FIG 1.

Setup of the study using integration events targeted by different plasmid designs and linearization. (A) A reporter plasmid bearing sequences homologous to the GUT1 locus (GUT1 5′ and 3′) is linearized with SwaI or SacI. Linearization with SwaI results in overhangs suitable for an omega/ends-out-type recombination event (38) via double crossover at the GUT1 locus in the genome. Correct integration will result in a replacement of the GUT1 gene with the heterologous expression cassette (i.e., Δgut1). Linearization of the same vector with SacI targets a recombination event via the ends-in (38) at the AOX1 promoter in the genome. (B) A SacI integration event was also performed with a control vector lacking GUT1 integration sequences. The reporter plasmid bears an enhanced GFP (eGFP) gene under the control of the AOX1 promoter (pAOX1) and the AOX1 transcription terminator (AOX1TT). The zeocin resistance (ZeoR) cassette consists of an ILV5 promoter for expression in P. pastoris, an EM72 promoter for expression in E. coli, the Sh ble gene, and a terminator (details not shown). The gray sequence between pUC ORI and GUT1 3′UTR/PAOX1 is a remnant present in typical pPpT4-derived vectors (11). Panels A and B are not drawn on the same scale (although elements within panels A or B are at correct relative scale).