4.5 Primer Extension

DIPLOMARBEIT Titel der Diplomarbeit Analysis of novel small non-coding RNAs in Streptococcus pyogenes angestrebter akademischer Grad Magister/Magistra der Naturwissenschaften (Mag. rer.nat.) Verfasserin / Verfasser: Doris Veit Matrikel-Nummer: 0203387 Studienrichtung Molekulare Biologie (lt. Studienblatt): Betreuerin / Betreuer: Dr. Emmanuelle Charpentier Wien, am 13.08.2008 Table of contents Table of contents .......................................................................................................... 2 1. Abstract ...................................................................................................................... 5 2. Zusammenfassung ................................................................................................... 7 3. Introduction .............................................................................................................. 9 3.1 Streptococcus pyogenes ............................................................................................. 9 3.1.1 S. pyogenes virulence factors ............................................................................ 10 3.1.1.1 Cell‐associated virulence factors .................................................................. 10 3.1.1.2 Secreted virulence factors ............................................................................. 11 3.1.2 Regulation of virulence factors ....................................................................... 12 3.1.2.1 Stand alone response regulators .................................................................. 13 3.1.2.1.1 Multiple gene regulator (Mga) .................................................................. 13 3.1.2.1.2 RofA‐like protein (RALP) .......................................................................... 13 3.1.2.1.3 Rgg/RopB (Regulation of proteinase) ...................................................... 14 3.1.2.2 Two component systems (TCS) .................................................................... 14 3.1.2.2.1 CovR/S (control of virulence genes) ......................................................... 14 3.1.2.2.2 FasBCAX (fibronectin/fibrinogen binding/hemolytic ............................ 15 activity/streptokinase regulator) .............................................................................. 15 3.1.2.2.3 Ihk‐Irr (isp‐adjacent histidine kinase/response regulator) .................... 15 3.1.2.3 Regulatory sRNAs in S. pyogenes ................................................................. 15 3.1.2.3.1 pel RNA ......................................................................................................... 16 3.1.2.3.2 fasX RNA ...................................................................................................... 16 3.1.2.3.3 RivX RNA ..................................................................................................... 16 3.2 Regulatory RNAs in bacteria .............................................................................. 17 3.2.1 Mode of action of sRNAs ................................................................................. 19 3.2.1.1 Base‐pairing mechanism ............................................................................... 20 3.2.1.1.1 Cis‐encoded sRNAs .................................................................................... 20 3.2.1.1.2 Trans‐encoded sRNAs ................................................................................ 21 3.2.1.2 RNA‐Protein interaction ............................................................................... 22 3.2.1.3 RNAs with intrinsic enzymatic activity ...................................................... 22 3.2.2 Some additional examples of sRNAs involved in the control of bacterial pathogenicity .............................................................................................................. 23 3.2.2.1 RNAIII in Staphylococcus aureus ................................................................... 23 3.2.2.2 Qrr1‐Qrr4 in Vibrio cholerae ........................................................................... 23 3.2.2.3 RNAα in Vibrio anguillarum .......................................................................... 24 3.2.3 Regulatory RNA elements ............................................................................... 24 3.2.3.1 Riboswitches ................................................................................................... 24 3.2.3.2 T boxes ............................................................................................................. 27 3.2.3.3 Leader RNAs .................................................................................................. 27 3.2.3.4 Housekeeping RNAs ..................................................................................... 28 3.2.3.4.1 6S RNA ......................................................................................................... 28 3.2.3.4.2 tmRNA.......................................................................................................... 28 3.2.3.4.3 RNase P ........................................................................................................ 28 2 0BTable of contents 3.2.3.4.4 SRP ................................................................................................................ 29 4. Materials and Methods ......................................................................................... 30 4.1 Computational predictions and analysis of sRNAs ........................................ 30 4.1.1 RNA predictions using computational tools ................................................ 30 4.1.2 Preparation of probes for transcriptional expression analysis ................... 31 4.1.3 Secondary structure predictions ..................................................................... 31 4.1.4 mRNA target prediction .................................................................................. 31 4.2 Bacterial strains, growth conditions and media .............................................. 31 4.2.1 Bacterial strains ................................................................................................. 31 4.2.2 Growth conditions and media ........................................................................ 32 4.2.2.1 S. pyogenes ....................................................................................................... 32 4.2.2.2 Escherichia coli ................................................................................................. 32 4.2.2.3 Antibiotics ....................................................................................................... 33 4.3. DNA preparation ................................................................................................ 33 4.3.1 Plasmid preparation from E. coli .................................................................... 33 4.3.1.1 Quick gram‐negative plasmid preparation ................................................ 33 4.4 Standard DNA manipulation procedures ........................................................ 34 4.4.1 Polymerase chain reaction (PCR) ................................................................... 34 4.4.1.1 Taq‐DNA polymerase ................................................................................... 34 4.4.1.2 Phusion polymerase ...................................................................................... 34 4.4.2 Restriction digestions ....................................................................................... 34 4.4.3 Purification of DNA fragments ....................................................................... 35 4.4.4 Agarose gel electrophoresis ............................................................................ 35 4.4.5 General cloning steps ....................................................................................... 35 4.4.6 Preparation of competent E. coli cells............................................................. 36 4.4.7 Heat‐shock transformation of competent E. coli cells .................................. 36 4.4.8 Measurement of DNA concentration ............................................................. 36 4.5 Primer extension .................................................................................................. 37 4.5.1 PCR product sequencing ................................................................................. 37 4.5.2 γ‐32ATP Labelling of primers .......................................................................... 37 4.5.3 Primer extension ............................................................................................... 37 4.5.3.1 Primer extension using Promega AMV Reverse Transcriptase .............. 37 4.5.3.2 Primer extension using Invitrogen ThermoScript Reverse Transcriptase ...................................................................................................................................... 38 4.5.4 Sequencing gel................................................................................................... 38 4.6 RNA techniques ................................................................................................... 39 4.6.1 Preparation of total RNA from S. pyogenes ................................................... 39 4.6.2 PAA gel electrophoresis................................................................................... 40 4.6.3 Northern blot analysis using PAA gels ........................................................

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