CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.
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Transcript of CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.
![Page 2: CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.](https://reader036.fdocuments.in/reader036/viewer/2022081512/5a4d1b217f8b9ab0599955e2/html5/thumbnails/2.jpg)
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
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CRISPR Finder
H. mukohataei CRISPR 1_12
H. californiaeH. denitrificansH. mediterraneiH. mucosumH. sinaiiensisH. sulfurifontisH. volcaniiH. vallismortis*
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H. mukohataei direct repeats match hits in: • Haloarcula marismortui• Halorhabdus utahensis• Natronomonas pharaonis
Do different halophile species share similar direct repeat sequences?
![Page 5: CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.](https://reader036.fdocuments.in/reader036/viewer/2022081512/5a4d1b217f8b9ab0599955e2/html5/thumbnails/5.jpg)
CLUSTAL 2.0.12 multiple sequence alignment
Sulfurifontis_17_1 ----------GCTTCAATCCCACAAGGGTTCGTCTGAAAC---------- 30Denitrificans_10_x2 ----------GCTTCAATCCCACAAGGGTTCGTCTGAAAC---------- 30Mukohataei_2_1 ----------GCTTCAATCCCACAAGGGTCCGTCTGAAAC---------- 30Sinaiiensis_116_1 ----------GCTTCAATCCCACATGGGTTCGTCTGAAAC---------- 30Californiae_86_1 ----------GCTTCAACCCCACGAGGGTCCGTCTGTAAC---------- 30Utahensis_1_1 ----------GCTTCAACCCCACGAGGGTCCGTCTGAAAC---------- 30Marismortui_1_2 ----------GCTTCAACCCCACAAGGGTCCGTCTGAAAC---------- 30Californiae_86_2 ----------GCTTCAACCGCCCAAGGGTCCGTCTGAAAC---------- 30Mediterranei_5_x3 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30Mucosum_17_1 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAACC--------- 31Mediterranei_13_x2 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30Mucosum_10_x4 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30Mukohataei_1_12 ----------GTTTCAGACGGACCCTTGTGGGATTGAAGC---------- 30Californiae_65_1 ----------GTTTCAGACGGACCCTTGGGCGGTTGAAGC---------- 30Mucosum_4_x4 ----------GTTACAGACGAACCCTAGTTGGGTTGAAGC---------- 30Mucosum_11_1 ----------GTTACAGACGAACCCTAGTTGGGTTGAAGC---------- 30Californiae_108_1 ----------GTTACAGACGGACCCTCGTGGGGTTGAAGC---------- 30Denitrificans_5_x3 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30Npharaonis_1_1 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30Volcanii_16_x2 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30Sulfurifontis_12_2 ---------AGTTTCAGACGAACCCTTGTGGGATTGAAGC---------- 31Sinaiiensis_116_2 ----------GTTTCAGACGAACCCTTGTGGGATTGAAGC---------- 30Volcanii_72_2 ---------GGTTTCAGACGAACCCTTGTGGGTTTGAAGC---------- 31Sulfurifontis_21_1 ----------GTTTCAGACGAACCCTTGTTGGGTTGAAGT---------- 30Sulfurifontis_26_1 ----------GTTTCAATC---CCGTTCTGGGTTTCTACCGCATCGCGAC 37Sinaiiensis_46_1 ---------------AACCAGAGCGAACAGGGACCACC------------ 23Sulfurifontis_19_1 GTCGCGATGCGGTAGAAAC---CCAGAACGGGATTGAAAC---------- 37Sulfurifontis_26_2 GTCGCAGGGCAATAGAAAC---CCAGAACGGGATTGAAAC---------- 37Npharaonis_1_4 GTCGAGACGGACTGAAAAC---CCAGAACGGGATTGAAAC---------- 37Sulfurifontis_13_1 ------------CCGACACCGACGGCGACGGTCTCGACGACGG------- 31Californiae_37_1 -----------CTTGTCCTTGACCTCGGTCGTCTTGTCTTT--------- 30
Do different halophile species share similar direct repeat sequences?
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H. sinaiiensis 116-1
H. sinaiiensis 116-2
H. sinaiiensis 46-1
N. pharaonis 1-1
N. pharaonis 1-4
H. californiae 86-1H. 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 17-1H. 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 speciesDo different halophile species share similar direct repeat sequences?
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Do different halophile species share similar direct repeat sequences?
H. californiaeH. denitrificansH. marismortuiH. mediterraneiH. mucosumH. mukohataei
H. sinaiiensisH. sulfurifontisH. utahensisH. volcaniiN. pharaonisH. vallismortis
![Page 8: CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.](https://reader036.fdocuments.in/reader036/viewer/2022081512/5a4d1b217f8b9ab0599955e2/html5/thumbnails/8.jpg)
Can direct repeats indicate phylogeny?Phylogram based on direct repeat sequences
H. californiaeH. denitrificansH. marismortuiH. mediterraneiH. mucosumH. mukohataei
H. sinaiiensisH. sulfurifontisH. utahensisH. volcaniiN. pharaonisH. vallismortis
![Page 9: CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.](https://reader036.fdocuments.in/reader036/viewer/2022081512/5a4d1b217f8b9ab0599955e2/html5/thumbnails/9.jpg)
Can direct repeats indicate phylogeny?Phylogram based on 16S rRNA sequences
H. californiaeH. denitrificansH. marismortuiH. mediterraneiH. mucosumH. mukohataei
H. sinaiiensisH. sulfurifontisH. utahensisH. volcaniiN. pharaonisH. vallismortis
![Page 10: CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.](https://reader036.fdocuments.in/reader036/viewer/2022081512/5a4d1b217f8b9ab0599955e2/html5/thumbnails/10.jpg)
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
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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-------- 30Hmarismortui_2 -GCTTCAACCCCACAAGGGTCCGTCTGAAAC-------- 30NostocPCC_6 -GTTTCCATCCCCGTGAGGGGTA--AAGGAATTAAAAC- 35NostocPCC_12 -GTTTCCATCCCCGTGAGGGGTA--AGAGATTAAAAAC- 35NostocPCC_14 -GTTTCAATCCCTGATAGGGATTTTTGTTAGTTAAAAC- 37NostocPCC_15 -GTTTCAATCCCTGATAGGGATTTTTGTTAGTTAAAAC- 37Rxylan_2 -GTTTCAATCCCTTATAGGTAGGCTCAAAAC-------- 30Ecoli_4 -CGGTTTATCCCCGCTGGCGCGGGGAACTC--------- 29Ecoli_5 --GGTTTATCCCCGCTGGCGCGGGGAACAC--------- 28Hmarismortui_C1_2 ---GGCGGTCCCTGTTCGCTCTGGTT------------- 23NostocPCC_2 GTTACTTACCATCACTTCCCCGCAAGG-GGATGGAAAC- 37NostocPCC_18 -CTTTCAACCCTCCCATTACTGGAAGGAGGGTTGCAACG 38NostocPCC_7 -GTTTTAATTCCTTTACCCCT-CACGG-GGATGGAAAC- 35NostocPCC_8 GTTTCTATTAACACA-AATCCCTATCAGGGATTGAAAC- 37NostocPCC_17 -GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC- 37Rxylan_3 --TACCAGGCGTGGATCTTGCCCTCGGACAC-------- 29
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Is there any structure to the direct repeats for some potential function?
![Page 13: CRISPR Direct Repeat Sequences Olivia Ho-Shing 22 November 2009.](https://reader036.fdocuments.in/reader036/viewer/2022081512/5a4d1b217f8b9ab0599955e2/html5/thumbnails/13.jpg)
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