1 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

2 Introduction Vibrio parahaemolyticus

3 Introduction Vibrio parahaemolyticus chromosome 1chromosome 2

4 Introduction Chromosome 1Chromosome 2 Length of sequence:3,288,558 bp1,877,212 bp G+C ratio:45.4 % Open reading frame (ORF):3,0801,752 Protein coding region:86.9 % Average ORFs length:926.9 bp931.3 bp rRNA operons101 tRNA:11214

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 （ VpAQ ） RIMD （ VpKX ） RIMD （ VpI ） ATCC17802 T （ 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 Amplify 16S rRNA － VpD and VpAQ have polymorphism 16S rRNA

14 Result 161bp VpAQ and VpD have polymorphism in the same rrs region

15 Sequence differences of rrs 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 90bp

16 Result 利用 vpD 的多樣性來分析 operon ， 證明 gene conversion 16S23S spacer I-CeuI

17 DNA hybridization

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

19 Gene conversion in the 16S － 23S rDNA spacer regions Analysis of spacer sequence in VpD Class of rrs Size of neighbor spacer in bp 16S23S spacer

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