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no natural recombination system present (genes are not expressed) our attempts:

Myco lasma M tants. p. u. Isolation of M. pneumoniae Mutant Strains. no natural recombination system present (genes are not expressed) our attempts: Does recombination work in spite of the missing recombination system? Does the expression of an antisense RNA switch off

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no natural recombination system present (genes are not expressed) our attempts:

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  1. Myco lasma M tants p u Isolation of M. pneumoniae Mutant Strains • no natural recombination system present (genes are not • expressed) • our attempts: • Does recombination work in spite of the missing • recombination system? • Does the expression of an antisense RNA switch off • translation? • Does the expression of M. genitalium recombination • genes (ruvAB, recU) allow recombination? nothing works

  2. Myco lasma M tants p u Isolation of M. pneumoniae Mutants by Haystack Cloning Idea: Development of an efficient screening system for a large pool of transposon mutants Basic assumptions: 816 kb, 688 genes → 1011 bp/gene (Himmelreich et al. 1996) probably 180 – 215 non-essential genes (Hutchison et al. 1999) ergo: essential genome: ~ 600 kb non-essential genome: ~ 200 kb

  3. Myco lasma M tants p u Isolation of M. pneumoniae Mutants by Haystack Cloning How many individual random transposon insertion mutants have to be collected to obtain a desired mutant with a minimum probability of 99% ? 920 clones: 99% probability to find an insertion in any non-essential gene The tranposon mutant library: 2976 individual clones probability is about 99.999%

  4. Myco lasma M tants p u Isolation of M. pneumoniae Mutants by Haystack Cloning How do we find the needle? We need an ordered collection of the tranposon mutants! • 60 pools of each 50 clones were prepared • PCR with primers of goi and transposon to identify • positive pools • PCR with individual clones to identify the mutant • the system can be used for multiplex analysis

  5. Myco lasma M tants p u Isolation of M. pneumoniae Mutants by Haystack Cloning saturated transposon mutagenesis pMT85 pick 3000 transposants grow transposant library make pools of 50 transposants Tn search 60 pools by PCR for goi-Tn junction using primers specific for the goi and the Tn goi identify positive pools subscreen to identify the causative clone within a positive pool

  6. mini-Tn4001 SH29 Myco lasma M tants p glpD u control SH7 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 H2 A3 B3 C3 D3 E3 F3 G3 H3 wt 0,95 0,83 0,56 Isolation of a glpD Mutant Identification of the pool Identification of positive candidates

  7. Myco lasma M tants p u 26 bp inverted repeat 8 bp target duplication Isolation of a glpD Mutant glpD::Tn glpD::Tn WT WT [kb] 21,3 aac-ahpD 5,1 4,3 B 3,5 glpD MPN052 glpK A 2,0 1,6 EcoRV NdeI 1,4 probe B probe A SH29 →...GTGCCATGGGTTTTTACACAATTATACGGACTTTATCATCAACTTGCTTACTAAT... glpD pos 555

  8. Myco lasma M tants p u The Mutant Collection so far … Mutants isolated No mutants found glpD ?essential genes? hprK nox prpC glpF ldh glpK mpn474: surface protein mpn239 (GntR-like mpn372 (cytotoxin, regulator) ADP-ribosyltransferase) hrcA

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