Lecture 5: Challenges in the post-

genomic era

 The tiger leg leaf frogPhoto: Zig LeszccynskiImage: courtesy Rainforest Alliance

Are genome projects the end?

Functional Genomics Projects Genomics & Genetics Resources

Human Genome Project:

To access the Human Genome Project (HGP) research programme resources here at the WTSI

Copy Number Variation:

The CNV project examines copy number changes in the population

Cancer Genome Project:

Identifying somatically acquired sequence variants/mutations and hence genes critical in the development of human cancers

Genomic Microarrays:

Detailed analysis of genomic copy number changes in tumours

Immunogenomics:

Genetics and epigenetics of the immune subgenome and their roles in evolution and disease

Microarray Facility:

Development and use of high-density arrays for expression profiling

Molecular Cytogenetics:

Chromosome organisation and structure in human disease

Molecular Genetics & Proteomics:

Uses of human chromosome 22 as a model system for genomic

Molecular Cytogenetics:

Chromosome organisation and structure in human disease

Molecular Genetics & Proteomics:

Uses of human chromosome 22 as a model system for genomic analysis

Mouse Genomics:

Investigations of the function of genes using the mouse as model genetic system

Genetrap:

Sanger Institute Gene Trap Resource

Fission Yeast Functional Genomics:

Research focuses on genome-wide gene expression profiles and regulatory networks using DNA microarrays and molecular genetics

Atlas of Gene Expression:

Describing different cell types in adult/embryonic tissues alongside a description of expression level/localisation of protein products

Human Epigenome Project (HEP):

Provides an epigenetic resource of genome-wide DNA methylation reference profiles in human tissues and cell lines.

Malaria:

Understanding malaria from basic biology to practical applications

Pathogen Arrays:

Comparative genomics and expression profiling of small genomes

MICER:

Mutagenic Insertion and Chromosome Engineering Resource

ENCODE:

Encyclopedia Of DNA Elements project sets out to identify all functional elements in the human genome sequence

DECIPHER:

DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources

http://www.sanger.ac.uk/PostGenomics/

Functional genomics(A) Identifying genes from the sequence (B) Gene expression profiling

(transcriptomics)(C) Model systems

Proteomics

Systems biology

Post-genomics

(A) Hunting genes from the sequence

2 broad approaches1) Ab initio method (computational)

Codon analysis Regulatory regions (TF binding sites, CpG

islands etc) Exon-intron boundaries

2) Experimental method Hybridisation approaches – Northern

Blots, cDNA capture / cDNA select, Zoo blots

Transcript mapping: RT-PCR, exon trapping etc

Northern Blot Zoo Blot

Transcriptomecomplete collection of transcribed

elements of the genome (global mRNA profiling)

transcriptome maps 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

The transcriptome

Advantages: high-throughput information Gene expression profile of the

cell/tissue

problems false –positives data analysisCost

Analysed by DNA Microarrays

Microarrays….

gene inactivation methods (knockouts, RNAi, site-directed mutagenesis, transposon tagging, genetic footprinting etc)

Gene overexpression methods (knock-ins, transgenics, reporter genes)

(C) 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 – ‘

Genomics – CNV’s in medicine

‘Physiome’

Translational medicine

• General Reading– Chapter 19- HMG3 by Strachan and Read

Reference • Science 9 Feb 2007 Vol. 315. no. 5813, pp. 848 – 853

(CNV report)

• Nature (13 March 2003). Proteomics insight articles from Vol. 422, No. 6928 pgs 191-197

• Crit Rev Biotechnol. 2007 Apr-Jun;27(2):63-75 (good current review on challenges in transcriptomics /proteomics)

Resource:http://www.sanger.ac.uk/PostGenomics/