Fig. 1. Genetic separation of function screen. (A) The `checkpoint arrest' assay measures colony growth at the semi-permissive temperature of 27.3°C. In this assay, RAD9 cdc13-1 cells do not form colonies because they enter a G2/M arrest. By contrast, rad9 cdc13-1 cells cannot arrest and do form colonies. The rapid death assay measures colony growth after three periods of growth at the restrictive temperature of 36°C for 4 hours and finally a return to growth at 23°C. In this assay RAD9 cdc13-1 cells retain viability and form colonies, whereas rad9 cdc13-1 cells lose viability in this assay. In practice, to distinguish better between the growth of RAD9⁺ and rad9 cells, plates were subjected to three 4-hour periods at the restrictive temperature of 36°C, separated by 4-hour periods of recovery at the permissive temperature of 23°C. Colonies were then allowed to form at 23°C. (B) Novel alleles rad9i-1 and rad9s-2 have opposite phenotypes in the two assays. pRS416 plasmids containing the RAD9 (lanes 1,2), rad9i-1 (lanes 5,6) and rad9s-2 (lanes 7,8) alleles were transformed into a rad9 cdc13-1 strain. An empty vector was transformed into lanes 3 and 4. A 1:5 dilution series was prepared and spotted onto SC-uracil (left two) or YEPD (right) plates, which were incubated as shown. (C) Wild-type RAD9 is 1309 amino acids, contains an [S/T]Q cluster domain (SCD) and two BRCT domains. (D) The rad9i-1 allele contains an in-frame 5 amino acid insertion of GMFKH after C853. (E) The rad9s-2 allele contains a transposon insertion into the 5' half of the RAD9 gene. The first 711 bp of the RAD9 coding sequence have been removed and replaced by 335 bp of transposon sequence (shown in black). The longest open reading frame remaining encodes an N-terminal truncation of Rad9 using the ATG at methionine M243 as a start codon.