Figure 2

Shuttle transposon mutagenesis of Physcomitrella patens cDNA clones. The structure of a representative moss cDNA clone (ID: S_PP015059353; 808 bp) in the pUCMinIV minimal vector is shown. This defined plasmid was subjected to shuttle mutagenesis, and the transposon insertion sites for 72 resulting clones were mapped by DNA sequencing to assess the distribution of insertions. 41 insertions in "forward" orientation (nptII resistance marker on transposon and bla marker on vector transcribed in same orientation) are indicated by blue lines within the circle, 31 "reverse" insertions by red lines outside. Most of the insertions (66 / 72, corresponding to 92%) occurred throughout the cDNA, without apparent strong bias for insertion site or orientation. For production of the gene-disruption library used afterwards for the moss transformation, cDNA clones were mutagenised in pools; here about 70% of the mutagenised plasmids had insertions in the cDNA.