Figure 5

Integration of the marked Pch PRP8 inteins at CMD1 locus. A. Tetrad analysis of diploids that are heterozygous for the insertion of the marked inteins at the CMD1 locus (CMD1/CMD1::GFP-Pch PRP8 marker-intein). Individual haploid spores were grown on non-selective YPD medium. Segregation of the mutant alleles was 2:2 and the slow growing colonies contain CMD1 with the G418^R-and HygB^R-intein insertions as determined by plating the haploids on selective medium (not shown). B. Western blot analysis of protein extracts from haploids containing the Pch PRP8 marked-inteins at the CMD1 locus. Lane 1, 100 ng purified calmodulin; Lane 2 LAY18-2C expressing Cmd1-GFP (constructed using the classic PCR method [41]); Lane 3, extract from a haploid CMD1::GFP-Pch PRP8-HIS5-intein isolate; Lanes 4, 5 extracts from two separate haploid CMD1::GFP-Pch PRP8-G418^R-intein isolates. C. Fluorescence microscopy of the haploid CMD1::GFP-Pch PRP8-HIS5-intein isolate. Numbers 1-4 refer to images showing unbudded, small budded, medium budded or large budded cells, respectively.