Fig. 2. Leu^–/Ura^– clones are viable calnexin-independent (Cin) cells. PCR, Southern blot, northern blot and western blot analyses demonstrate the existence of Cin cells. (A) Total DNA from a representative Leu^–/Ura^– clone (lanes 1 and 5), SP556 + pREP41 (lanes 2 and 6), SP3222: cnx1 + phcd_cnx1 (lanes 3 and 7), purified pREP41cnx1⁺ plasmid DNA (lanes 4 and 8) was subjected to PCR analysis with two sets of primers. The primer set 3/4 amplifies the nmt1 coding sequences in the genome and sequences inserted in the multicloning sites of the pREP41 or pREP42. The primer pair 3/2 specifically detects episomal constructs with cnx1⁺ or cnx1 mutants. Positions of certain DNA size markers (in kb) (lane 9) are indicated on the right. (B) Schematic representation of the cnx1 + pcnx1 haploid strains with the annealing sites for the primers used. (C) PCR analyses of cnx1 and his3 sequences. Total DNA from a representative Leu^–/Ura^– clone (lanes 4 and 9), SP556 + pREP41 (lanes 1 and 6), SP3220: cnx1 + pcnx1⁺ (lanes 2 and 7), SP3222: cnx1 + phcd_cnx1 (lanes 3 and 8), or purified empty pREP41 vector DNA (lanes 5 and 10), were subjected to PCR analysis with two sets of primers. The primer set 1/2 amplifies the cnx1 coding sequences in the genome of WT cells, or the his3⁺ marker in the cnx1 (cnx1::his3⁺) strains. The primer pair 5/6 specifically detects genomic or episomal cnx1 sequences. Positions of DNA size markers are indicated on the left. (D) Southern blot analysis showing the absence of genomic integration of cnx1 coding sequences. DNA from cnx1 + phcd_cnx1 (SP3222; lanes 1), a representative Leu^–/Ura^– clone (Cin; lanes 2), or the control for genomic cnx1⁺ strain SP556 + pREP41 (lanes 3) was digested with either BglII or ClaI. After transfer, the membranes were hybridised with either the mini_cnx1 or the pREP41 probes. Positions of DNA size markers are indicated on the right. (E) Schematic representation of the cnx1⁺ locus relevant to the Southern blot analyses. The cnx1⁺ open reading frame is indicated by a box with hatching to the right. The 5' and 3' untranslated regions are denoted by boxes with hatching to the left. Only relevant restriction sites and distances in kilobases (kb) are shown. (F) Northern blot analysis probing cnx1. Total RNA from strains SP556 (genomic cnx1⁺; lane 1), SP3220: cnx1 + pcnx1⁺ (lane 2), SP3222: cnx1 + phcd_cnx1 (lane 3), SP7188: Cin (lane 4) and SP7202: Cin + pcnx1⁺ (lane 5), were hybridised as described by Jannatipour and Rokeach (Jannatipour and Rokeach, 1995) with a ³²P-labelled DNA probe encompassing the entire cnx1⁺ coding sequence. (G) Western blot analyses using rabbit anti-Cnx1p or anti-BiP polyclonal antibodies, as indicated. The anti-Cnx1p antibodies detect epitopes throughout the entire calnexin/Cnx1p molecule. Log phase cultures of SP556 (genomic cnx1⁺; lane 1), SP3220: cnx1 + pcnx1⁺ (lane 2), SP3222 SP cnx1 + phcd_cnx1 (lane 3), 7188: Cin (lane 4), and SP7202: Cin + pcnx1⁺ (lane 5) cells were used to prepare total protein extracts as described in Materials and Methods, and 20 µg of material was loaded for fractionated by SDS-PAGE, and incubated with antibodies as described by Elagöz et al. (Elagöz et al., 1999). Perpendicular, thick black arrows in A, C, D, F and G indicate the lanes corresponding to analyses of the Leu^–/Ura^–(Cin) strain.