Figure 5.

 Disruption of the S. cerevisiae PIA1 gene results in lethality. (A) A schematic diagram of the disruption of the PIA1 gene. Numbers indicate nucleotide positions. Primers a and b were both 60 nucleotides in length; each contained 40 nucleotides homologous to the locus, and 20 nucleotides homologous to pRS (indicated by open arrows). These two primers were used to amplify the TRP1 gene from pRS304 and the PCR product was transformed into a wild-type strain. The 40-bp region flanking TRP1 allowed homologous recombination at the PIA1 locus and deletion clones were first identified by PCR analysis (using primers a and d or primers b and c), then verified by Southern blot analysis (probed with oligonucleotide c). (B) The heterozygous pia1 deletion strain was sporulated and the tetrads were dissected by micromanipulation. The four spores from individual asci were aligned vertically and allowed to germinate on a YPD plate at 30°C. The picture was taken 5 days after dissection.