Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in <em>Saccharomyces cerevisiae</em>

Juan de Dios Barba Tena; Etienne Schwob; Nicolas Talarek

doi:10.3791/64490

Bestimmung der S-Phasendauer mittels 5-Ethynyl-2'-Desoxyuridin-Einbau in Saccharomyces cerevi...

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

Determination of S-Phase Duration Using 5-Ethynyl-2′-deoxyuridine Incorporation in Saccharomyces cerevisiae

Bestimmung der S-Phasendauer mittels 5-Ethynyl-2'-Desoxyuridin-Einbau in Saccharomyces cerevisiae

Full Text

1,956 Views

08:40 min

October 21, 2022

DOI: 10.3791/64490-v

Juan de Dios Barba Tena¹, Etienne Schwob¹, Nicolas Talarek¹

¹Institut de Génétique Moléculaire de Montpellier,Université de Montpellier, CNRS

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study describes a precise method to determine S-phase duration in the model organism Saccharomyces cerevisiae. The protocol utilizes EdU, a thymidine analog, combined with Click chemistry to identify DNA synthesis and potentially overlooked replication defects in yeast mutants.

Key Study Components

Research Area

Cell cycle regulation
DNA replication
Mutant analysis

Background

Budding yeast as a model organism
Significance of S-phase duration
Previous limitations of classical protocols

Methods Used

EdU incorporation and Click chemistry
Saccharomyces cerevisiae
Microscopy and flow cytometry

Main Results

Successfully measured S-phase duration of approximately 25 minutes
Identified 29% of cells in the S-phase during analysis
Detected mild synthesis defects not apparent in classical methods

Conclusions

The protocol provides a reliable approach for studying S-phase duration
Implications for further research on cell cycle dynamics in yeast

Frequently Asked Questions

What is EdU?

EdU is a thymidine analog used to label cells undergoing DNA synthesis.

Why use budding yeast for this study?

Saccharomyces cerevisiae is a well-established model for studying eukaryotic cell biology.

How does Click chemistry work in this protocol?

Click chemistry is used to label EdU incorporated in DNA, allowing detection via microscopy and flow cytometry.

What are the advantages of this method?

The method is quick, easy, reproducible, and sensitive enough to detect slight replication defects.

What does S-phase duration indicate?

S-phase duration is critical for understanding DNA replication timing and its regulation during the cell cycle.

Can this method be applied to other organisms?

While this method is optimized for yeast, it may be adapted for other cell types with appropriate adjustments.

What future applications may arise from this research?

Insights from this study may facilitate research in cancer biology, genetics, and cell cycle regulation.

Wir beschreiben zwei komplementäre Protokolle zur genauen Bestimmung der S-Phasendauer in S. cerevisiae unter Verwendung von EdU, einem Thymidinanalogon, das in vivo eingebaut und mittels Click-Chemie durch Mikroskopie und Durchflusszytometrie nachgewiesen wird. Es ermöglicht die einfache Charakterisierung der Dauer der DNA-Replikation und übersehener Replikationsdefekte in Mutanten.

Dieses Protokoll ermöglicht eine präzise Bestimmung der S-Phasendauer in synchronisierten Knospungshefezellen. Es ist eine schnelle, einfache und reproduzierbare Methode. Darüber hinaus ist es empfindlich genug, um leichte Synthesedefekte zu erkennen, die von klassischen Protokollen nicht erkannt werden.

Diese Methode wird für viele Forscher nützlich sein, die an der Regulation des Zellzyklus in knospenden Hefen arbeiten. Dieses Protokoll wird von Nicolas Talarek, einem leitenden Forscher, und Juan De Dios Barba Tena, einem Doktoranden im Labor, demonstriert. Impfen Sie Saccharomyces cerevisiae-Zellen in 10 Milliliter SC-Medium bei niedriger Zellkonzentration für eine Nachtkultur bei 30 Grad Celsius mit orbitaler Bewegung bei 130 Umdrehungen pro Minute.

View the full transcript and gain access to thousands of scientific videos