Transcript PPT

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%
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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
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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
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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
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 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
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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 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
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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
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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)
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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