Fig. 6. The Cin state is plasmid independent, maintained in the cnx1⁺ genomic background and transmitted in a non-Mendelian fashion. (A) Phenotypic analysis of tetrads resulting from sporulation of the cnx1⁺/Cin diploid SP7501 (SP247/SP7188; see Table 1), in which deletion of the cnx1 genomic copy in the SP7188 haploid is marked with his3⁺. Cin spores bear the his3⁺ maker (i.e. cnx1:: his3=cnx1), thus they can grow on medium lacking histidine (MMAUL), while cnx1⁺ spores grow only on medium containing histidine (MMaULH). The his3⁺ marker was inherited in 2:2 ratio. Spores bearing the ade6-210 allele produce dark pink colonies on low adenine medium (MMaULH), while the ade6-210 allele produces light pink colonies. The ade6 markers were inherited in 2:2 ratio. To assess whether the factor encoding calnexin independence was present in the cnx1⁺ progeny, extracts form the his3^– germinated spores were transformed into the cnx1 + pcnx1⁺ strain (SP3220; Table 1), and their capacity to generate Cin cells was examined by plasmid segregation. All cnx1⁺/his3^– extracts produced Cin cells, as symbolised by [cif] within a circle. The putative [cif] factor was then inherited in a 4:0 ratio. Tetrads are labelled 1-4, and spores A-D. (B) Western blotting with anti-Cnx1p antibodies confirmed that the his3⁺ germinated spores were viable in the absence of calnexin. As positive control, the same extracts were immunoblotted with anti-BiP antibodies. Tetrads are labelled 1-4, and spores A-D.