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CRISPR Direct Repeat Sequences

CRISPR Direct Repeat Sequences. Olivia Ho-Shing olhoshing@davidson.edu 22 November 2009. degenerate DR. QUESTIONS: Do different halophile species share similar direct repeat sequences? Can direct repeats indicate phylogeny?

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CRISPR Direct Repeat Sequences

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

  2. degenerate DR 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----

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

  5. Do different halophile species share similar direct repeat sequences? 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

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

  7. Do different halophile species share similar direct repeat sequences? H. sinaiiensis H. sulfurifontis H. utahensis H. volcanii N. pharaonis H. vallismortis H. californiae H. denitrificans H. marismortui H. mediterranei H. mucosum H. mukohataei

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

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

  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

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