1. Functionality of pack-mule sequences in Rice genome Kousuke Hanada 9/21/’06

2. Mutator-like transposable elements (MULEs) are DNA transposons that can be classified as either autonomous (transposase-encoding) or non- autonomous (not encoding but requiring transposase). Transposase insert Duplication Genomic region target target site duplications Interestingly, about 5,000 loci are duplicated in rice by this mechanism.

3. Sequence1 ATG AGG TGC TCT CCC CAC ・・・ Sequence2 ATG AGA TAC TCT CCC CAC ・・・ Met MetArgArg Cys Tyr Ser Ser Pro Pro His His # nonsynonymous substitution / nucleotide site (Ka) # synonymous substitution / nucleotide site (Ks) Selective pressure= Purpose: To examine functionality of duplicated exon in pack-mule sequences Test of functionality : We can use selective pressure Ka Ks <<1 : Functional constrain is strong Ka Ks = 1 : Functional constrain is week (Neutral) = 1 : Functional constrain is week (Neutral)

4. Genomic sequences Pack-mule sequences Out group sequence Infer ancestor sequence Selective pressure (ka/ks ratio) between inferred ancestor sequence (●) and genomic sequence Selective pressure (ka/ks ratio) between inferred ancestor sequence (●) and pack- mule sequence Two selective pressures If Ka/Ks ratio between inferred ancestor sequence and a pack-mule sequence is significantly less than 1, the pack-mule sequence has undergone strong functional constrain.  the pack-mule sequence seems to be functional.

5. Genomic sequences Pack-mule sequences 1.Check whether genomic sequence includes coding region or not? If yes  next stepif no  end 2.Blastx between the protein sequence of genomic sequence and Pacl-mule sequences Pack-mule sequences has six kids of frames for coding. I defined a frame with the highest e-value as the frame of pack-mule. Blastx automatically produces the best alignments between coding region of genomic sequence and pack-mule sequence.(If stop codons existed in genomic sequence, I just removed the stop codon sites from both genomic and pack- mule sequences.) If the total length of the aligbment is over 150bp (50aa)  next step if no  end Pipeline PackMule GCG GGA TAG GCG ACG GENOMIC GCG AGA TCG GCG ACG Remove PackMule GCG GGA GCG ACG GENOMIC GCG AGA GCG ACG

6. 3. Blastp search to all plant proteins (the protein sequence of genomic sequence as query) to get out group sequence 4. If the hit sequence is exactly match to genomic or pack-mule sequence, I did not use the sequence as outgroup 5. Construct alignment in genomic, pack-mule and putative outgroup sequences 5. Estimate synonymous distance (G-O, G-P, P-O) if(the distances do not have required conditions and remove the outgroup, go back to 3) If(the distances have required condition)  we can get outgroup sequence Genomic Sequences(G) Pack-mule sequences(P) Out group Sequence(O) Required condition for outgroup Distance(G-O) > Distance(G-P) && Distance(P-O) > Distance (G-P)

7. Genomic Sequences(G) Pack-mule sequences(P) Out group Sequence(O) Infer ancestor sequence I estimate Ka and Ks distance in genomic lineage and pack-mule lineage. Although I got pipeline, I do not have all the data.