期刊
JOURNAL OF FUNGI
卷 9, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/jof9101033
关键词
yeast; cell cycle; histone; Ogataea; Saccharomyces; flow cytometry
In this study, a method called Histone Abundance Quantification (HAQ) was developed to quantitatively measure the level of histones in live yeast cells. The HAQ method showed consistent results with DNA levels and can be used in conjunction with other fluorescent markers. This method provides a simple approach for directly monitoring the effects of various stimuli on the cell cycle.
Assaying changes in the amount of DNA in single cells is a well-established method for studying the effects of various perturbations on the cell cycle. A drawback of this method is the need for a fixation procedure that does not allow for in vivo study nor simultaneous monitoring of additional parameters such as fluorescence of tagged proteins or genetically encoded indicators. In this work, we report on a method of Histone Abundance Quantification (HAQ) of live yeast harboring a GFP-tagged histone, Htb2. We show that it provides data highly congruent with DNA levels, both in Saccharomyces cerevisiae and Ogataea polymorpha yeasts. The protocol for the DNA content assay was also optimized to be suitable for both Ogataea and Saccharomyces yeasts. Using the HAQ approach, we demonstrate the expected effects on the cell cycle progression for several compounds and conditions and show usability in conjunction with additional fluorophores. Thus, our data provide a simple approach that can be utilized in a wide range of studies where the effects of various stimuli on the cell cycle need to be monitored directly in living cells.
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