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Expression of the Genome The transcriptome Decoding the Genetic Information The information is 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 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 Strand on which the promoter is located 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 Transcription - Translation Template strand 3’ Coding strand 5’ Sense strand 5’ NH3— — COOH ORFs All double stranded sequences necessarily have 6 reading frames GCCGATTAGAGA> TGCCGATTAGAG> ATGCCGATTAGA> 5’-ATGGCGATTAGAGACAGCCATTAA-3’ 3’-TACTGCTAATCTCTGTCGGTAATT-5’ <CTGTCGGTAATT <TCTGTCGGTAAT <CTCTGTCGGTAA How many ORFs does this sequence have? 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 9 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 10 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% 11 Homologues Orthologues : Homologues found in different organisms which have a common ancestor Duplication Paralogues followed by speciation : Homologues Duplication found within the same species prior to speciation 12 Mutations Point Mutations Missense - Neutral Synonymous/Silent : Base change that does NOT change the amino acid coded Ex. AGG → CGG both Arg Missense - Non-Synonymous - Conserved: Base change results in a different but similar amino acid Same charge and shape Ex. AAA → AGA Lys to Arg both basic amino acids Point Mutations Missense - Non-Synonymous-Semi conserved: Base change resulting in a different but similar amino acid Same shape but different charge Ex. CGC → CUC Arg (Polar) to Leu (Non-polar) Missense - Non-Synonymous - Non conserved Base change resulting in totally different amino acids Different shape different charge Point Mutations Nonsense point mutation: Base change resulting in the creation of a premature stop codon within the ORF Causes premature translation termination Truncated protein Indel – Insertion or deletion of a single base within the ORF Changes reading frame Changes protein sequence May cause premature termination 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 19 Comparaison of MethodsNorthern 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 THE SEQUENCE MUST BE EXPRESSED Yes No YES YES 20 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 22 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 23 Constitutive expression Normalization 24 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 25 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 26 Reverse Transcriptase Reaction Gene NonSpecific 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 27 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 28 RT PCR cDNA Collection cDNA of mRNA of interest PCR with primers specific to sequence of interest Analysis on gel 29 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 30 Sequences and their Properties Nucleotides DNA A, T, G, C RNA A, U, G, C Annealing Nucleic acids can base pair with their reverse complement sequence Two opposing forces affect annealing Hydrogen bonds favours annealing Phosphate groups favours denaturation Annealing-Melting Point (Tm) The Tm is the temperature at which 50% of the nucleic acid molecules are in a single stranded state (or double stranded) The Tm is a function of: Percentage G:C Ionic composition of the environment The percentage of complementarity Estimate of Tm =2(#A:T) + 4(#G:C) 34 Tm Vs percentage G:C % Double stranded (38%) G+C 0 (52%) (58%) 50 (66%) 100 70 80 90 100 Temperature (C) 35 Tm Vs Conc. of Positive Ions % Double stranded (0.1M NaCl) 0 (0.2M NaCl) 50 (0.5M NaCl) 100 70 80 90 100 Temperature (C) 36 Tm Vs percentage of Complementarity % Double stranded (25%) 0 (50%) 50 (100%) 100 70 80 90 100 Temperature (C) 37 Stringency Percentage of complementarity required to allow the formation of stable duplexes The Tm influences the stringency conditions required to allow annealing A high stringency requires a high level of complementarity GATCCGGTTATTA vs GATCCGGTTATTA CTAGGCCAATAAT CTTGGACGATAAT 38 Parameters that Influence Stringency [salt] = High stringency Temperature = High stringency [salt] = ? Temperature = ? 39 4. Hybridization with Free Probe Wash Detection: Autoradiography 41 Properties of the Probe Complementarity Complete or partial? Complete; ideal; 100% complementarity Partial continuous; acceptable 100% complementarity Partial discontinuous; more difficut Partial complementarity Hybridization Stringency 43 The Probe Labelled Single DNA or RNA molecule stranded Strand Double specific (sense specific) stranded Strand non-specific (sense non specific) Digoxygenin Labelled Probe Indiret detection X ray film S S ENZ ENZ S S ENZ ENZ D D D D D D D D Peroxidase Ab-Dig conjugated Probe+ Dig Target Membrane Hybridization Signals Hybridization Specific Non specific Background Binding of probe to membrane Binding of Ab to membrane