A Fluorescent Based System to Monitor 2‐micron Copy Number in Saccharomyces cerevisiae

Abstract

The 2‐micron plasmid is a small genetic element that persists in Saccharomyces cerevisiae with chromosome like stability. It encodes for four proteins (Rep1, Rep2, Raf1, and Flp) that appear to provide no advantage for the host cell, but instead have a role in replicating and maintaining a steady state concentration of the plasmid‐‐ 1. Flp recombinase is known to promote amplification of the 2‐micron; disruption of the FLP gene is a valid way of preventing the necessary amplification of the plasmid to pass itself on. The 2‐micron is a selfish DNA element that is able to maintain a copy number of 40–60, however its ability to maintain these levels is not completely understood. To study this regulation we have established a quantitative and efficient means to monitor copy number. We used CRISPR‐Cas9 to replace ADE2 (encoded on 2‐micron/ADE22) with mCherry, enabling copy number to be monitored using flow cytometry. Preparation of the strains required no additional staining, allowing for quick processing and analysis. To establish the sensitivity of the system, mCherry expression was monitored in strains that had ectopic expression of the flp‐H305L mutant, which destabilizes the 2‐micron plasmid 2. We observed a decrease in mCherry expression in these strains, consistent with the role Flp plays in 2‐micron amplification. This reduction in copy number was confirmed using quantitative PCR to examine expression of mCherry from the 2‐micron. Current studies are employing this system to monitor how copy number is affected by two long non‐coding RNAs (lncRNA), which are expressed from the plasmid and regulated by Rrp6p and the TRAMP complex, components of the nuclear RNA surveillance pathway. Support or Funding Information SJFC internal funds K.-M.ChanY.-T.LiuC.-H.MaM.JayaramS.SauPlasmid701217E. L.TsalikM. R.GartenbergYeast14847852