Expression of the Genome The transcriptome

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

The Genes Transcription initiation site Termination sequence 3’ Promoter/ Regulatory sequences 5’ Introns RNA Transcript 5’ untranslated region 3’untranslated region Exon 2Exon 3 Int. 2 Exon 1 Int. 1 Exons Only one of the two strands is coding!

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 the promoter is located 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 DNA: 5’ TAG 3’ 3’ ATC 5’ Translation Leu Protein: Genetic code : CUA = Leu UAG = Stop Transcription RNA: ?5’3’

Template strand 3’ Coding strand 5’ Sense strand 5’ NH 3 — — COOH Transcription - Translation

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

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 10 77% identity  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 * * * * * * * *

Protein homologues  Protein sequences with greater than 25% identity  Ex. A protein homologue of the human hemoglobin is found in soya 11 Percentage identity: 28% 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

Homologues  Orthologues :  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 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 Transcriptome Collection of RNA from genes that code for proteins Collection of RNA that represents the fraction of the genome that is expressed Proteome Collection of proteins derived from the transcriptome Transcription Translation

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  Northern Hybridization Analysis  RT-PCR  RT-PCR 19

Comparaison of Methods 20 Northern RT-PCR Sequence must be known Presence or absence of a transcript Allows to determine size Sensitivity Compare relative abundance Obtain sequence of transcript Determine which strand is transcribed Determine how many transcripts are made from a single sequence No Yes Yes Yes No Low High Yes No Yes Yes Yes No THE SEQUENCE MUST BE EXPRESSED YES Northern RT-PCR

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

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  Constitutive expression 23

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 25 Tissues: F C R P ActinFos

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 Non-Specific 27 AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT Annealing of polyT primer Collection of complementary DNAs to RNAs expressed at a given time under given conditions AAAAAAA mRNA AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT AAAAAAA TTTT Transcription to cDNA RT

Reverse Transcriptase Reaction Gene Specific 28 AAAAAAA Synthesis of cDNA RT DNA complementary to one mRNA of interest AAAAAAA Annealing of gene specific primer AAAAAAA

RT  PCR 29 cDNA CollectioncDNA of mRNA of interest Analysis on gel PCR with primers specific to sequence of interest

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

35 Tm Vs percentage G:C % Double stranded Temperature (C) (38%) G+C (52%) (58%) (66%)

36 Tm Vs Conc. of Positive Ions % Double stranded Temperature (C) (0.1M NaCl) (0.2M NaCl) (0.5M NaCl)

37 Tm Vs percentage of Complementarity % Double stranded Temperature (C) (25%) (50%) (100%)

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 38 GATCCGGTTATTA vs GATCCGGTTATTA CTAGGCCAATAAT CTTGGACGATAAT

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 DNA or RNA molecule  Single stranded  Strand specific (sense specific)  Double stranded  Strand non-specific (sense non specific)

Digoxygenin Labelled Probe  Indiret detection SSSS Membrane Target DDDDDDDD Probe+ Dig ENZ Ab-Dig conjugated Peroxidase X ray film

Hybridization Signals  Hybridization  Specific  Non specific  Background  Binding of probe to membrane  Binding of Ab to membrane