1. Expression of the Genome
The transcriptome

2. Decoding the Genetic Information
The information is 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 Only one of the two strands is coding! Exons
Transcription initiation site
3’untranslated region
5’ untranslated region
Introns
Promoter/
Regulatory sequences 5’
Termination sequence 3’
RNA Transcript
Exon 2
Exon 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 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
Strand on which the promoter is located 5

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

7. Transcription - Translation
Template strand 3’
Coding strand 5’
Sense strand 5’
NH3—
— COOH

8. ORFs Most double stranded sequences have 6 reading frames
GCCGATTAGAGA>
TGCCGATTAGAG>
ATGCCGATTAGA>
5’-ATGGCGATTAGAGACAGCCATTAA-3’
3’-TACTGCTAATCTCTGTCGGTAATT-5’
<CTGTCGGTAATT
<TCTGTCGGTAAT
<CTCTGTCGGTAA
How many ORFs does this sequence have?

9. Homologues Gene sequences that possess a common ancestor
Homologues share a high level of identity
Identity
Percentage of bases or amino acids that are the same between different sequences

10. Nucleotide Homologues
DNA sequences with greater 70% identity
Ex. A homologue of the human hemoglobin gene is found in soya
G.G.T.G.A.G.G.G.T.A.T.C.A.T.C.C.C.A.T.C.T.G
G.G.T.C.A.G.G.A.T.A.T.G.A.T.T.C.C.A.T.C.A.C
* * * * * * * * * * * * * * * *
77% identity

11. Protein homologues Protein sequences with greater than 25% identity
Ex. A protein homologue of the human hemoglobin is found in soya
G A R G G W L
G.G.T.G.A.G.G.G.C.A.T.C.A.T.C.C.C.A.T.C.T
G.G.T.C.A.G.G.A.C.A.T.G.A.T.T.C.C.A.T.C.A
G T P M I W E
Percentage identity: 28%

12. Homologues Orthologues : Paralogues :
Homologues found in different organisms which have a common ancestor
Duplication followed by speciation
Paralogues :
Homologues found within the same species
Duplication prior to speciation

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

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

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

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

17. 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

18. 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

19. 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

20. Normalization

21. 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
Tissues: F C R P
Actin
Fos

22. 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

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

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

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

26. 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