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. 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

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

11. Does a Sequence Code for a Transcript?
RT-PCR
Allows the amplification of an RNA sequence
Two steps
Step I: Reverse transcriptase reaction
Transcribe RNA into cDNA with reverse transcriptase
Step 2: Amplify sequence of interest by PCR

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

13. RT-PCR method Isolate total RNA from cells or tissue Verify quality
Separate RNA according to their sizes on denaturing agarose gel
Formaldehyde + Formamide
rRNA
tRNA

14. First strand synthesis Gene Non-Specific Reverse Transcriptase Reaction
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

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

16. 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

17. 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

18. Normalization

19. Problem
RT-PCR using RNA isolated from different tissues was preformed to compare the expression of the Fos gene. Simultaneous reactions were done for the house keeping gene; Actin. Explain the results obtained.
Tissues: F C R P
Actin
Fos

20. Related sequences Homologues
Sequences that share a high level of identity
Nucleotide
DNA squences that share more than 70% identity
Protein
Protein sequences that share more than 25% identity

21. 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

22. 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%

23. 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