Single-Molecule mRNA Detection in Live Yeast.
Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also exhibits random variability over time even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this chapter, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loops sequences that are introduced into the gene of interest. We give detailed protocols for the construction of the necessary yeast strains, for image acquisition, and for validation.
Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
Tineke L
TL
Lenstra
Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
Daniel R
DR
Larson
RNA; Single-molecule; fluorescence; live cell; microscopy; transcription