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Expression of the Genome
The transcriptome
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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
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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!
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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
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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
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Codant Vs Non-coding DNA: 5’ TAG 3’ 3’ ATC 5’ Transcription RNA: ? 5’
Translation Leu Protein: Genetic code : CUA = Leu UAG = Stop
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Transcription - Translation
Template strand 3’ Coding strand 5’ Sense strand 5’ NH3— — COOH
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ORFs All double stranded sequences may have up to 6 reading frames
GCCGATTAGAGA> TGCCGATTAGAG> ATGCCGATTAGA> 5’-ATGGCGATTAGAGACAGCCATTAA-3’ 3’-TACTGCTAATCTCTGTCGGTAATT-5’ <CTGTCGGTAATT <TCTGTCGGTAAT <CTCTGTCGGTAA How many ORFs does this sequence have?
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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
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One Genome Is the transcriptome the same in all the cells of an organism? Is the transcriptome always the same in a given cell?
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Does a Sequence Code for a Transcript?
Northern Hybridization Analysis RT-PCR
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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
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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
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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
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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
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Normalization
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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
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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
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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
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Reverse Transcriptase Reaction Gene Specific
AAAAAAA AAAAAAA Annealing of gene specific primer DNA complementary to one mRNA of interest AAAAAAA Synthesis of cDNA RT
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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
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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
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