Figure 7. Loss of CTNNBL1 delays quiescence exit in S. pombe. (A) Targeting the yeast SPAC1952.06c (ctnnbl1) locus. The SPAC1952.06c gene was replaced by homologous recombination with a cassette comprising a kanamycin resistance marker under the control of translation elongation factor 1A regulatory signals as described by Bahler et al. (1998). Correct targeting was confirmed by PCR using primers whose locations are indicated. (B) Δctnnbl1 S. pombe grow similarly to controls after spotting on to YES plates. Viability of cells in log-phase growth in either yeast extract (YE) or minimal medium (EMM) from serial 10-fold dilutions spotted onto YE plates containing 5 mg/ml phloxine B. (C) Nitrogen-starved Δctnnbl1 S. pombe showed delayed initiation of growth compared with controls after transfer into rich medium. Growth curves after release from nitrogen starvation (time zero) are shown for wild type S. pombe (wt; black line), two-independent Δctnnbl1 mutants (red and orange lines) in which the ctnnbl1 locus had been replaced by a kan^r cassette as well as of S. pombe carrying a control ctnnbl1 targeting in which the ctnnbl1 locus had been replaced by 3HA-tagged CTNNBL1 driven from the nmt1 promoter (blue dashed line). Similar results were obtained in four independent experiments. Growth of serial 10-fold dilutions of 3 week-starved S. pombe cultures on YE plates revealed that CTNNBL1 deficiency did not affect their viability. (D) Starved CTNNBL1-deficient S. pombe exhibit delayed exit from G₀ following release from nitrogen starvation. Quiescent S. pombe, which adopt a small, round shape on nitrogen starvation, elongate prior to their first cell division on release into rich medium.²³ Cells were fixed in 70% ethanol at the times indicated and visualized by phase-contrast microscopy.