of regulation. This approach was used to analyze differential expression
in the oyster
C
.
gigas
subjected to pesticides (Tanguy et al. 2005). The authors revealed
137 sequences differentially expressed between contaminated versus uncontaminated
oysters; among them, 81 were new markers. The 56 identified sequences were linked to
different processes: detoxification, energy metabolism. Brown et al. (2006), analyzing
the responses of the mussel
Mytilus edulis
to PAHs, by SSH, showed different biomark-
ers compared to those observed for vertebrates. Sheader et al. (2004) explored by SSH the
transcriptomic responses of the flatfish
Platichtys flesus
to intraperitoneal contamination
by benzo[
a
]pyrene and cadmium; they revealed 184 differentially expressed genetic frag-
ments. The same SSH method allowed the identification of new candidate genes involved
in the responses of
P. l e su s
to experimental cocktails of pesticides; the induction or the
inhibition of particular genes observed in the previous experiments was confirmed by
comparison of fish from pesticide-impacted versus “pristine” estuaries (Marchand et al.
2006).
The target genes revealed by these global approaches could be potentially subjected to
selective pressure induced by contaminants; they could constitute good candidate genes:
1. To analyze their differential expression levels in relation to the nature of the chem-
ical stress
2. To explore the possible functional role of their polymorphism in the ability of
organisms or populations to resist contamination
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