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Fig. 3. cif1⁺ is unessential for vegetative growth. (A) Tetrad analysis of a cif1⁺ x cif1::kanMX4 (cif1) diploid. Dissection was performed on MM+AULH, and colonies from germinated spores were streaked on YE+150 µg/ml G418 to verify the presence of the cif1::kanMX4 marker, and on MM+low adenine+ULH to verify the segregation of the ade6 marker. All the tetrads showed 2:2 segregation of both cif1 and ade6 alleles. (B) Northern blot analysis of Cin and cif1 cells. Samples of 5 µg of total RNA prepared from exponential phase cells were loaded on a 1.2% agarose-formaldehyde gel. RNA was probed with a ³²P-labeled DNA probe encompassing the entire cif1⁺ or cnx1⁺ coding sequence. Photograph of the ethidium-bromide (EtBr) staining shows the 18S and 25S rRNAs as loading control. Cells harboring calnexin on a plasmid have higher levels of cnx1⁺ RNA; however, these cells contain the same amount of Cnx1p, thus confirming that the levels of plasmid-encoded Cnx1p are equivalent to genomic expression. (C) Western blotting of Cin and cif1 cells. Cell extracts were made as described in the Materials and Methods, and 10 µg of material was loaded for fractionation on a 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane; Western blotting was carried out using rabbit polyclonal anti-Cif1p serum at a 1:5000 dilution, and with anti-Cnx1p polyclonal antibodies at a 1:35,000 dilution. As a loading control, the same membrane was immunoblotted with and anti-tubulin monoclonal antibodies at a 1:5000 dilution.

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