1. CRISPR Direct Repeat Sequences Olivia Ho-Shing olhoshing@davidson.edu 22 November 2009

2. QUESTIONS: Do different halophile species share similar direct repeat sequences? Can direct repeats indicate phylogeny? Is there any structure to the direct repeats for some potential function? 21 – 37 bp in length Surround spacers that may contain viral sequences Not palindromic, some dyad symmetry ----GACTAC----CTG----GTAGTC---- degenerate DR

3. CRISPR Finder H. mukohataei CRISPR 1_12 H. californiae H. denitrificans H. mediterranei H. mucosum H. sinaiiensis H. sulfurifontis H. volcanii H. vallismortis*

4. H. mukohataei direct repeats match hits in: Haloarcula marismortui Halorhabdus utahensis Natronomonas pharaonis Do different halophile species share similar direct repeat sequences?

5. CLUSTAL 2.0.12 multiple sequence alignment Sulfurifontis_17_1 ----------GCTTCAATCCCACAAGGGTTCGTCTGAAAC---------- 30 Denitrificans_10_x2 ----------GCTTCAATCCCACAAGGGTTCGTCTGAAAC---------- 30 Mukohataei_2_1 ----------GCTTCAATCCCACAAGGGTCCGTCTGAAAC---------- 30 Sinaiiensis_116_1 ----------GCTTCAATCCCACATGGGTTCGTCTGAAAC---------- 30 Californiae_86_1 ----------GCTTCAACCCCACGAGGGTCCGTCTGTAAC---------- 30 Utahensis_1_1 ----------GCTTCAACCCCACGAGGGTCCGTCTGAAAC---------- 30 Marismortui_1_2 ----------GCTTCAACCCCACAAGGGTCCGTCTGAAAC---------- 30 Californiae_86_2 ----------GCTTCAACCGCCCAAGGGTCCGTCTGAAAC---------- 30 Mediterranei_5_x3 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30 Mucosum_17_1 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAACC--------- 31 Mediterranei_13_x2 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30 Mucosum_10_x4 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30 Mukohataei_1_12 ----------GTTTCAGACGGACCCTTGTGGGATTGAAGC---------- 30 Californiae_65_1 ----------GTTTCAGACGGACCCTTGGGCGGTTGAAGC---------- 30 Mucosum_4_x4 ----------GTTACAGACGAACCCTAGTTGGGTTGAAGC---------- 30 Mucosum_11_1 ----------GTTACAGACGAACCCTAGTTGGGTTGAAGC---------- 30 Californiae_108_1 ----------GTTACAGACGGACCCTCGTGGGGTTGAAGC---------- 30 Denitrificans_5_x3 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30 Npharaonis_1_1 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30 Volcanii_16_x2 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30 Sulfurifontis_12_2 ---------AGTTTCAGACGAACCCTTGTGGGATTGAAGC---------- 31 Sinaiiensis_116_2 ----------GTTTCAGACGAACCCTTGTGGGATTGAAGC---------- 30 Volcanii_72_2 ---------GGTTTCAGACGAACCCTTGTGGGTTTGAAGC---------- 31 Sulfurifontis_21_1 ----------GTTTCAGACGAACCCTTGTTGGGTTGAAGT---------- 30 Sulfurifontis_26_1 ----------GTTTCAATC---CCGTTCTGGGTTTCTACCGCATCGCGAC 37 Sinaiiensis_46_1 ---------------AACCAGAGCGAACAGGGACCACC------------ 23 Sulfurifontis_19_1 GTCGCGATGCGGTAGAAAC---CCAGAACGGGATTGAAAC---------- 37 Sulfurifontis_26_2 GTCGCAGGGCAATAGAAAC---CCAGAACGGGATTGAAAC---------- 37 Npharaonis_1_4 GTCGAGACGGACTGAAAAC---CCAGAACGGGATTGAAAC---------- 37 Sulfurifontis_13_1 ------------CCGACACCGACGGCGACGGTCTCGACGACGG------- 31 Californiae_37_1 -----------CTTGTCCTTGACCTCGGTCGTCTTGTCTTT--------- 30 Do different halophile species share similar direct repeat sequences?

6. H. sinaiiensis 116-1 H. sinaiiensis 116-2 H. sinaiiensis 46-1 N. pharaonis 1-1 N. pharaonis 1-4 H. californiae 86- 1 H. californiae 86- 2 H. californiae 65- 1 H. californiae 108-1 H. californiae 37-1 H. mukohataei 1-12 H. mukohataei 2-1 H. mediterranei 13(x2) H. mediterranei 5(x3) H. mucosum 10(x4) H. mucosum 17-1 H. mucosum 11-1 H. mucosum 4(x4) H. denitrificans 10(x2) H. denitrificans 5(x3) H. volcanii 16(x2) H. volcanii 72- 2 H. sulfurifontis 17-1 H. sulfurifontis 19-1 H. sulfurifontis 26-2 H. sulfurifontis 13-1 H. sulfurifontis 26-1 H. sulfurifontis 12-2 H. sulfurifontis 21-1 ClustalW alignment grouped by species Do different halophile species share similar direct repeat sequences?

7. H. californiae H. denitrificans H. marismortui H. mediterranei H. mucosum H. mukohataei H. sinaiiensis H. sulfurifontis H. utahensis H. volcanii N. pharaonis H. vallismortis

8. Can direct repeats indicate phylogeny? Phylogram based on direct repeat sequences H. californiae H. denitrificans H. marismortui H. mediterranei H. mucosum H. mukohataei H. sinaiiensis H. sulfurifontis H. utahensis H. volcanii N. pharaonis H. vallismortis

9. Can direct repeats indicate phylogeny? Phylogram based on 16S rRNA sequences H. californiae H. denitrificans H. marismortui H. mediterranei H. mucosum H. mukohataei H. sinaiiensis H. sulfurifontis H. utahensis H. volcanii N. pharaonis H. vallismortis

10. Is there any structure to the direct repeats for some potential function? Characterizing a halophile consensus direct repeat sequence G T T T C A A A C G A A C C [AC] [GT] G G T G G G T T T G A A [AG] C

11. Is there any structure to the direct repeats for some potential function? Comparing consensus halophile sequence to other species CLUSTAL 2.0.12 multiple sequence alignment Consensus_Halophile -GTTTCAAACGAACCCGGGTGGGTTTGAAAC-------- 30 Hmarismortui_2 -GCTTCAACCCCACAAGGGTCCGTCTGAAAC-------- 30 NostocPCC_6 -GTTTCCATCCCCGTGAGGGGTA--AAGGAATTAAAAC- 35 NostocPCC_12 -GTTTCCATCCCCGTGAGGGGTA--AGAGATTAAAAAC- 35 NostocPCC_14 -GTTTCAATCCCTGATAGGGATTTTTGTTAGTTAAAAC- 37 NostocPCC_15 -GTTTCAATCCCTGATAGGGATTTTTGTTAGTTAAAAC- 37 Rxylan_2 -GTTTCAATCCCTTATAGGTAGGCTCAAAAC-------- 30 Ecoli_4 -CGGTTTATCCCCGCTGGCGCGGGGAACTC--------- 29 Ecoli_5 --GGTTTATCCCCGCTGGCGCGGGGAACAC--------- 28 Hmarismortui_C1_2 ---GGCGGTCCCTGTTCGCTCTGGTT------------- 23 NostocPCC_2 GTTACTTACCATCACTTCCCCGCAAGG-GGATGGAAAC- 37 NostocPCC_18 -CTTTCAACCCTCCCATTACTGGAAGGAGGGTTGCAACG 38 NostocPCC_7 -GTTTTAATTCCTTTACCCCT-CACGG-GGATGGAAAC- 35 NostocPCC_8 GTTTCTATTAACACA-AATCCCTATCAGGGATTGAAAC- 37 NostocPCC_17 -GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC- 37 Rxylan_3 --TACCAGGCGTGGATCTTGCCCTCGGACAC-------- 29

12. Is there any structure to the direct repeats for some potential function?

13. Conclusions Halophile direct repeats are similar to each other (significant e-values) – swapping, functionality Interesting triplet motifs in dyad symmetry of direct repeats – Binding site for CRISPR-associated proteins? – Folding site for siRNA? Direct repeats may be more indicative of phylogeny in a larger more widespread group of species