1. Gene Discovery & Genome Browsing
Background – myself
NsGene – DTU satellite
Parkinson Disease (Affymetrix GeneChip)
Analysis of fetal brain tissue
Genome browsing
Introduction to the UCSC BLAT browser

2. Background Thomas Nordahl Petersen
Chemist, Ph.D protein Crystallography, University of Copenhagen
Computational Scientist, SBI-AT
Prediction of protein structure, secondary structure, fold recognition, homology modeling
Bioinformatics - Gene discovery, NsGene
Devolop novel cell and gene based products for the
treatment of neurological diseases.

4. ECT Products ECT for Parkinson’s Disease
Growth of cells in a capsule matrix
The therapeutic protein be released directly in the relevant brain area
Safe delivery across the blood-brain-barrier
Michael J. Fox foundation granted US $3 million to support a clinical “proof-of-concept” (May 2004)

5. Factor Products Identification of novel genes by use of bioinformatics
Neublastin (GDNF family – potent neuroprotective effects)
Scanning the human genome or assembled protein sets for different features of interest

6. A case study Affymetrix GeneChip experiments Fetal brain tissue
Search for Parkinson related gene(s)
Affymetrix GeneChip experiments
Fetal brain tissue

7. Parkinson Disease Degenerative central nervous system (CNS) disorder

8. Parkinson Disease Loss of dopamine producing brain cells

9. Parkinson’s Disease
Dopamine from Substantia nigra activates neurons in Striatum/Basal ganglia
Important for initiation of movement

10. Cure for Parkinson’s Disease ?
Parkinson disease may be cured provided that new dopamine
producing cells replace the dead ones.
Dopamin producing brain cells from aborted foetuses have been
operated into the brain of parkinson patients and ín some cases
cured the disease. Brain tissue from approx 6 foetuses were needed.
Major ethical problems !
Search for a protein drug is the only valid option

11. Parkinson Disease Dopamine producing cells
Dopaminergic neurons can be found in the ventral part of the mesencephalon (VM) from approximately 6 weeks
No dopaminergic neurons can be found in the neighbouring dorsal part (DM).
Dopaminergic differentiation by use of GeneChips to compare the expression profiles of VM and DM

12. Fetal brain tissue Midbrain mesencephalon
- Dopamine producing
cells
+ Dopamine producing
cells Vm Dm
Aborted feotus brain tissue – Karolinska hospital
Feotus of age 6-10 weeks, 2 cases

13. Midbrain mesencephalonDm Vm
- Dopamine
producing
cells
+ Dopamine
producing
cells
Dopamine producing cells at the interface ?
Isolate the two samples (Vm/Dm)
RNA purification + amplification
Affymetrix genechip analysis

14. GenePublisher (program by Steen Knudsen)
Scale, normalize the Affymetrix GeneChip experiments
A1 A2 A2 B1 B2 B2 P-value
e-07
e-05
e-05
e-05

15. Vulcano plot
P-value
Log2 Fold change

16. Assigning Affymetrix GeneChip probes to a protein sequence
~ probes on each of the A/B Affymetrix chips. The
probes are normally not a part of a protein sequence.
Unigene sequence (cDNA) 5’ 3’
IPI protein sequence
Blast
Affymetrix probe
Blast
inferred

17. Internal database

18. Signal Peptide prediction

19. Conclusion – so far
The most up-regulated genes include several ‘known’ genes like dopamine transporter (good positive control)
The most interesting genes are the ‘unknowns’ that were up-regulated in Vm. Futher analysis is ongoing.
Roland JR et al., Exp Neur (2006) Vol 198,2,
“Identification of novel genes regulated in the developing human ventral mesencephalon”

20. BLAT The Blast Like Alignment Tool
Smith-Waterman (1980)
Local alignment (best alignment)
FASTA (1988)
Fast alignment
BLAST-programs (1990,1997)
Query seq versus an indexed database
MegaBLAST (2000)
Fast alignment of DNA sequences
SSAHA (2001)
Maps sequence reads to genome

21. BLAT Blast Like Alignment Tool
Very fast searches (MySQL database)
Handle introns in RNA/DNA alignments
Data for more that 30 genomes (human, mouse, rat…)
Exon Intron Exon
Splice sites

22. BLAT genome Browser

23. BLAT genome Browser
Using a search term or position eg Chr1:10,234-11,567

24. BLAT genome Browser

25. BLAT genome Browser
Using a protein or DNA sequence

26. Blat genome Browser

27. BLAT genome Browser ”Details”

28. Blat genome Browser

29. BLAT genome Browser ”Browser”

30. BLAT genome Browser Zoom in ’base’

31. BLAT genome Browser ”Browser”
Trembl
SwissProt
Hypothetical

32. BLAT genome Browser ”Description & Page Index”

33. BLAT genome Browser ”Description & Page Index”

34. Blat Genome Browser ”tracks”

35. BLAT genome Browser ”SNPs”

36. BLAT genome Browser ”SNPs”

37. Gene Sorter

38. Gene sorter