Strain |
sic1Δ
a
|
hct1Δ
b
| +c
|
|---|
tab1-1
| - | + | + |
tab2-1
| +/- | + | + |
tab3-1
| - | + | + |
TAB5-1
| - | + | + |
TAB6-1
| +/- | + | + |
TAB7-1
| - | + | + |
- aTo assay if bypass required SIC1, cdc15Δ::TRP1 tab [pMET3-CDC15/URA3] cells were transformed with an SIC1-deletion cassette (S. pombe his5+ DNA fragment whose termini were engineered to be homologous to SIC1 5' and 3' untranslated regions). All transformants were tested for their ability to bypass cdc15Δ using the 5-FOA growth assay (sample size n = 4–17). They were also screened for the absence of SIC1 as the result of homologous integration. In the case of tab2 and TAB6, about half of sic1Δ transformants could bypass. This incomplete penetrance is not well understood. There are at least two sources of variability in this experiment. First, sic1Δ transformants of tab cdc15Δ [CDC15, URA3] were pregrown in rich media to accumulate cells that had lost the [CDC15, URA3] plasmid. Because plasmid-loss is a low-probability event and because cells without the plasmid are at a growth-disadvantage, the number of plasmid-free cells spotted on a 5-FOA plate probably was small and varied among different transformants. Second, mutant individuals can exhibit more variability than wild-type individuals, a phenomenon frequently observed in worms and flies. Therefore, it is possible that in only a fraction of plasmid-free cells, the amount of Cdc14 activity reached a level high enough to tolerate sic1Δ. Thus, one can imagine that for some transformants, the number of plasmid-free cells spotted on 5-FOA was small and that by chance they or their limited numbers of progeny could not exit mitosis, resulting in the absence of bypass colonies. In other transformants, the opposite could occur, giving rise to mixed results in the plate assay. bSimilar assay was conducted for HCT1. Sample size n = 1–7. cTransformants from a and b that resulted from non-homologous integration (and hence no deletion of SIC1 or HCT1) were used as a control. Sample size n = 12–86.
