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Speaker ：連家翎 Date ： 94/12/13

Polymorphism and gene conversion of the 16S rRNA genes in the multiple rRNA operons of Vibrio parahaemolyticus Narjol Gonza’lez-Escalona, Jaime Romero, Romilio T. Espejo First published online 22 April 2005 Edited by J.M. Ketley. Speaker ：連家翎 Date ： 94/12/13. Introduction.

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Speaker ：連家翎 Date ： 94/12/13

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  1. Polymorphism and gene conversion of the 16S rRNA genesin the multiple rRNA operons of Vibrio parahaemolyticusNarjol Gonza’lez-Escalona, Jaime Romero, Romilio T. EspejoFirst published online 22 April 2005Edited by J.M. Ketley Speaker：連家翎 Date：94/12/13

  2. Introduction Vibrio parahaemolyticus

  3. Introduction Vibrio parahaemolyticus chromosome 1 chromosome 2

  4. Introduction

  5. Introduction • Difference of Vibrio. between 16S rRNA genes（rrs） • VpKX cotains almost identical rrs • VpD cotains two rrs classes： • differing 10 nucleotide site of 25bp sequence • 11 rrn：7 contain class1，4 contain class 2

  6. rrs of VpD and VpKX

  7. Introduction • Gene conversion • The possibility that two or more rrs might independently acquire the same sequence by randon mutation is zero • Resulting in the differences of rrs

  8. Gene conversion

  9. Heteroduplexes

  10. Bacterial strain • RIMD 2210856（VpAQ） • RIMD 2210633（VpKX） • RIMD 2210086（VpI） • ATCC17802T（VpD）

  11. methods Chromosome extraction PCR amplification PCR purification Vector purification（pGEMT） Restriction enzyme digestion（I-CeuI） cloning Sequence confirmation

  12. Primer of amplify 16S－23S rDNA spacer • Amplify 16S rDNA • 27F（5’ AGAGTTTGATCCTGGCTCAG 3’） • 1492R（5 ’GGTTACCTTGTTACGACTT 3’） • Amplify shoter segment 0f 16S rDNA • 357F（5’ CTCCTACGGGAGGCAGCA 3’） • 518R（5’CGTATTACCGCGGCTGCTGG3’） • Amplify 16S－23S rDNA gene spacer • G1F（5’ GAAGTCGTAACAACG 3’） • L1R（5’ AAGGCATCCACCGT 3’）

  13. Result of Amplify 16S rDNA 16S rRNA 27 1492 Amplify 16S rRNA－ VpD and VpAQ have polymorphism

  14. Result 357 518 27 1492 161bp VpAQ and VpD have polymorphism in the same rrs region

  15. Sequence differences of rrs 90bp VpI 和VpKX的rrs sequence是相同的， VpAQ的rrs sequence差異很大， VpD含有兩種rrs class

  16. Result spacer 16S 23S I-CeuI 利用vpD的多樣性來分析operon，證明gene conversion

  17. DNA hybridization

  18. Result of hybridization 利用class1和class2作為probe Class 1 Class 2 Class 1

  19. Gene conversion in the 16S－23S rDNA spacer regions • Analysis of spacer sequence in VpD spacer 16S 23S Size of neighbor spacer in bp Class of rrs

  20. Heteroduplexes

  21. Summary • VpKX contains 11 rrn with identical rrs • VpD have two classes rrs Class 1－7 Class 2－4 • Gene conversion of rrs cause polymorphism

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