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Related Concept Videos

Yeast Signaling01:28

Yeast Signaling

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Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
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Related Experiment Video

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Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast
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Calling Card Analysis in Budding Yeast.

David Mayhew1, Robi D Mitra1

  • 1Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63110.

Cold Spring Harbor Protocols
|February 3, 2016
PubMed
Summary
This summary is machine-generated.

Calling card analysis is a high-throughput method to identify where transcription factors bind in yeast genomes. This technique uses Ty5 retrotransposon insertions and Illumina sequencing to map multiple transcription factor binding sites simultaneously.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Yeast Genetics

Background:

  • Transcription factors regulate gene expression by binding to specific DNA sequences.
  • Identifying transcription factor binding sites is crucial for understanding gene regulation.
  • Existing methods can be low-throughput or analyze only one factor at a time.

Purpose of the Study:

  • To develop and validate a high-throughput method for identifying genomic transcription factor binding sites in budding yeast.
  • To enable the simultaneous analysis of multiple transcription factors in a single experiment.

Main Methods:

  • Calling card analysis utilizes a DNA-binding protein tagged with a targeting domain that directs Ty5 retrotransposon insertion.
  • Genomic binding sites are marked by retrotransposon insertions.
  • Transposition locations are identified using high-throughput Illumina sequencing.
  • Barcodes are cloned into the Ty5 transposon to uniquely identify each transcription factor.

Main Results:

  • The calling card method successfully identifies genomic binding sites for transcription factors in budding yeast.
  • The protocol allows for the simultaneous mapping of binding sites for multiple transcription factors.
  • The method can be adapted for transcription factors expressed from native loci or plasmids.

Conclusions:

  • Calling card analysis is an efficient and scalable method for mapping transcription factor binding sites in yeast.
  • This technique facilitates a deeper understanding of gene regulatory networks.
  • The method offers flexibility in transcription factor expression for binding site analysis.