1. Expression of the Genome The transcriptome

2. Decoding the Genetic Information  Information encoded in nucleotide sequences contained in discrete units  The genes  The information contained in the genes is transcribed to generate the RNAs and then decoded to generate the proteins

3. The Genes Transcription initiation site Termination sequence 3’ Promoter/ Regulatory sequences 5’ Introns RNA Transcript 5’ untranslated region 3’untranslated region Exon 2Exon 3 Int. 2 Exon 1 Int. 1 Exons Only one of the two strands is coding!

4. Coding  Coding strand  Positive strand  Sense strand  Strand which is complementary to the template strand  Strand of which the sequence is the same as that of the RNA transcript  Strand on which is located the promoter 4

5. Non Coding  Non coding strand  Negative strand  Antisense strand  Template strand  Strand of which the sequence is complementary to that of the RNA transcript 5

6. Codant Vs Non-coding DNA: 5’ TAG 3’ 3’ ATC 5’ Translation Leu Protein: Genetic code : CUA = Leu UAG = Stop Transcription RNA: ?5’3’

7. Genome Transcriptome Collection of RNA from genes that code for proteins Collection of RNA that represents the fraction of the genome that is expressed Proteome Collection of proteins derived from the transcriptome Transcription Translation

8. One Genome  Is the transcriptome the same in all the cells of an organism?  Is the transcriptome always the same in a given cell?

9. Does a Sequence Code for a Transcript?  Northern Hybridization Analysis  Northern Hybridization Analysis  RT-PCR  RT-PCR 9

10. Comparaison of Methods 10 Northern RT-PCR Sequence must be known Presence or absence of a transcript Allows to determine size Sensitivity Compare relative abundance Obtain sequence of transcript Determine which strand is transcribed Determine how many transcripts are made from a single sequence No Yes Yes Yes No Low High Yes No Yes Yes Yes No THE SEQUENCE MUST BE EXPRESSED YES

11. Northern Analysis  Isolate total RNA from cells or tissue  Separate RNA according to their sizes on denaturing agarose gel  Formaldehyde + Formamide  Hybridization with complementary probe rRNA tRNA

12. Northern Hybridization  Requires a probe  Hybridization= the probe has sequences of the gene  The sequence is expressed  Intensity of hybridization signal = relative abundance  Number of hybrids= number of transcripts  Possibly number of genes 12

13. Northern Hybridization  Allows to compare the relative quantity of a transcript  Low sensitivity  Requires an internal control  Gene whose abundance is constant under the different conditions examined –Controls for variations in the amount of RNA loaded –Use housekeeping genes :  Genes that ensure indispensable functions for the survival of all cell types  Constitutive expression 13

14. Normalization 14

15. Problem  A Northern of ARN isolated from different tissues was probed with the Fos gene as well as a house keeping gene; Actin. Explain the results obtained 15 Tissues: F C R P ActinFos

16. RT-PCR  Allows the amplification of an RNA sequence  Isolate total RNA from cells or tissues  Transcribe RNA into cDNA with reverse transcriptase  Amplify sequence of interest by PCR 16

17. Reverse Transcriptase Reaction Gene Non-Specific 17 AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT Annealing of polyT primer Collection Collection of complementary DNAs to RNAs expressed at a given time under given conditions AAAAAAA mRNA AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT Transcription to cDNA RT

18. Reverse Transcriptase Reaction Gene Specific 18 AAAAAAA Synthesis of cDNA RT one DNA complementary to one mRNA of interest AAAAAAA Annealing of gene specific primer AAAAAAA

19. RT  PCR 19 cDNA CollectioncDNA of mRNA of interest Analysis on gel PCR with primers specific to sequence of interest

20. RT-PCR  The sequence must be known in to design primers  Amplification product =  The primer sequences are part of the gene  The sequence is expressed  Intensity proportional = relative abundance  The size of the amplification product is not equal to the size of the transcript 20