post-genomics functional genomics (a) identifying genes from the sequence (b) gene expression...

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

Analysis can be done by either

Microarray technology

SAGE (serial analysis of gene expression) technology

(a) Schematic drawing of a DNA chip.

Microarray(chip) Segment of

a chip Spot containing copiesof a single DNAmolecule

Part of oneDNA strand

AG

GACGT

DNAbases

AATTCGC

Examplesof reactions

TT

AAGCG

AAT

TCGC

cCNA fromuntreatedcells

chip DNA

TT

AAGCG

cDNAfromtreatedcells

Pair ofcomplementary

basesA

GGACGT

GG

GACTA

CC

CGGAT

TC

CTGCA

(b) The analysis of the hybridization process identifies genes that respond in specific ways.

Cell samples are stabilizedand fluorescent labelsare added.

Gene that strongly increasedactivity in treated cells

Gene that strongly decreasedactivity in treated cellsGene that was equally activein treated and untreated cells

Gene that was inactivein both groups

(c) Computer analysis of the binding of complementary sequences can identify genes that respond to drug treatment.

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

C:\Documents and Settings\bssmvh\My Documents\0055\med_res RNAi movie.mov

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 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


High throughput approach

1)Mass- spectrometry

2) Array based

proteomics

3)Structural proteomics

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


Electrospray ionisation (ESI)matrix-assisted laser desortion/ionisation (MALDI)

MALDI-MS - simple peptide mixtures whereas ESI-MS - for complex samples.

2) Array-based proteomics


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….


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….


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

Identification of protein-protein

interactionsaffinity capture/mass

spectrometry

Fig. 10. 31

Identification of protein-protein interactionsPhage display

Fig. 10.32

Systems Biology – the global study of multiple components of biological systems and their

interactions

• New approach to studying biological systems has made possible– Sequencing genomes– High-throughput platform development– Development of powerful computational

tools– The use of model organisms– Comparative genomics

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 form perturbations

• Formulate hypothesis to explain disparities between model and experimental data

• Refine the model after integrating data

19_20.jpg

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

The systems biology approach to studying how yeast turns genes on and off in the

utilization of galactose

Fig. 10.34

Fig. 10.35

Human Genome Project has changed the potential for predictive/preventive

medicine

• Provided access to DNA polymorphisms underlying human variability– Makes possible identification of genes

predisposing to disease– Understanding of defective genes in context of

biological systems– Circumvent limitations of defective genes

• Novel drugs• Environmental controls• Approaches such as stem-cell transplants or

gene therapy

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