DNA Sequence Controls Expression of Gene Involved in Cancer

2. DNA Sequence Controls Expression of Gene Involved in Cancer MDG 5101 Group 1

3. Two Major Apoptotic Pathways Mitochondrial pathway Death-receptor pathway Nature 407:770-5, 2000

4. Susceptible Cell Bcl-2 Protected Cell Death Survival Bcl-2/Bax: A Threshold Regulator The EMBO Journal 17(14):3878-85, 1998

5. causes constitutive decay of Bcl-2 mRNA, thereby maintaining appropriate Bcl-2levels in cells recruits proteins that cause degradation of Bcl-2 mRNA translation Over-expression Bcl-2 Protein CANCER Inhibition of apoptosis The Journal of Biological Chemistry 279(41):42758–64, 2004

6. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning) Bioinformatics in a Multi-step Biological Problem

7. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning)

8. To Identify Gene Sequence Using Genbank 1 gcgcccgccc ctccgcgccg cctgcccgcc cgcccgccgc gctcccgccc gccgctctcc 61 gtggccccgc cgcgctgccg ccgccgccgc tgccagcgaa ggtgccgggg ctccgggccc 121 tccctgccgg cggccgtcag cgctcggagc gaactgcgcg acgggaggtc cgggaggcga 181 ccgtagtcgc gccgccgcgc aggaccagga ggaggagaaa gggtgcgcag cccggaggcg 241 gggtgcgccg gtggggtgca gcggaagagg gggtccaggg gggagaactt cgtagcagtc 301 atccttttta ggaaaagagg gaaaaaataa aaccctcccc caccacctcc ttctccccac 361 ccctcgccgc accacacaca gcgcgggctt ctagcgctcg gcaccggcgg gccaggcgcg … 4621 aatgattcta atttttaagc aaaatattat tttatgaaag gtttacattg tcaaagtgat 4681 gaatatggaa tatccaatcc tgtgctgcta tcctgccaaa atcattttaa tggagtcagt 4741 ttgcagtatg ctccacgtgg taagatcctc caagctgctt tagaagtaac aatgaagaac 4801 gtggacgttt ttaatataaa gcctgttttg tcttttgttg ttgttcaaac gggattcaca 4861 gagtatttga aaaatgtata tatattaaga ggtcacgggg gctaattgct agctggctgc 4921 cttttgctgt ggggttttgt tacctggttt taataacagt aaatgtgccc agcctcttgg 4981 ccccagaact gtacagtatt gtggctgcac ttgctctaag agtagttgat gttgcatttt 5041 ccttattgtt aaaaacatgt tagaagcaat gaatgtatat aaaagc http://www.ncbi.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=M13994&doptcmdl=GenBank

9. Search for Bcl-2 Gene across Species Using SwissProt BCL2_BOVIN (O02718) Apoptosis regulator Bcl-2. {GENE: Name=BCL2} - Bos taurus (Bovine) BCL2_CHICK (Q00709) Apoptosis regulator Bcl-2. {GENE: Name=BCL2; Synonyms=BCL-2} - Gallus gallus (Chicken) BCL2_CRIGR (Q9JJV8) Apoptosis regulator Bcl-2. {GENE: Name=BCL2} - Cricetulus griseus (Chinese hamster) BCL2_HUMAN (P10415) Apoptosis regulator Bcl-2. {GENE: Name=BCL2} - Homo sapiens (Human) BCL2_MOUSE (P10417) Apoptosis regulator Bcl-2. {GENE: Name=Bcl2; Synonyms=Bcl-2} - Mus musculus (Mouse) BCL2_RAT (P49950) Apoptosis regulator Bcl-2. {GENE: Name=Bcl2; Synonyms=Bcl-2} - Rattus norvegicus (Rat) http://ca.expasy.org/uniprot/p10415

10. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning)

11. To Identify CAR and ARE Regions 2161 tatctgagcc acaagtgaag tcaacatgcc tgccccaaac aaatatgcaa aaggttcact 2221 aaagcagtag aaataatatg cattgtcagt gatgtaccat gaaacaaagc tgcaggctgt 2281 ttaagaaaaa ataacacaca tataaacatc acacacacag acagacacac acacacacaa 2341 caattaacag tcttcaggca aaacgtcgaa tcagctattt actgccaaag ggaaatatca • To obtain the CA-rich (CAR) region and AU-rich (ARE) region, sequence alignment can be performed starting from the first nucleotide of the stop codon up to the last nucleotide of the CA (or AU) repeats. • The CAR and ARE can then be derived from the shared nucleotides among species using multiple sequence alignments such as ALGGEN, EMBOSS and 3DCoffee.

12. To Obtain mRNA Structure Using mFold Input mRNA sequence http://www.bioinfo.rpi.edu/applications/mfold/old/rna/

13. mFold Result Partial structure from Bcl-2 mRNA (a segment of 800 nt) http://www.bioinfo.rpi.edu/applications/mfold/

14. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning)

15. To Obtain Protein Sequence Using SwissProt http://ca.expasy.org/uniprot/p10415

16. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning)

17. To Obtain Protein Functional Domain Using PFAM/SwissProt Database 1g5mApoptosis Human Bcl-2, isoform 1Domain Chain Start Residue 7 End Residue 33 BH4 (green) http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?acc=PF02180&pdb=1g5m

18. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning)

19. To Identify Restriction Endonuclease Sites UsingSMS http://bioinformatics.org/sms2/rest_summary.html

20. To identify Bcl-2 gene in the genome of multiple organisms in silico To obtain Bcl-2 mRNA sequence, including CAR & ARE, and secondary structure To obtain Bcl-2 protein sequence To identify the functional domain in Bcl-2 To identify restriction endonuclease site on Bcl-2 in vitro To design PCR primer for Bcl-2 (for cloning)

21. To Design PCR Primer http://bioinformatics.org/sms2/primer_map.html

22. In Addition… Much more additional information can be found from various bioinformatics tools to assist the Bcl-2 case study: • Protein 3D structure and interactions visualization • Multiple Sequence Alignment (MSA) using ClustalW • Gene map • Gene expressions • … • and much more!!!

23. Summary • Biological databases represent an invaluable resource in support of biological research.• We can learn much about a particular molecule by searching databases and using available analysis tools.• A large number of databases and tools are available for such task - BLAST, SWISSPROT, PDB, EMBOSS, STADEN, SMS, PHD, RASMOL & WHATIF. For best results we often need to access multiple databases.• Common database search methods include keyword matching, sequence similarity, motif searching & class searching.

24. Future Work • Bioinformatics allows research to built upon results from all over the world, crossing timeandspace • Several subfields in bioinformatics are advancing rapidly - Computational Physiology, Computational Cancer Research, etc. • Still, there are problems using biological databases include scattered information, repeating information, dead links, constant change, etc. • No matter, bioinformatics has given us eyes that see deep into the nano scale, brains that compute as fast as light travels, and memory that collect everything from every corner of the world. • Bioinformatics will have a dramatic impact on biomedical research!