1 polymorphism and gene conversion of the 16s rrna genes in the multiple rrna operons of vibrio...
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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