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Limitations of genome projects
Windowjhgjhddoorhubbahubbastairduh
107 3.109
What do proteins do for a living?
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(A) Identifying genes from the sequence
(B) Gene expression profiling
(C) Genome activity studies
Genomes2 by TA Brown; chapter 7
Post-genomics
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(A) Hunting genes from the sequence
2 broad approaches
1) Ab initio method (computational)
2) Experimental method
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Ab initio method (computational) Scanning ORFs (open reading frames) –
initiation or termination codons Codon bias found in specific species Exon-intron boundaries Upstream control sequences – e.g
conserved motifs in transcription factor binding regions
CpG islands
Homology searches
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Ab initio method (computational)…..
Software for automated annotation of genes like GENSCAN, Genie, GENEBUILDER etc are being used. These scan for special features like
1) Scanning ORFs (open reading frames) – initiation or termination codons
5’- ATGACGCATGATCGAGGAT –3’
3’ – TACTGCGTACTAGCTCCTA –5’
AACTAA
ATG
CCTCTA
TCC
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Ab initio method (computational)… Codon bias found in specific species
Not all codons used at same frequency e.g.human leucine mainly coded by CTG and rarely by TTA or CTA
Exon-intron boundaries (splice sites)5’-AG GTAAGT-3’ hit and miss affair
Upstream control sequences – e.g conserved motifs in transcription factor
binding regions CpG islands
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experimental method
Experimental evaluation based on the use of transcribed RNA to locate exons and entire genes from DNA fragment.
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experimental method 2 main strategies Hybridisation approaches – Northern
Blots, cDNA capture / cDNA select, Zoo blots
Transcript mapping: RT-PCR, exon trapping etc In this method, known DNA databases are searched to find out whether the test sequence is similar to any other known genes, suggesting an evolutionary relationship.
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Northern Blot Zoo Blot
Fig 7.4: Genomes 2 Fig 7.5: Genomes 2
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RT-PCR Exon trapping
Fig 7.: Genomes 2 Fig 7.8: Genomes 2
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(B) Gene expression profiling
• COMPUTATIONAL APPROACH
Homology searches for either
- Orthologous genes (homologues in
different organisms with common
ancestor)
- Paralogous genes (genes in the same
organism, e.g. multigene families)
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(B) Gene expression profiling…..
• EXPERIMENTAL APPROACH
gene inactivation methods (knockouts,
RNAi, site-directed mutagenesis,
transposon tagging, genetic
footprinting etc)
Gene overexpression methods (knock-
ins, transgenics, reporter genes etc)
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(C) Genome activity studies
Gene expression needs to be complemented by
Transcriptome analysis
Proteome analysis
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The transcriptome
mRNA
Pre-r RNA Pre-t RNA sn RNA
sno RNA
sc RNA
t RNA tm RNA etc
hn RNA
Non-coding RNA(96%)
coding RNA(4%)
Total RNA
r RNA
All organisms eukaryotes bacteria
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The transcriptome
complete collection of transcribed elements of the genome
transcriptome maps will provide clues on
Regions of transcription • Transcription factor binding sites • Sites of chromatin modification • Sites of DNA methylation • Chromosomal origins of replication
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The transcriptome
Analysis can be done by either
SAGE (serial analysis of gene expression) technology
Microarray technology
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SAGEShortcut to doing cDNA library screeningSAGE tags identify • mRNAs derived from known genes • anonymous mRNAs, also known as expressed
sequence tags (ESTs) • mRNAs derived from currently unidentified genes
Advantages• Analyzes all transcripts (Transcriptome) without prior
selection of known genes • Provides quantitative data on both known and
unknown genes • Ideally suited for determining changes on gene
expression as consequence of an experimental treatment (e.g. carcinogen, hormone)
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SAGE
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Microarrays – allows comparisons
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Microarrays….
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Proteomics
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Proteomics
Nature (2003) March 13: Insight articles from pg 194
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Proteomics
Proteome projects - co-ordinated by the HUPO (Human Protein Organisation)
Involve protein biochemistry on a high-throughput scale
Problems limited and variable sample material, sample degradation, abundance, post-translational modifications, huge tissue, developmental and temporal
specificity as well as disease and drug influences.
Nature (2003) March 13: Insight articles from pgs 191-197.
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Approaches in proteomics
Nature (2003) March 13: Insight articles from pgs 191-197.
High throughput approach
1)Mass- spectrometry
based
2)Array based
3)Structural
proteomics
4)Informatics
5)Clinical proteomics
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High throughput approaches in proteomics
1) Mass spectrometry-based proteomics: relies on the discovery of protein ionisation techniques.
used for protein identification and
quantification, profiling, protein interactions and modifications.
Nature (2003) March 13: Insight articles from pgs 191-197
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Mass spectrometry (MS)
Nature (2003) March 13: Insight articles from pgs 191-197
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Principle of MS
Nature (2003) March 13: Insight articles from pgs 191-197.
oion source, omass analyser that measures mass-to-charge ratio (m/z)odetector that registers the number of ions at each m/z value
Electrospray ionisation (ESI)matrix-assisted laser desortion/ionisation (MALDI)
MALDI-MS - simple peptide mixtures whereas
ESI-MS - for complex samples.
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Principle of MALDI-TOF
Fig 7.24 Genomes 2 by TA Brown pg 210
Matrix assisted laser desorption/ionisation – time of flight
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2) Array-based proteomics
Nature (2003) March 13: Insight articles from pgs 191-197.
Based on the cloning and amplification of identified ORFs into homologous (ideally used for bacterial and yeast proteins) or sometimes heterologous systems (insect cells which result in post-translational
modifications similar to mammalian cells). A fusion tag (short peptide or protein
domain that is linked to each protein member e.g. GST) is incorporated into the plasmid construct.
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Array based proteomics….
Nature (2003) March 13: Insight articles from pgs 191-197.
a. Protein expression and purification b. Protein activity: Analysis can be done using
biochemical genomics or functional protein microarrays. c. Protein interaction analysis two-hybrid analysis (yeast 2-hybrid), FRET (Fluorescence resonance energy transfer), phage display etc d. Protein localisation: immunolocalisation of epitope-tagged products. E.g the use of GFP or luciferase tags
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3) Structural proteomics !
Nature (2003) March 13: Insight articles from pgs 191-197.
a. Protein expression and purification b. Protein activity: Analysis can be done using
biochemical genomics or functional protein microarrays. c. Protein interaction analysis two-hybrid analysis (yeast 2-hybrid), FRET (Fluorescence resonance energy transfer), phage display etc d. Protein localisation: immunolocalisation of epitope-tagged products. E.g the use of GFP or luciferase tags
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PROTEIN INTERACTION MAPS FOR MODEL ORGANISMS
Nature Reviews Molecular Cell Biology 2; 55-63 (2001); doi:10.1038/35048107
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Challenges for the future – ‘physiome’
Nature Reviews Molecular Cell Biology 4; 237-243 (2003)