EFFECTS.

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 with PAHs.

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

Genomic Results

Array effects summary

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 binding protein

Genespring summary list

Q-PCR data

A real time PCR procedure for flounder CYP 1A has been worked out a tested by Tom Dixon

Published PDF file

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.

Proteomics effects summary

Expression of CYP1A was measured by activity determination using EROD activity, metallothionein was estimated by a radiocadmium saturation assay.

Protein 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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