Figure 3

Integration of pTcINDEX-Luc and pTcINDEX-RFP into the ribosomal non- transcribed spacer. (A) Configuration of correctly targeted constructs showing relevant restriction sites. R-NTS/P represents the ribosomal non-transcribed spacer/promoter region with the white flag indicating the promoter [24]. The targeting fragment is designed to integrate upstream of the rRNA transcription start. The dotted line represents the position of the Spe I site introduced into the spacer to facilitate linearization. This site is absent from the genomic DNA. The crossed lines indicate the sites of homologous recombination. The double headed arrow shows the region of the 18S rRNA gene used as a probe when assessing integration. The other symbols are as in Fig. 2. The configurations for integration of pTcINDEX-Luc and pTcINDEX-RFP are shown. The expected fragment size following a targeted integration is illustrated below each map. (B) Southern analysis of the pTcINDEX-Luc and pTcINDEX-RFP transformants Arrowheads indicate fragments specific to the transformants following hybridisation with the 18S rRNA probe. These bands also hybridise specifically to the full-length luciferase or RFP probes. Lanes 1,3,5,7 contain DNA from CL-Brener [pLEW13], lanes 2 and 4 from CL-Brener:pTcINDEX-Luc [pLEW13]. Lanes 6 and 8 contain DNA from CL-Brener:pTcINDEX-RFP [pLEW13]. DNA in lanes 1–4 was digested with Kpn I and in lanes 5–8 with Nco I. The probes used are indicated below each autoradiograph. A second smaller band (1.6 kb) which hybridises to the 5' end of the RFP probe (lane 8) migrated off the bottom of this gel. Molecular sizes are shown in kb.