speaker ：連家翎 date ： 94/12/13

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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 2005Edited by J.M. Ketley

Speaker：連家翎Date： 94/12/13

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Introduction

Vibrio parahaemolyticus

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Introduction

Vibrio parahaemolyticus

chromosome 1 chromosome 2

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Introduction

Chromosome 1 Chromosome 2

Length of sequence: 3,288,558 bp 1,877,212 bp G+C ratio: 45.4 % 45.4 %

Open reading frame (ORF):

3,080 1,752

Protein coding region: 86.9 % 86.9 %

Average ORFs length: 926.9 bp 931.3 bp rRNA operons 10 1

tRNA: 112 14

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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

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rrs of VpD and VpKX

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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

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Gene conversion

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Heteroduplexes

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Bacterial strain

• RIMD 2210856 （ VpAQ ）

• RIMD 2210633 （ VpKX ）

• RIMD 2210086 （ VpI ）

• ATCC17802T （ VpD ）

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methods

Chromosome extraction

PCR amplification

PCR purification Vector purification （ pGEMT ）

Restriction enzyme digestion （ I-CeuI ）

cloning

Sequence confirmation

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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’ ）

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Result of Amplify 16S rDNA

Amplify 16S rRNA －

VpD and VpAQ have polymorphism

16S rRNA

27 1492

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Result

161bp

VpAQ and VpD have polymorphism in the same rrs region

27 1492

357 518

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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

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Result

利用 vpD 的多樣性來分析 operon ，證明 gene conversion

16S 23Sspacer

I-CeuI

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DNA hybridization

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Result of hybridization

利用 class1 和 class2 作為 probe

Class 1

Class 1

Class 2

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Gene conversion in the 16S－ 23S rDNA spacer regions

• Analysis of spacer sequence in VpD

Class of rrs

Size of neighbor spacer in bp

16S 23Sspacer

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Heteroduplexes

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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