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1.
Figure 2.

Figure 2. From: A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe.

Excision of PB was nearly always precise. (A) More than 95% of the FOA-resistant derivatives of an ade6::PB strain were Ade+ and had lost the PB sequence through precise excision. Representative examples of gel electrophoresis results of colony PCR products were shown. PCR was performed with primers flanking the insertion site (oligo 111 and 112). (B) In the two Ade derivatives, a 2-kb PB sequence proximal to the ade6 ORF was deleted (dashed rectangle). The deleted region was flanked by two TTAAs: one was at a PB-genome junction, and the other was inside the ura4 ORF.

Jun Li, et al. Nucleic Acids Res. 2011 March;39(6):e40-e40.
2.
Figure 1.

Figure 1. From: A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe.

Transposition of PB in S. pombe. (A) A schematic view of the binary transposition system. pDUAL-PBase, a plasmid integrated into the S. pombe genome provided PBase under the control of the nmt1 promoter. The donor plasmid pPB[ura4] provided a modified PB transposon lacking transposase but containing a ura4+ selection marker. Pnmt1, promoter of nmt1. Amp, ampicillin-resistant cassette. ITR, inverted terminal repeat, is composed of a 13-bp terminal repeat and a 19-bp internal repeat. At the left end, ITR-L, two repeats are separated by a 3-bp spacer, and at the right end, ITR-R, by a 31-bp spacer. (B) Images of plates selecting for Ura+ transformants of pPB[ura4]. When transformed with pPB[ura4], cells with PBase formed more colonies than those without on plates lacking uracil.

Jun Li, et al. Nucleic Acids Res. 2011 March;39(6):e40-e40.
3.
Figure 4.

Figure 4. From: A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe.

Profiling of PB insertion sites in S. pombe genome by Illumina sequencing. (A) Statistics of PB insertion target sequences. TTAA was the predominant target. Reverse-complement pairs (TTAT and ATAA, TTAG and CTAA) were highlighted with the same background. (B) Distribution of TTAA-targeting PB insertions along the three chromosomes. Each bar represents the number of independent PB insertion sites (red) or the number of reference TTAAs (blue) in a 10 kb-window. (C) Comparison of the genomic context of TTAA-targeting PB insertions and reference TTAAs. The proportions of insertion sites and reference TTAAs falling into different types of IGRs and ORFs are plotted. IGRs are classified into three types: divergent, tandem and convergent, based on the transcription orientations of the flanking genes. ORFs are classified based on whether their deletion mutants are lethal (essential), viable (non-essential) or with unknown phenotype (lacking dispensability information).

Jun Li, et al. Nucleic Acids Res. 2011 March;39(6):e40-e40.
4.
Figure 5.

Figure 5. From: A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe.

Reversion analysis. (A) A diagram of strain layout on TBZ plates. The TBZ-resistant parent strain and eight independent FOA-resistant derivatives (F1–F8) induced by remobilization of PB were streaked on plates containing 30 mg/l of TBZ. A klp6Δ deletion strain and a wild-type (WT) strain were also included as controls. (B) Phenotype differences of the FOA-resistant derivatives of two TBZ-resistant mutants isolated in a pilot PB mutagenesis screen. DY3039 had one copy of PB inserted at klp6 and all its derivatives were as sensitive to TBZ as WT. In contrast, all the derivatives of DY3038, which contained a single copy of PB at arg6, were resistant to TBZ. The plates were photographed after 7 days at 30°C. (C and D) Excision of PB from non-TTAA sites was also precise. In a TBZ-resistant mutant DY2552, PB targeted a TTAG site in the klp5 ORF. Both the colony PCR result using primers flanking the insertion site in klp5 (C) and the reversion of TBZ resistance phenotype (D) suggested that no footprint was left after excision.

Jun Li, et al. Nucleic Acids Res. 2011 March;39(6):e40-e40.
5.
Figure 3.

Figure 3. From: A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe.

An excision marker allowed the transposition events to be monitored and enriched. (A) In the starting strain, a PB insertion in the arg6 locus resulted in auxotrophy for arginine. Upon transposition, PB relocated from arg6 to a new site, reverting cells from Arg Ura+ to Arg+ Ura+. (B) Transposition efficiency at the arg6 locus. The percentage of Arg+ Ura+ cells was calculated as the number of colonies on EMM plates lacking arginine and uracil divided by the number of colonies on YES plates. Three independent strains with PBase-integration, DY1432, DY1433 and DY1434, were tested. (C) An illustration of how the PB copy number may be influenced by the choice of different DNA repair pathways. If transposition occurs at S or G2 phase, the repair pathway choice of non-homologous end joining (NHEJ) or homologous recombination (HR) could contribute to the copy number fluctuation. (D) The copy numbers of PB in Arg+ Ura+ derivatives were determined by monitoring the levels of PB versus act1+ using qPCR (n = 24).

Jun Li, et al. Nucleic Acids Res. 2011 March;39(6):e40-e40.
6.
Figure 6.

Figure 6. From: A piggyBac transposon-based mutagenesis system for the fission yeast Schizosaccharomyces pombe.

PB-mediated genetic screens. (A) Genetic screen strategy. Expression of PBase was induced in cells containing a single copy of PB at arg6. Next, transposition events were enriched by spreading cells on plates lacking arginine and uracil. Then, the cells were replica-plated to selection plates or shifted to screening conditions. (B) A schematic view of insertion sites of PB in klp5, klp6 and dam1 mutants isolated from the screen for TBZ-resistant mutants. The numbers in parentheses indicate the total numbers of mutants associated with each gene. Open boxes represent protein-coding regions. All TTAA sites are marked with green vertical lines. Primers used to check insertions by colony PCR are shown above the klp5 and klp6 ORFs as arrows. Smaller arrows below each gene denote PB insertions. The directions of the arrows indicate the directions of transcription of the ura4+ marker in PB. Two insertions in klp5 hit TTAG rather than TTAA. (B) Spot assay showed the TBZ resistance of an isolated dam1 allele in comparison with the wild-type and the klp6 null allele strains. Four-fold serial dilutions of cells were spotted on plates containing indicated concentrations of TBZ. The plates were photographed after 2 days at 30°C. (C) wee1 was hit by PB in the cdc25-22 suppressor screen at 37°C. Labels are the same as in (B).

Jun Li, et al. Nucleic Acids Res. 2011 March;39(6):e40-e40.

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