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1.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  7.—

F igure 7.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

Role of WHI3 in suppression of bck2Δ swi6-4. (A) Inactivation of WHI3 suppresses bck2Δ swi6-4. bck2 swi6-4 (left) and bck2 swi6-4 whi3 (right) cells carrying a control plasmid were spotted onto −URA plates as described and incubated for 2 days at the temperature indicated. (B) Multicopy PAB1 depends upon WHI3 for suppression of bck2Δ swi6-4. bck2 swi6-4 (left) and bck2 swi6-4 whi3 (right) cells carrying YEpPAB1 were analyzed as described in A.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.
2.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  6.—

F igure 6.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

Suppression of bck2Δ swi6-4 by RNA-binding motif mutants of PAB1. bck2 swi6-4 cells were transformed with control plasmid or with plasmids containing mutant forms of PAB1 depicted on the left side of the table. The relative translational efficiency observed in Pab1-depleted in vitro translation extracts supplemented with the indicated Pab1 mutant proteins is derived from Kessler and Sachs (1998). Cells were spotted as described and the minimum restrictive temperature of the strain is shown.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.
3.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  3.—

F igure 3.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

The ubiquitin moiety of RPL40a suppresses bck2Δ swi6-ts. (A) UBI1 is the suppressing moiety of RPL40a. bck2 swi6-4 cells containing either control plasmid (YEp24) or YEpRPL40a/UBI1 were grown and spotted as described in Figure 1. bck2 swi6-4 cells were transformed with control plasmid (pRS416) or a plasmid expressing UBI1 from the inducible CUP1 promoter. Cells were then grown to log phase at permissive temperature in −TRP medium, spotted onto −TRP plates containing 100 μm CuSO4, and incubated at the indicated temperatures for 3 days. (B) Suppression by UBI1 is not dependent upon SIC1. bck2 swi6-4 sic1 cells were transformed with control plasmid (pRS416) or a plasmid expressing UBI1 from the inducible CUP1 promoter. The spotting assay was performed as described in A.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.
4.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  5.—

F igure 5.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

Analysis of G1 cyclin transcripts and protein in bck2Δ swi6-4 mutants containing multicopy PAB1. (A) Overexpression PAB1 leads to an increase in CLN1 and CLN2 transcript. Wild-type and bck2 swi6-4 cells were transformed with either control plasmid or plasmid overexpressing PAB1. Cells were grown to log phase in −Ura medium. The cells were then split and either grown at permissive temperature or shifted to 37° for 6 hr. CLN1, CLN2, and ACT1 transcript levels were determined by Northern blotting. CLN1 and CLN2 RNA levels were normalized to ACT1 RNA and are presented as the proportion of the RNA level in wild-type cells at the permissive temperature.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.
5.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  2.—

F igure 2.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

Overview of bck2Δ swi6-ts suppressors. (A) Dosage suppressors of bck2Δ swi6-ts mutants. After screening ∼1 × 105 transformants at 1° higher than maximal restrictive temperature, plasmids SS6/78, SS6/395, SS6/396, and SS6/406 were isolated as dosage suppressors of bck2 swi6-ts. bck2 swi6-4 cells were retransformed with these plasmids, grown to log phase at 25° in −URA medium and spotted as described for Figure 1. The suppressing genes were identified by subcloning as PAB1, GIN4, FBA1, and RPL40a/UBI1, respectively. (B) Bypass of bck2Δ swi6Δ. FBA1, GIN4, and PAB1 not only are able to suppress the temperature sensitivity of bck2 swi6-ts, but also can bypass a swi6 deletion. A bck2Δ swi6Δ strain kept alive by GAL-CLN2 was transformed with the dosage suppressors shown in A. Cells were grown to log phase in −URA medium containing galactose, spotting was done as described above onto plates containing galactose or glucose, and plates were incubated at the indicated temperatures.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.
6.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  4.—

F igure 4.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

Suppression of bck2Δ swi6-ts by GIN4. (A) Morphogenetic and septin ring defects in bck2Δ swi6-ts mutants with and without multicopy GIN4. bck2 swi6-4 strains carrying either control plasmid (YEplac181) or YEpGIN4 were transformed with YCpCDC3-GFP. Cells were grown to log phase at permissive temperature and then shifted to 35° for a time course; the 10-hr point is shown here. For microscopic analysis, cells were fixed in methanol at −70°. DIC and fluorescence microscopy were performed using an Eclipse E800 microscope with a ×100 objective. (B) Suppression by GIN4 is not dependent upon SWE1. bck2 swi6-12 cells (top) and bck2 swi6-12 swe1 (bottom) were transformed with a control plasmid (Yep24) or YEpGIN4. Spotting assays were done as described in Figure 1. (C) Suppression by GIN4 is not dependent upon a functional protein kinase domain. bck2 swi6-4 cells were transformed with a control plasmid (YEplac181), YEpGIN4, or YEpgin4K48M. YEpgin4K48M harbors a mutation that renders GIN4 kinase inactive (Longtine et al. 1998). Cell growth and spotting assays were performed as described above.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.
7.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  1.—

F igure 1.—. From: Multiple Pathways for Suppression of Mutants Affecting G1-Specific Transcription in Saccharomyces cerevisiae.

Characterization of bck2Δ swi6-ts mutants. (A and B) Thermosensitivity of bck2Δ swi6-4 and bck2Δ swi6-12 mutants. bck2 swi6-4 (A) or bck2 swi6-12 (B) with either control plasmid (Yep24) or YCp SWI6 plasmid were grown to log phase in −URA medium at 25°. Cells were counted and 10-fold serial dilutions with a starting dilution of ∼5000 cells were spotted onto −URA plates and incubated at the temperatures indicated for 2–3 days. (C) Swi6 protein is thermolabile in swi6-ts mutant. Cells were grown to log phase and then split and either grown at permissive temperature or shifted to 37° for 6 hr. After separation on a 10% SDS gel and Western blotting, Swi6 was detected with a polyclonal anti-Swi6 antibody. Cdc28 is shown as loading control. (D) Cell size of bck2 and swi6 mutants. Strains carrying the indicated SWI6 and BCK2 alleles were grown at 25° in YEPD and then either left untreated or shifted to 37° for 3 hr. Approximately 1.5 × 105 cells were sonicated and diluted into 20 ml of physiological saline solution and the mean cell volume in femtoliters was determined with a Coulter Z2 Channelyzer. Cell number in each size class is presented as a percentage of the total population. (E) Morphology of bck2 and swi6 mutants. Cells carrying the indicated BCK2 and SWI6 alleles were grown to log phase at 25° in YEPD and then either left untreated or shifted to 37° for 3 hr. Cells were imaged by phase-contrast microscopy on an Axiostar plus microscope (Zeiss) with a ×40 objective.

Karin Flick, et al. Genetics. 2005 January;169(1):37-49.

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