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Genome annotation searching for group I and group II introns

Yan Zhou & Mohamed Tikah M. BIN6002 Summer, 2005. Genome annotation searching for group I and group II introns. Assembling a mitochondrial Genome. Assembled 1002 reads using phred/phrap/consed into one contiguous sequence of 69586 base pairs. Sequencing output was of high quality.

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Genome annotation searching for group I and group II introns

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  1. Yan Zhou & Mohamed Tikah M. BIN6002 Summer, 2005 Genome annotation searching for group I and group II introns

  2. Assembling a mitochondrial Genome • Assembled 1002 reads using phred/phrap/consed into one contiguous sequence of 69586 base pairs. • Sequencing output was of high quality. • Software tools did 99% of the job.

  3. Genome annotation • Blasting the assembled sequence made it obvious that the sequence has a high similarity with the mitochondrial genome of (the previously annotated) Reclinomonas americana nz strain ATTC 50395. • Further (exact) alignments allowed the identification of (at least) 95 genes (26 of them encode for tRNAs)

  4. Genome annotation • Freshwater protozoan. • Belongs to the protozoan group called ‘Jakobids’. • Gene rich mitochondrial genome. Thought to most resemble the ancestral mitochondrion. GOBASE (2005)

  5. Genome annotation

  6. Genome annotation • nadn, coxn, rnl, rns, rrn5 … were all identified. • In most cases, we obtained a high similarity score (over 85% identity) between the coding sequences from the two genomes. • Order of genes is (so far) conserved. • In one case the overlapping between adjacent genes is also conserved.

  7. Genome annotation • Using tRNAscan-SE identified 25 cove confirmed tRNAs candidates. • Folding some of this candidates using mfold gave the expected tRNA secondary structure. • 1 tRNAW was not detected by tRNAscan-SE

  8. Genome annotation • Is the group II intron r. americana nz also present? • Probably yes. • Allowed the identification of the 26th tRNA tRNAW(cca)

  9. Genome annotation • 70% similar to the group II intron in r. americana nz. • Identified ‘GUGCG’ in 5’ end and other ‘consensus sequences • Doesn’t contain an ORF N. Toor et al (2001)

  10. Genome annotation • RNAse P is an RNAse that cleaves an extra sequence of tRNA (it can act alone as a catalyst in vitro). • rnpB encodes for RNAse P is found in r. americana nz and also in the new genome. • Secondary structure resolved. The new RNAse P has probably a similar secondary structure.

  11. Genome annotation QuickTime™ et un décompresseur TIFF (LZW) sont requis pour visionner cette image. GOBASE (2005)

  12. Genome annotation

  13. Genome annotation

  14. Genome annotation

  15. Whole sequence Blastn GeneScan mRNA for each gene blastx Exon + Intron

  16. Our results for sequence II

  17. Group I intron Self-splicing intron that requires an external guanine-containing nucleotide for splicing; releases the intron in a linear form.

  18. Group I intron

  19. Find group I intron • Locate the exons, then obtain the splicing sites, the U|G, N’N’G • Using citron to locate the P7, P4,P5,P6, thereafter P3 • Manually draw the structure, and to verify it.

  20. Group II intron Self-splicing intron that does not require an external nucleotide for splicing; releases the intron in a lariat form.

  21. Self-splicing reaction

  22. Protein-assisted splicing reaction

  23. Group II intron Group II introns fold into a conserved secondary structure consisting of six domains arranged around a central wheel. Domain 1 is the largest domain by far, while domain 5 is most conserved in sequence and is considered the active site of the ribozyme. 

  24. The ORF contains the seven domains common to all RTs, and also domain X and the En domain. Domain X is probably analogous to the thumb domain of other polymerases, and is associated with the maturase (splicing) activity of the protein. The D domain is a DNA binding region, and the En domain has a nuclease activity utilized in the mobility reaction. 

  25. Does the unknown sequence contain an ORF closely related to group II intron RTs? (Blast search) Is the unknown sequence >80% identical to another group II intron DNA sequence? (alignement)  What is the closest relative of the intron? Can intron domains 5 and 6 be located? Identification of the 5’ end.

  26. Conclusion • We have almost finished annotatingboth sequences • We have identified some of the group I and group II introns in the two sequences and also predicted their secondary structure.

  27. Future work • Try to obtain all introns’ secondary structure in sequence 2. • Convert the annotation to a master file format.

  28. Thanks to everyone. QUESTIONS ?

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