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Cyanobacteria’s Repeat Sequences … where did they come from?

Cyanobacteria’s Repeat Sequences … where did they come from?. Molly Sergio. Overview of Presentation. unique DNA repeats Cyanobacteria genomes Similarities? Research !. Eureaka! Repeated Sequences!. Dispersed Repeats. Tandem Repeats. CRISPRs.

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Cyanobacteria’s Repeat Sequences … where did they come from?

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  1. Cyanobacteria’s Repeat Sequences… where did they come from? Molly Sergio

  2. Overview of Presentation unique DNA repeats Cyanobacteria genomes Similarities? Research !

  3. Eureaka! Repeated Sequences! Dispersed Repeats Tandem Repeats CRISPRs

  4. Jumping Genes Barbara McClintock Dispersed Repeats Transposons!

  5. Tandem Repeats “Slippage”

  6. ~ 35 to 37 nucleotides Exact count of nuclotides Jump? Non-CRISPR sequence would be duplicated Slippage? CRISPRs Clustered Short Regularly Spaced Palindromic Repeat Where do CRISPRs come from? Picture credit: James Godde

  7. Example of CRISPRs in Npun

  8. 58ATTGCAATTTCTCAAAATCCCTATTAGGGATTGAAAC 42 ATTGCAATTCATCAAAATCCCTATCAGGGATTGAAAC 19 ATTGCAATTCATCAAAATCCCTATTAGGGATTGAAAC 9 ATTGCAATTTATCAAAATCCCTATTAGGGATTGAAAC 12 GTGGCAACAACCCTCCAGGTACTGGGTGGGTTGAAAG Nostoc punctiforme Escherichia coli 2-4 CGGTTTATCCCCG GCGGG-GAACAC CTGGC CRISPR Sequences Found In Cyanobacteria Copy # 28GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC16GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC13GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC4GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC4GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120

  9. Nostoc PCC 7120 28GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC 16GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC 13GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc punctiforme 58 ATTGCAATTTCTCAAAATCCCTATTAGGGATTGAAAC 42 ATTGCAATTCATCAAAATCCCTATCAGGGATTGAAAC 19 ATTGCAATTCATCAAAATCCCTATTAGGGATTGAAAC 9 ATTGCAATTTATCAAAATCCCTATTAGGGATTGAAAC 12GTGGCAACAACCCTCCAGGTACTGGGTGGGTTGAAAG Escherichia coli2-4 CGGTTTATCCCCG GCGGG—GAACAC CTGGC Similarity Between CRISPRs

  10. Anabaena 7120 compare ? ? ? ? ? ? ? ? ? Anabaena variabilis ? ? How did CRISPRS arise in cyanobacteria genomes? Any matching CRISPR in Anabaena variabilis? Anynon-CRISPRsequences match?

  11. Locating a CRISPR in a7120 136 • (SEQUENCE-SIMILAR-TO "CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC" IN a7120) QueryQ-startQ-endSubjectS-startS-endID value 1. "Seq1" 1 36 #$A7120.CHROMOSOME5647145 5647110 100% 2. "Seq1" 1 36 #$A7120.CHROMOSOME5647076 5647041 100% 3. "Seq1" 1 36 #$A7120.CHROMOSOME5646995 5646960 100% 4. "Seq1" 1 36 #$A7120.CHROMOSOME 5646922 5646887 100% 5. "Seq1" 1 36 #$A7120.CHROMOSOME5646849 5646814 100% 6. "Seq1" 1 36 #$A7120.CHROMOSOME5646774 5646739 100% 7. "Seq1" 1 36 #$A7120.CHROMOSOME5646703 5646668 100% 8. "Seq1" 1 36 #$A7120.CHROMOSOME5646635 5646600 100% 9. "Seq1" 1 36 #$A7120.CHROMOSOME5646562 5646527 100% 10."Seq1" 1 36 #$A7120.CHROMOSOME5646490 5646455 100%

  12. Locating in between an a7120 CRISPR • (FOR-EACH coord-pair IN * AS left-coord = (+ (FIRST coord-pair) 1) AS right-coord = (- (SECOND coord-pair) 1) AS seq = (SEQUENCE-OF A7120.chromosome FROM left-coord TO right-coord) COLLECT seq) Non-coding sequences (in between CRISPRs) 1-2. "GAGAGACTGGAAACAATTTCTATAGCGATGTCGGAT" 2-3. "TCAATCATCTTTGCATTATATCCTGAAATTACAAGAT" 3-4. "CTCCCTTAGCCACTCTAGGATTTGTGACTGTT" 4-5. "ATGATTATCGAGGAATCTCGAAAATAGGACGTCGA" 5-6."AAGCGACCATCGCTTTTTGCACGAACAGCAGATGGAACG" 6-7. "TCCTGCTCCAAGCATTAGTCCTTCGGAGATTAAAAAC" 7-8. "TAACTACTGCAAGCGTTGTGCAAGCAGCAATACCTGC" 8-9. "TGCACATTGATATAAACGAAGCTAAAAAAGCCTCTACCAATATAA" 9-10."TCTTGCATACAAAGCTGCATTTCTAGATGACAA"

  13. Compare CRISPR similarity Compare similarity Anabaena variabilis How did CRISPRS arise in cyanobacteria genomes? GAGAGACTGGAAACAATTTCTATAGCGATGTCGGAT Anabaena 7120 CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC

  14. 1 2 3 4 5 6 Anabaena variabilis ? Blast CRISPR Sequences Found In Cyanobacteria Copy # 28 GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC16 GTTTTAACTAACAAAAATCCCTATCAGGGATTGAAAC13 GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC 9 GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC 4 GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC 4 GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120

  15. (SEQUENCE-SIMILAR-TO "CTTTCCGATCACATCACCCCGAAAGGGGATGGAAAC" IN avar) • Nil • (SEQUENCE-SIMILAR-TO "**********************************************" IN avar) • Nil How did CRISPRS arise in cyanobacteria genomes? Use a different CRISPR and its non-CRISPR sequences from a7120… • (SEQUENCE-SIMILAR-TO "GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC" IN avar) • QueryQ-startQ-endSubjectS-startS-endE-value • 1. "Seq1" 1 35 #$A29413.Contig256360180 360074 1.0d-13 • 2. "Seq1" 1 35 #$A29413.Contig256360039 360005 1.0d-13 • (SEQUENCE-SIMILAR-TO "**********************************************" IN avar) • Nil

  16. Anabaena variabilis ? Blast Sequences Found In Cyanobacteria Copy # 28GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC16GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC13GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC9GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC4GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC4GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG Nostoc PCC 7120 1 2 3 4 5 6

  17. Anabaena variabilis ? Blast Sequences Found In Cyanobacteria Copy # 28GTTACTTACCATCACTTCCCCGCAAGGGGATGGAAAC16GTTTTAATTCCTTTACCCCTCACGGGGATGGAAAC13GTTTCTATTAACACAAATCCCTATCAGGGATTGAAAC9GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC4GTTAAAACCCTCTAAAATCCCTATCAGGGATTGAAAC4GTTGCAACCCTCCTTCCAGTAATGGGAGGGTTGAAAG 0 2 147 35 146 0 0 0 0 0 0 0 Nostoc PCC 7120 1 2 3 4 5 6

  18. Six matching CRISPRs inserted What conclusions can be drawn? • Anabaena 7120 has at least four unique CRISPRs that were found in Anabaena variabilis common ancestor of cyanobacteria retained all six CRISPRs lost two CRISPRs Anabaena 7120 Anabaena variabilis

  19. common ancestor of cyanobacteria six CRISPRs inserted four CRISPRs inserted into A.var Anabaena variabilis Anabaena 7120 What conclusions can be drawn? • Anabaena 7120 has at least four unique CRISPRs that were found in Anabaena variabilis

  20. The End The End Special thanks to Dr. James Godde • Assistant Professor at Monmouth, Illinois • Contact number: 309-457-2350 • Email:jgodde@monm.edu

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