Limitations of genome projects Windowjhgjhddoorhubbahubbastairduh 10 7 3.10 9 What do proteins do...

Limitations of genome projects

Windowjhgjhddoorhubbahubbastairduh

107 3.109

What do proteins do for a living?

(A) Identifying genes from the sequence

(B) Gene expression profiling

(C) Genome activity studies

Genomes2 by TA Brown; chapter 7

Post-genomics

(A) Hunting genes from the sequence

2 broad approaches

1) Ab initio method (computational)

2) Experimental method

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

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

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

experimental method

Experimental evaluation based on the use of transcribed RNA to locate exons and entire genes from DNA fragment.

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.

Northern Blot Zoo Blot

Fig 7.4: Genomes 2 Fig 7.5: Genomes 2

RT-PCR Exon trapping

Fig 7.: Genomes 2 Fig 7.8: Genomes 2

(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)

(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)

(C) Genome activity studies

Gene expression needs to be complemented by

Transcriptome analysis

Proteome analysis

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

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

The transcriptome

Analysis can be done by either

SAGE (serial analysis of gene expression) technology

Microarray technology

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)

SAGE

Microarrays – allows comparisons

Microarrays….

Proteomics

Proteomics

Nature (2003) March 13: Insight articles from pg 194

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

based

2)Array based

3)Structural

proteomics

4)Informatics

5)Clinical 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

Mass spectrometry (MS)

Nature (2003) March 13: Insight articles from pgs 191-197

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.

Principle of MALDI-TOF

Fig 7.24 Genomes 2 by TA Brown pg 210

Matrix assisted laser desorption/ionisation – time of flight

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

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

PROTEIN INTERACTION MAPS FOR MODEL ORGANISMS

Nature Reviews Molecular Cell Biology 2; 55-63 (2001); doi:10.1038/35048107

Challenges for the future – ‘physiome’

Nature Reviews Molecular Cell Biology 4; 237-243 (2003)