Lecture 5 Post-genomics. Functional genomics (A) Identifying genes from the sequence (B) Gene expression profiling (transcriptome) (C) Model systems

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

http://upload.wikimedia.org/wikipedia/en/3/32/Microarray-schema.gif

Microarrays….

(a) Schematic drawing of a DNA chip.

Microarray(chip)


Microarray(chip)


Microarray(chip)

Segment ofa chip


Microarray(chip)

Segment ofa chip

Spot containing copiesof a single DNAmolecule


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


AAT

TCGC

chip DNA

TT

AAGCG

cDNAfromtreatedcells

Pair ofcomplementary

bases

GG

GACTA

AATTCGC

CC

CGGAT

AG

GACGT

AATTCGC


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



Gene that strongly decreasedactivity in treated cells




Gene that was equally activein treated and untreated cells




Gene that was equally activein treated and untreated cellsGene that was inactivein both groups


High-throughput microarrays

C:\Documents and Settings\bssmvh\My Documents\0055\microarray_mov.zip

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 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


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


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

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