1. Volume 17, Issue 1, Pages 145-151 (January 2005)
A Kinase-Independent Function of Cks1 and Cdk1 in Regulation of Transcription 
Veronica P.C.C. Yu, Chris Baskerville, Björn Grünenfelder, Steven I. Reed 
Molecular Cell 
Volume 17, Issue 1, Pages (January 2005)
DOI: /j.molcel

2. Figure 1 Defective Expression of GAL1 in cks1 and cdc28 Mutants
(A) Expression of GAL1 under inducing or noninducing conditions. Wild-type (Wt) cells or three independently derived clones of cks1 null (Δcks1) cells were cultured in YPRaffinose to log phase at 30°C. Cells were then induced by incubation in 2% galactose for 1 hr (or raffinose in control samples). Total mRNA was extracted and reverse transcribed, and cDNA was analyzed by real-time PCR using primers to the GAL1 gene (primer set 3 in Figure 3). All values were normalized to ACT1 levels in each sample.
(B) Expression of GAL1 under inducing condition in a temperature-sensitive mutant of CDC28. Wild-type (Wt) or cdc28-4 cells were cultured at either the restrictive (37°C) or the nonrestrictive (30°C) temperatures for 3 hr in YPRaffinose. Cells were then induced in 2% galactose for 1 hr at the respective temperatures. cDNA was analyzed using real-time PCR as in Figure 1A.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Transcriptional Role of Cdc28 Does Not Depend on Kinase Activity
(A) GAL1 expression is not cell cycle regulated. Wild-type cells were grown to an OD600 of 0.1 in YPGalactose. Cells were synchronized with α factor for 1 hr at 30°C before being released into fresh warm media. Samples were collected at time points indicated. mRNA was extracted and reverse transcribed into cDNA. cDNA was amplified using primers to either the GAL1 gene or an abundantly expressed control gene, TCM1. Similar data was observed when ACT1 was used as control (data not shown).
(B) Cell cycle parameters after α factor release. Wild-type cells were treated under identical conditions as in Figure 2A. Samples were collected at time points indicated and scored for budding and nuclear division.
(C) Effect of Cdc28 kinase inhibition on GAL1 transcription. Wild-type or cdc28-as1 mutant cells were induced with galactose as described in Figure 1A, and GAL1 expression was assessed by real-time PCR either in the presence or absence of the high-affinity kinase inhibitor 1NM PP1 at a concentration of 1 μM.
(D) Cell division is inhibited at 1 μM 1NMPP1 in the cdc28-as1 background but not in wild-type controls. Wild-type or cdc28-as1 mutant cells were grown in YPGalactose at 30°C and seeded out in equal numbers. 1NM PP1 was added to a concentration of 1 μM. Samples were taken at time points indicated and counted for cell number.
(E) The K40L plasmid cannot rescue viability of cdc28 null cells. A heterozygous diploid carrying a deletion of one of its CDC28 alleles (Δcdc28:: LEU2/ CDC28) was transformed with a centromeric plasmid carrying cdc28-K40L and TRP1. It was sporulated on 1% KAc plates for 5 days at room temperature. 30 asci were dissected with a micromanipulator (Singer MSM) onto YPD plates and incubated 3 days at 30°C. Plates were replica plated onto selective medium (SC-Trp) to identify colonies carrying the cdc28-K40L allele.
(F) The cdc28-K40L allele cannot rescue temperature sensitivity in the cdc28-4 background. The cdc28-4 mutant is inviable at the restrictive temperature of 37°C due to cell cycle arrest. Transformation of the mutant with a kinase-dead cdc28 allele (K40L) did not reverse the temperature sensitivity.
(G) The cdc28-K40L allele can rescue GAL1 expression in cdc28-4 cells. Expression of GAL1 was assessed under inducing condition in wild-type, cdc28-4, or cdc28-4 cells transformed with a plasmid carrying the kinase-dead cdc28 K40L allele at either the permissive (30°C) or restrictive (37°C) temperature. cDNA was amplified as in previous experiments by real-time PCR.
(H) The cdc28-K40L allele can rescue GAL1 expression in a strain carrying a CDC28 allele with a heat-inducible degron (cdc28td). As in Figure 2G, cdc28td and cdc28td, transformed with a plasmid containing the cdc28-K40L allele, were induced with galactose either at the permissive (30°C) or restrictive (37°C) temperature and cDNA was amplified using primers to GAL1.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Transcription-Dependent Recruitment of Cks1 and Cdc28 to the GAL1 ORF
(A) Recruitment of Cks1 to GAL1. Genomically HA-tagged Cks1 in the wild-type or cdc28-1n backgrounds were analyzed by chromatin immunoprecipitation under induced (galactose) or repressed (dextrose) conditions. Cells were cultured for 3 hr at 37°C before induction with galactose. (Wild-type samples cultured at either 30°C or 37°C showed a similar pattern of recruitment, data not shown). DNA precipitated was amplified using primer sets on the GAL1 locus as depicted at the top of the figure and analyzed using real-time PCR.
(B) Recruitment of Cdc28 or Cdc28-IN to GAL1. Genomically HA-tagged Cdc28 in the wild-type and cks1 null backgrounds or HA-tagged Cdc28-IN in the cdc28-1n background were analyzed by chromatin immunoprecipitation under induced (galactose) or repressed (dextrose) conditions. Samples were cultured at 30°C, except those in the cdc28-1n background, which were cultured at 37°C prior to induction.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Proteasome Recruitment to the GAL1 ORF Depends on Cks1 and Cdc28
(A) Expression of GAL1 under inducing condition in a proteasome mutant. Wild-type (Wt) or sug1-25 (rpt6 mutated) cells were cultured at 37°C or 30°C for 3 hr in YPRaffinose. Cells were then induced in 2% galactose for 1 hr at the respective temperatures. The resultant cDNA was analyzed by real-time PCR with the GAL1 primers (primer set 3).
(B) Efficient recruitment of Rpt1 to GAL1 requires Cks1. Genomically (FLAG)-tagged Rpt1 in the wild-type or cks1 null backgrounds were analyzed by chromatin immunoprecipitation under induced (galactose) or repressed (dextrose) conditions. Cells were cultured at 30°C prior to induction.
(C) Efficient recruitment of Rpt1 to GAL1 requires Cdc28. Identical experimental conditions were used as in Figure 4B except that cells were cultured at 37°C. Rpt1 was genomically tagged with the FLAG tag in both the wild-type (Wt) or the cdc28-4 backgrounds.
Molecular Cell  , DOI: ( /j.molcel )