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Alpha factor

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Alpha factor is a yeast mating pheromone that can be used to synchronize the growth of yeast cultures. It functions by arresting yeast cells in the G1 phase of the cell cycle, allowing for the synchronization of the culture.

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Lab products found in correlation

Pronase e, by Merck Group (1 mentions) Bx51 fluorescence microscope, by Olympus (1 mentions) Speedvac, by Thermo Fisher Scientific (1 mentions) Ultrapure h2o, by Thermo Fisher Scientific (1 mentions) Tetracycline, by NZYTech (1 mentions)

5 protocols using alpha factor

1

Cell Synchronization and DNA Damage

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Exponentially growing cells were arrested in G1 by addition of 10–8 M alpha factor (Genscript) at 30°C for 2 h or until >95% of cells were arrested in G1. Cells were then treated with 0.01% MMS (SIGMA) for 30 min to induce a pulse of alkylation damage, and cultures were released by washing cells three times and re-suspension in media containing 0.1 mg/ml pronase E (SIGMA). Synchronic cultures were routinely checked by FACS analysis. DNA was stained using 4,6,-Diamidino-2-phenylindole (DAPI) at 1 μg/ml final concentration in the presence of mounting solution and 0.4% Triton X-100 to permeablize cells. For fluorescence microscopy, series of z-focal plane images were collected with a DP30 monochrome camera mounted on an upright BX51 Olympus fluorescence microscope.
Bermúdez-López M., Pociño-Merino I., Sánchez H., Bueno A., Guasch C., Almedawar S., Bru-Virgili S., Garí E., Wyman C., Reverter D., Colomina N, & Torres-Rosell J. (2015). ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair. PLoS Biology, 13(3), e1002089.
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2

Cell Cycle Regulation by SPT6

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Saturated cultures of the wild type and spt6tSH2Δ strains were inoculated into YPD at an OD600 of 0.1 and allowed to grow to an OD600 ~0.6. Alpha-factor (25 nM; GenScript USA, RPO1002) was added to cultures to arrest cells in the G1-phase of cell cycle. G1 arrest was confirmed by microscopy. G1-arrested cells were washed three time in water and released into fresh YPD medium. Samples were collected at indicated time points for propidium iodide staining to assess the population distribution across different phases of cell cycle, and additional samples were collected in parallel for RNA extraction and cDNA synthesis. FRB-tagged SPT6 strains were treated with α-factor to induce G1-arrest for 2 h, followed by the addition of 1 μg/ml of rapamycin for 90 minutes. After the treatments, cells were washed and released into fresh YPD medium. At indicated time intervals, cells were collected and flash frozen in liquid nitrogen for RNA extraction and qRT-PCR. Cells were also collected at the same time intervals and fixed in 70% ethanol to measure their cell cycle distribution by flow cytometry of propidium iodide-stained nuclei.
Dronamraju R., Hepperla A.J., Shibata Y., Adams A.T., Magnuson T., Davis I.J, & Strahl B.D. (2018). Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription. Molecular cell, 70(6), 1054-1066.e4.
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3

Yeast Shmoo Induction and Survival

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Cells were grown to late exponential phase in liquid SC medium. Shmooing and apoptotic cell death was induced by the addition of 100 µg/mL alpha-factor (GenScript, Piscataway, NJ, USA). After incubation for 4 h at 28 °C under constant shaking, the appearance of shmoos was evaluated by light microscopy. Cell survival was calculated by plating 500 cells on YEPD medium and counting the number of colonies after 3 days of incubation at 28 °C.
Weber M., Basu S., González B., Greslehner G.P., Singer S., Haskova D., Hasek J., Breitenbach M., W.Gourlay C., Cullen P.J, & Rinnerthaler M. (2021). Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways. Antioxidants, 10(2), 322.
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4

Synchronized Yeast Cell Metabolite Extraction

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Cells grown to stationary phase were transferred to acidic media (pH 3.5) and grown to logarithmic phase. Cells were synchronized in G1 phase over two hours with the addition of 2 μg/ml alpha-factor (Genscript) every hour. Cells were washed twice with sterile water. 2.5×108 yeast cells were pelleted, resuspended in 1 ml 60% methanol, and disrupted by 5 consecutive freeze and thaw cycles using liquid nitrogen and warm water, followed by incubation at -20°C for 90 minutes and boiling at 100°C for 3 minutes. The lysate was centrifuged at 19000×g for 15 minutes and the supernatant frozen in liquid nitrogen. Methanol was evaporated in a SpeedVac (Thermo Scientific) and the residue was rehydrated in 100 μl Ultra-pure H2O (Invitrogen, GIBCO). Determination of cellular dNTP concentration was performed as earlier described [86 (link)]. Each extraction was performed at least in triplicate.
Syed S., Desler C., Rasmussen L.J, & Schmidt K.H. (2016). A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression. PLoS Genetics, 12(12), e1006451.
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5

Yeast Cell Cycle Synchronization and Drug Sensitivity Assay

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Yeast strains were grown in YPD medium containing 2% glucose as carbon source at 25 °C. For cell cycle synchronization, exponentially grown cells at 25 °C were synchronized either in G1 phase by adding alpha factor (Genscript) to a final concentration of 3–5 mg/ml for 2 h or in G2/M phase with Nocodazole to a final concentration of 10 µg/ml together with DMSO to a final concentration of 1% for 2 h at 25 C. The cells were released from the respective arrests by washing them with YP medium (YP + 1%DMSO in case of Nocadazole arrest) followed by its release into fresh YPD medium containing 0.033% MMS at 28 °C. To inhibit de-novo Sgs1 translation in cells expressing pADH1-Tc-3HA, tetracycline (NZYTech) was added to a final concentration of 1 mM at the indicated times. For drug sensitivity assays, cells from overnight cultures were counted and diluted before being spotted on YPD plates containing the indicated concentrations of drugs and incubated at 28 °C for 2–3 days.
Joseph C.R., Dusi S., Giannattasio M, & Branzei D. (2022). Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase. Nature Communications, 13, 2480.
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