lecture 5 post-genomics. functional genomics (a) identifying genes from the sequence (b) gene...
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Lecture 5Post-genomics
Post-genomics
Functional genomics(A) Identifying genes from the sequence (B) Gene expression profiling
(transcriptome)(C) Model systems
Proteomics
Systems biology
Post-genomics
(A) Hunting genes from the sequence
2 broad approaches
1) Ab initio method (computational)
2) Experimental method
1) Ab initio method (computational)
Scanning ORFs (open reading frames)
5’- ATGACGCATGATCGAGGAT –3’
3’ – TACTGCGTACTAGCTCCTA –5’
AACTAA
ATG
CCTCTA
TCC
Ab initio method (computational)
initiation or termination codons 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
2) experimental method
Experimental evaluation based on the use of transcribed RNA to locate exons and entire genes from DNA fragment.
experimental method Some 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.
Northern Blot Zoo Blot
Transcriptomecomplete collection of transcribed
elements of the genome (global mRNA profiling)
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
(B) Gene expression profiling
Homology searches (BLAST searches)
- Orthologous genes (homologues in different
organisms with common ancestor) – comparative
genomics
- Paralogous genes (genes in the same organism, e.g.
multigene families)
- orphan genes / families
COMPUTATIONAL APPROACH
The transcriptome
Advantages: high-throughput information Gene expression profile of the
cell/tissue
problems false –positives data analysisCost
Analysed by DNA Microarrays
Microarrays….
(a) Schematic drawing of a DNA chip.
Microarray(chip)
(a) Schematic drawing of a DNA chip.
Microarray(chip)
(a) Schematic drawing of a DNA chip.
Microarray(chip)
Segment ofa chip
(a) Schematic drawing of a DNA chip.
Microarray(chip)
Segment ofa chip
Spot containing copiesof a single DNAmolecule
(a) Schematic drawing of a DNA chip.
Microarray(chip)
Segment ofa chip
Spot containing copiesof a single DNAmolecule
Part of oneDNA strand
AG
GACGT
DNAbases
(b) The analysis of the hybridization process identifies genes that respond in specific ways.
Cell samples are stabilizedand fluorescent labelsare added.
Examplesof reactions
AAT
TCGC
AATTCGC
AG
GACGT
GG
GACTA
chip DNA
Examplesof reactions
AAT
TCGC
chip DNA
TT
AAGCG
cDNAfromtreatedcells
Pair ofcomplementary
bases
GG
GACTA
AATTCGC
CC
CGGAT
AG
GACGT
AATTCGC
Examplesof reactions
TT
AAGCG
AAT
TCGC
cCNA fromuntreatedcells
chip DNA
TT
AAGCG
cDNAfromtreatedcells
Pair ofcomplementary
basesA
GGACGT
GG
GACTA
CC
CGGAT
TC
CTGC
A
(c) Computer analysis of the binding of complementary sequences can identify genes that respond to drug treatment.
Gene that strongly increasedactivity in treated cells
(c) Computer analysis of the binding of complementary sequences can identify genes that respond to drug treatment.
Gene that strongly increasedactivity in treated cells
Gene that strongly decreasedactivity in treated cells
(c) Computer analysis of the binding of complementary sequences can identify genes that respond to drug treatment.
Gene that strongly increasedactivity in treated cells
Gene that strongly decreasedactivity in treated cells
Gene that was equally activein treated and untreated cells
(c) Computer analysis of the binding of complementary sequences can identify genes that respond to drug treatment.
Gene that strongly increasedactivity in treated cells
Gene that strongly decreasedactivity in treated cells
Gene that was equally activein treated and untreated cellsGene that was inactivein both groups
(c) Computer analysis of the binding of complementary sequences can identify genes that respond to drug treatment.
High-throughput microarrays
gene inactivation methods (knockouts, RNAi, site-directed mutagenesis, transposon tagging, genetic footprinting etc)
Gene overexpression methods (knock-ins, transgenics, reporter genes)
MODEL SYSTEMS
RNAi
RNAi mimics loss-of-function mutations
Non-inheritable
Lack of reproducibility
How does RNAi work?
http://www.nature.com/focus/rnai/animations/index.html
Gene overexpression methods (knock-ins, transgenics, reporter genes etc)
MODEL SYSTEMS
Proteomics
Nature (2003) March 13: Insight articles from pg 194
Analysis of protein expressionProtein structure and function Protein-protein interactions
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.
Approaches in proteomics
Nature (2003) March 13: Insight articles from pgs 191-197.
High throughput approach
1)Mass- spectrometry
2) Array based
proteomics
3)Structural proteomics
High throughput approaches in proteomics
1) Mass spectrometry-based proteomics:Nobel prize in Chemistry (2002)
John B. Fenn
"for the development of methods for identification and structure analyses of biological macromolecules"
"for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules"
"for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological
macromolecules in solution
Koichi Tanaka Kurt Wüthrich
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
two dimensional gels and mass spectrometry
Identification of proteins in complex mixtures
19_09.jpg
two dimensional gels
Mass spectrometry (MS)
Nature (2003) March 13: Insight articles from pgs 191-197
ionizer source: converts analyte to gaseous ions mass analyser: measures mass-to-charge ratio
(m/z)detector: registers the number of ions at each m/z
Principle of MS
Types of ionizer sources
Nature (2003) March 13: Insight articles from pgs 191-197.
Electrospray ionisation (ESI)matrix-assisted laser desortion/ionisation (MALDI)
MALDI-MS - simple peptide mixtures whereas ESI-MS - for complex samples.
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.
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
Array based proteomics….
Nature (2003) March 13: Insight articles from pgs 191-197.
Protein chips
Antibody chips – arrayed antibodiesAntigen chips – arrayed antigensFunctional arrays – arrayed proteinsProtein capture chips – arrayed capture agents that interact with proteins e.g. BIAcoreSolution arrays – nanoparticles
19_14.jpg
3) Structural proteomics
8HDF / MTHF?
FAD
Modelling of a novel photolyase based on sequence Winnie Wu
Identification of protein-protein
interactions
affinity capture/mass spectrometry
Fig. 10. 31
Identification of protein-protein interactionsPhage display
Fig. 10.32
Systems Biology Systems Biology the global study of multiple components of the global study of multiple components of
biological systems and their interactionsbiological systems and their interactions
– Sequencing genomes– High-throughput platform development– Development of powerful computational
tools– The use of model organisms– Comparative genomics
New approaches to studying biological systems
19_20.jpg
Six steps in systems approach
• Formulate computer based model for the system
• Discovery science to define as many of the system’s elements as possible
• Perturb the system genetically or environmentally
• Integrating levels of information from perturbations
• Formulate hypothesis to explain disparities between model and experimental data
• Refine the model after integrating data
Nitin S. Baliga et al. Genome Res. 2004; 14: 1025-1035
Systems biology approach to studying how Halobacterium NRC-1 transcriptome responds to uv radiation
Challenges for the future – ‘physiome’?
Nature Reviews Molecular Cell Biology 4; 237-243 (2003)
• General Reading– Chapter 19- HMG3 by Strachan and Read
• Reference
• - Nature (13 March 2003). Proteomics insight articles from Vol. 422, No. 6928 pgs 191-197