Initial field comparisons
have been made with livers of flounders from 2 UK estuaries, one
the Alde has no industrial inouts and a small domestic seqage input,
it is in an agricultural area; the Tyne flows through a major industrial
area with historically a large shipbuilding industry and a huge
domestic/industrial sewage input , the sediments are heavily polluted
RNA samples of male fish
were compared with a minarray of candidate genes and real time PCR
for CYP1A, UGT, MT and VTG.
Protein expression in
five male fish were compared by 2D gel electrophoresis
First round pilot experiments
have been carried out by Tim, on an array of the Birmingham set
of cloned PCR products of flounder candidate genes. This was probed
with cDNA from Alde (reference) vs. Tyne (polluted) flounders.
The results showed that female fish (as predicted) were pretty
hopeless, with very variable results. Interesting results were
found with the males.
Upregulated > 1.6-fold in Tyne Males
CYP1A; UDPGT; a-2HS glycoprotein; dihydripyrimidine dehydrogemase;
CU/ZN SOD; Aldehyde dehydrogenase; Paraoxonase
Downregulated > 1.6-fold in Tyne Males
Ferritin H/M chain; Ornithine decarboxylase EF 1 delta ; EF-2; Glucose-6-phosphatase;
Complement C3; Metallothionein; Int-6; Ribosomal 40S; Chitin
A real time PCR procedure
for flounder CYP 1A has been worked out a tested by Tom Dixon
Real time PCR
methods for MT, UGT and VTG have also been optimised and
applied to the RNA samples from the two estuaries. These data
showed that CYP1A, UGT and VTG expression were increased in most
Tyne fish compared with the Alde and that MT mRNA levels were
only elevated in few fish.
Real Time QPCR methods
have now been developed for other genes ...EF1, AHRR, Glutathione
Reductase, Cu/Zn and Mn-SOD's, Thioredoxin and are normalised
to 28S RNA.
Expression of CYP1A was
measured by activity determination using EROD activity, metallothionein
was estimated by a radiocadmium saturation assay.
expression patterns on 2D gels were remarkably similar between individuals
with charactersitic patterns for the two estuaries, many differentially
expressed proteins were identified and these are being characterised.
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