α factor

For G₁ arrest and release 6x10⁶ cells/ml were treated with α-factor (Zymo Research) for 3 hrs at 37°C in YPGal media containing 50 μg/ml doxycycline. Following extensive washes and addition of 50 μg/ml Pronase (Calbiochem) to fresh YPGal+Doxycycline, cells were further incubated at 37°C for the indicated time. Co-immunoprecipitation was performed as described [29 (link)]. Cells were collected and lysed at 4°C with glass beads in IP buffer (100 mM HEPES-KOH pH 7.9, 100 mM potassium acetate, 10 mM magnesium acetate, 2 mM sodium fluoride, 1 mM PMSF, 0.1 mM Na3VO4, 20 mM β-gycerophosphate, 1% Triton X-100, leupeptin, pepstatin, 1x complete protease inhibitor cocktail without EDTA (Roche)). Lysed material was treated with 200 U of Benzonase nuclease (Novagen 70746–3) at 4°C for 1 hour. Clarified extract was then mixed with 2 μl of specified antibody and rotated for 2 hours at 4°C. Following this, 7 μl of Dynabeads Protein A (Invitrogen 100.01D) beads equilibrated with IP buffer were added to the extract and further rotated for 1 hr at 4°C. Beads were washed twice with 500 μl of IP buffer and finally resuspended in SDS-sample buffer. Western analysis was performed and blots were scanned using the LI-COR Odyssey Infrared Imager and analyzed in the Image Studio 4.0 Software. The mean ± standard deviation is shown for three independent experiments.

GFP reporter expression analysis basically followed previous methods [32] (link), [35] (link) with some modifications. The engineered yeast a-cells were grown in 5 mL of SD-His, -Leu, -Ura media (for affinity enhancement) or SD-Leu, -Ura media (for affinity attenuation) at 30°C overnight. The cultured cells were inoculated into fresh 2 mL of SD-His, -Leu, -Ura or SD-Leu, -Ura media containing 5 µM α-factor (Zymo Research, Orange, CA, USA) to give an initial OD₆₀₀ of 0.1. Then, the expression of FIG1-EGFP fusion reporter gene was stimulated by growing at 30°C for 6 hours.
Fluorescence intensities of cultured cells were measured using a BD FACSCanto II flow cytometer equipped with a 488-nm blue laser (BD Biosciences, San Jose, CA, USA) [52] (link). The EGFP fluorescence signal was collected through a 530/30-nm band-pass filter. The mean of fluorescence intensity was defined as the GFP-A mean of 10,000 cells. The data were analyzed using BD FACSDiva software (version 5.0, BD Biosciences).
After washing the cultured cells three times, the cells were resuspended in distilled water and observed under a BIOREVO BZ-9000 fluorescence microscope (KEYENCE, Osaka, Japan). Green fluorescence images were acquired with a 470/40 band-pass filter for excitation and a 535/50 band-pass filter for emission.

Each strain carrying an individual deletion (or a double deletion, as in Figure 5) was transformed with a domain-rearrangement variant (or a combination of two, as in Figure 5) that effectively replaced the deleted gene(s). Samples were induced with 1 µM α-factor (Zymo Research), while controls were left untreated. Cultures were grown for two more hours, upon which protein synthesis was stopped by addition of cyclohexamide. GFP fluorescence was measured by flow cytometry, using a Miltenyi MACSQuant VYB flow cytometer. The GFP signal was averaged for all duplicates and standard errors were calculated. All experiments were repeated at least twice (total number of colonies analyzed: n≥4) and found to be in good agreement.

Yeast cells were grown at 30 °C in 2 % glucose-containing YP or in synthetic-defined (SD) dropout media to an optical density at 600 nm of 0.8. To synchronize yeast cells in G1 phase for a total of 3 h, α-factor (10 µg ml⁻¹; Zymo Research) was added as described [57 (link)]. One ml culture was removed to prepare spheroplasts, followed by nuclei isolation by using EZ Nucleosomal DNA Prep Kit (Zymo Research). Nuclei were treated with 0.25 U/µl of micrococcal nuclease. Reactions were stopped after 2, 5, or 10 min, and pure nucleosome DNA was isolated and subsequently resolved in 2 % agarose gels. Nucleosomal DNA was also probed by PCR at loci enriched for HMO1 (18S rDNA, MAT, 0.2 kb upstream of MAT) and a locus at which HMO1 was not detected (KRE5). Primer sequences were previously reported [47 (link)] or are available on request.

Cell cycle arrest was performed according to our standard procedures [6 (link), 21 (link)]. Hydroxyurea (Sigma) was used at 0.3 M for approximately 3 hours for S phase arrest. For mitotic arrest, Nocodazole (Calbiochem) was used for approximately 3 hours, with 5 μg/ml added initially and then an additional 2.5 μg/ml added after 90 minutes. For G1 arrest of BAR1 strains, cells were first washed 2X with fresh YPD, and then resuspended in YPD at pH 3.5 containing 2 μg/ml of α-factor (Zymo Research) and cultured for approximately 3 hours, with an additional 2 μg/ml added after 90 min. Cultures were considered arrested once >90% of the cells were in the desired phase of the cell cycle according to bud morphology. To release cells from arrest, cultures were washed twice with fresh media. To stop protein synthesis Cycloheximide (CHX; Calbiochem) was used at 10 μg/ml. Arrests were confirmed by flow cytometry (Saskatoon Cancer Center/Pharmacy & Nutrition), as performed previously [5 (link), 26 (link)].

For G₁ arrest and release 6x10⁶ cells/ml were treated with α-factor (Zymo Research) for 3 hrs at 37°C in YPGal media containing 50 μg/ml doxycycline. Following extensive washes and addition of 50 μg/ml Pronase (Calbiochem) to fresh YPGal+Doxycycline, cells were further incubated at 37°C for the indicated time. Chromatin immunoprecipitation was performed as described [33 (link)]. We performed PCR with [³²P-α]-dCTP as a component of the PCR reaction to quantify the amplified product. Formaldehyde cross-linked cells were lysed with glass beads in a Bead Beater. DNA was fragmented by sonication (Branson 450). RPA, Cdc45, or GINS antibody and magnetic protein A beads (Dynabeads protein A, Invitrogen 100.02D) were added to the cleared lysate to immunoprecipitate the DNA. Immunoprecipitates were washed extensively to remove nonspecific DNA. Eluted DNA was then subjected to PCR analysis using primers directed against ARS305, ARS306 or a site midway between ARS306 and ARS305 as described [34 (link)]. The radioactive band in the native gel, representing specific PCR amplified DNA product was quantified by phosphorimaging and normalized by a reference standard run in the same gel. The reference standard was a PCR reaction with a known quantity of template DNA replacing the immunoprecipitate. The mean ± standard deviation is shown for three independent experiments.