Transcript YES YES
Expression of the Genome The transcriptome Decoding the Genetic Information Information encoded in nucleotide sequences contained in discrete units The The genes information contained in the genes is transcribed to generate the RNAs and then decoded to generate the proteins The Genes Transcription initiation site 3’untranslated region 5’ untranslated region Introns 5’ Exon 1 Promoter/ Regulatory sequences Int. 1 Exon 2 Int. 2 Exon 3 Exons RNA Transcript Only one of the two strands is coding! 3’ Termination sequence 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 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 Codant Vs Non-coding 5’ TAG 3’ 3’ ATC 5’ Transcription DNA: 5’ RNA: Protein: Genetic code ? 3’ Translation Leu : CUA = Leu UAG = Stop 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 One Genome Is the transcriptome the same in all the cells of an organism? Is the transcriptome always the same in a given cell? Does a Sequence Code for a Transcript? Northern Hybridization Analysis RT-PCR 9 Comparaison of Methods 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 THE SEQUENCE MUST BE EXPRESSED Yes No YES YES 10 Northern Analysis Isolate total RNA from cells or tissue Separate RNA according to their sizes on denaturing agarose gel Formaldehyde Hybridization probe + Formamide with complementary rRNA tRNA 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 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 13 Constitutive expression Normalization 14 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 15 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 Reverse Transcriptase Reaction Gene Non-Specific AAAAAAA AAAAAAA AAAAAAA AAAAAAA mRNA AAAAAAA Annealing of polyT primer TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA Collection of complementary DNAs to RNAs expressed at a given time under given conditions 17 Reverse Transcriptase Reaction Gene Specific AAAAAAA AAAAAAA AAAAAAA AAAAAAA AAAAAAA Annealing of gene specific primer AAAAAAA AAAAAAA AAAAAAA AAAAAAA AAAAAAA DNA complementary to one mRNA of interest AAAAAAA Synthesis of cDNA RT AAAAAAA AAAAAAA AAAAAAA AAAAAAA 18 RT PCR cDNA Collection cDNA of mRNA of interest PCR with primers specific to sequence of interest Analysis on gel 19 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