2. Post-genomics

3. Post-genomics Functional genomics
(A) Identifying genes from the sequence
(B) Gene expression profiling (transcriptome)
(C) Model systems
Proteomics
Systems biology

4. (A) Hunting genes from the sequence
2 broad approaches
1) Ab initio method (computational)
2) Experimental method

5. 1) Ab initio method (computational)
Scanning ORFs (open reading frames)

6. 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

7. 2) experimental method
Experimental evaluation based on the use of transcribed RNA to locate exons and entire genes from DNA fragment.

8. 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.

9. Northern Blot
Zoo Blot

10. (B) Gene expression profiling
Transcriptome
complete 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

11. Homology searches (BLAST searches)
COMPUTATIONAL APPROACH
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

12. The transcriptome Analysis can be done by either Microarray technology
SAGE (serial analysis of gene expression)
technology

13. Microarrays (a) Schematic drawing of a DNA chip.
bases
Microarray
(chip)
Segment of
a chip
Spot containing copies
of a single DNA
molecule
Part of one
DNA strand
(b) The analysis of the hybridization process identifies
genes that respond in specific ways.
Pair of
complementary
bases
cDNA
from
treated
cells T C G A A G C T
cCNA from
untreated
cells C G A T G A C T
chip DNA T A G C A T C G T A G C A T C G
Cell samples are stabilized
and fluorescent labels
are added.
Examples
of reactions
(c) Computer analysis of the binding of complementary
sequences can identify genes that respond to drug
treatment.
Gene that strongly increased
activity in treated cells
Gene that strongly decreased
activity in treated cells
Gene that was equally active
in treated and untreated cells
Gene that was inactive
in both groups

14. Gene overexpression methods (knock-ins, transgenics, reporter genes)
MODEL SYSTEMS
gene inactivation methods (knockouts, RNAi, site-directed mutagenesis, transposon tagging, genetic footprinting etc)
Gene overexpression methods (knock-ins, transgenics, reporter genes)

15. RNAi RNAi mimics loss-of-function mutations Non-inheritable
Lack of reproducibility

16. How does RNAi work?

17. MODEL SYSTEMS
Gene overexpression methods (knock-ins, transgenics, reporter genes etc)

18. Proteomics Analysis of protein expression Protein structure
Protein-protein interactions
Nature (2003) March 13: Insight articles from pg 194

19. 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

20. Approaches in proteomics
High throughput approach
Mass- spectrometry
Array based proteomics
Structural proteomics
Nature (2003) March 13: Insight articles from pgs

21. 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

22. two dimensional gels and mass spectrometry
Identification of proteins in complex mixtures
two dimensional gels and mass spectrometry

23. two dimensional gels
19_09.jpg
19_09.jpg

24. Mass spectrometry (MS)
Nature (2003) March 13: Insight articles from pgs

25. Principle of MS
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

26. Types of ionizer sources
Electrospray ionisation (ESI)
matrix-assisted laser desortion/ionisation (MALDI)
MALDI-MS - simple peptide mixtures whereas
ESI-MS - for complex samples.
Nature (2003) March 13: Insight articles from pgs

27. 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.
Nature (2003) March 13: Insight articles from pgs

28. 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
Nature (2003) March 13: Insight articles from pgs

29. Array based proteomics….
Protein chips
Antibody chips – arrayed antibodies
Antigen chips – arrayed antigens
Functional arrays – arrayed proteins
Protein capture chips – arrayed capture agents that interact with proteins e.g. BIAcore
Solution arrays – nanoparticles
Nature (2003) March 13: Insight articles from pgs

30. 3) Structural proteomics
19_14.jpg
19_14.jpg

31. 3) Structural proteomics
19_14.jpg
19_14.jpg

32. Identification of protein-protein interactions affinity capture/mass spectrometry
Figure 10.31
Fig

33. Identification of protein-protein interactions Phage display
Figure 10.32
Fig

34. New approach to studying biological systems has made possible
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

35. 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

36. 19_20.jpg
19_20.jpg

37. Systems biology approach to studying how Halobacterium NRC-1 transcriptome responds to uv radiation
Nitin S. Baliga et al. Genome Res. 2004; 14:

38. The systems biology approach to studying how yeast turns genes on and off in the utilization of galactose
Figure 10.34
Fig

39. Figure 10.35
Fig

40. 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

41. Challenges for the future – ‘physiome’
Nature Reviews Molecular Cell Biology 4; (2003)

42. General Reading Reference
Chapter 19- HMG3 by Strachan and Read
Reference
- Nature (13 March 2003). Proteomics insight articles from Vol. 422, No pgs