Toxicology in the omics era. Chris Evelo BiGCaT Bioinformatics Group – BMT-TU/e & UM

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Gene expression Genes are part of the chromosomes in the cell nucleus. Genes are transcribed to messenger RNA (mRNA). mRNA is processed further in the nucleus. Complete mRNA’s leave the nucleus and are translated to protein in the cytosol.

Figure The transfer of information from DNA to protein. The transfer proceeds by means of an RNA intermediate called messenger RNA (mRNA). In procaryotic cells the process is simpler than in eucaryotic cells. In eucaryotes the coding regions of the DNA (in the exons,shown in color) are separated by noncoding regions (the introns). As indicated, these introns must be removed by an enzymatically catalyzed RNA-splicing reaction to form the mRNA. Alberts et al. Molecular Biology of the Cell, 3rd edn.

mRNA processing Genes contain: – Expressed regions (exons) – Non expressed regions (introns) During gene splicing introns are removed and exons connected A poly-adenosine (poly-A) tail is added Complete mRNA’s leave the nucleus

Figure Control of the poly-A tail length affects both mRNA stability and mRNA translation. (A) Most translated mRNAs have poly-A tails that exceed a minimum length of about 30 As. The tails on selected mRNAs can be either elongated or rapidly cleaved in the cytosol, and this will have an effect on the translation of these mRNAs. (B) A model proposed to explain the observed stimulation of translation by an increase in poly-A tail length. The large ribosomal subunits, on finishing a protein chain, may be directly recycled from near the 3' end of an mRNA molecule back to the 5' end to start a new protein by special poly- A-binding proteins (red). Alberts et al. Molecular Biology of the Cell, 3rd edn.

Genes and vulnerability Genes can be: Absent (e.g. GST mu deletion) Broken (e.g. single nucleotide point mutations SNP’s) Differently expressed (e.g. P450 class of enzymes) – Intra individual differences (vulnerability) – As a result of exposure (BEM)

What about the human genome? Smart people copied chromosomal sequences to computer hard discs. So now you can read it (although I still prefer a good novel). If you are good at it (and care to read it 6 times over) you can even predict genes. But even if you are among the best you can’t predict proteins or their function

Tell me about your proteins Hard working biochemists and physiologists did spend a century to describe proteins, their function, structure and sequence. Molecular biologists used decades found huge amounts of expressed mRNA sequences (ESTs) and tried to relate them to function. And… they failed. Cluttering up the databases with things like “EST found in very seldom tumor so and so” (this could still be myoglobin mRNA)

So what can we do? Take the EST sequences and cluster them to full mRNA sequences (Unigene!) Build the full coding sequences from this (useful part of EMBL) Translate that into hypothetical proteins (trEMBL) Check whether that happens to be a known protein (Swissprot) Use all that to find microarray reporter sequences for known proteins

DNA useful after all? Yes, if you know from population genetics or animal experiments about loci important for trades. Your gene might be in such a locus. And to find regulatory sequences

Past, present and (near) future Toxicologists detect: Enzyme activity; classic clinical chemistry. Single gene DNA identity, PCR. Single gene expression at the mRNA level (RT-PCR) Transcriptomics. Full genome mRNA expression (microarray, expression libraries) Proteomics. Full genome protein expression (proteomics, 2D-gels with MS, antibody arrays)

Gene expression measurement Functional genomics/transcriptomics: Changes in mRNA – Gene expression microarrays – Proteomics: Changes in protein levels – – DNA  mRNA  protein

Gene expression arrays Microarrays: relative fluorescense signals. Identification. Macroarrays: absolute radioactive signal. Validation.

Gene expression microarrays Contain many immobilized unique cDNA sequences (20,000) Sample mRNA is transcribed to complementary DNA (cDNA) Sample cDNA is made fluorescent using 2 different dyes cDNA’s will bind (hybridize) specifically to their own complementary spotted cDNA Fluorescence is read using laser technology

Layout of a microarray experiment 1)Get the cells 2)Isolate RNA 3)Make fluorescent cDNA 4)Hybridize 5)Laser read out 6)Analyze image

Next slide shows data of one single actual microarray Normalized expression shown for both channels. Each reporter is shown with a single dot. Red dots are controls Note the GEM barcode (QC) Note the slight error in linear normalization (low expressed genes are higher in Cy5 channel)

Next slide shows same data after processing Controls removed Bad spots (<40% average area) removed Low signals (<2.5 Signal/Background) removed All reporters with <1.7 fold change removed (only changing spots shown)

Final slide shows information for one single reporter This signifies one single spot It is a known gene: an UDP glucuronyltransferase Raw data and fold change are shown

Microarrays can detect Differences in mRNA expression (that’s what they were made for) – Can compare the individual to “the population” – Or “the exposed group” with the “control group” Gene deletions SNP’s (single nucleotide point mutations)

We could now Isolate mRNA from an individuals white blood cells. Run a 10,000 gene mRNA expression array Put the results on a personal microchip or CD-ROM And know his vulnerabilities

And we could Sell the results to his insurance company…

Are you ready? Are you ready to hop on the genomics wagon? It may be a bit awkward But you will have to…

Hop, Step and … Jump

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