Figure 2: Regulation of reporter gene by polyA tracks in the single-cell prokaryotic and eukaryotic organisms.
(a) Percentage of mCherry fluorescence of tested LysAAG ((AAG)6–12) and LysAAA((AAA)3–12) insertion constructs compared with wild type fluorescence (WT, no insertion construct). mCherry fluorescence was assayed at excitation wavelength of 475±9 nm and emission was detected at 620±9 nm. Error bars indicate mean mCherry fluorescence values±s.d. for three individual E. coli colonies for each construct. Background levels of mCherry expression can be estimated from the fluorescence of the non-induced wild type construct (WT(NI)). (b) Western blot analysis of mCherry constructs expressed in E. coli cells. Equal amounts of E. coli cell lysates with Thioredoxin(Trx) fusion proteins were used for analysis. Fusion proteins were detected using HA-tag specific antibody. Positions of the fusion protein (Trx-HA-mCherry) and sizes of molecular weight markers (MWM) are indicated. (c) Representative differential interference contrast microscopy (left panel) and the corresponding fluorescence image (right panel −25 ms exposure) of a T. thermophila cell expressing the wild type (WT) MLP1-HA-YFP fusion. Arrowheads denote the position of the macronucleus. (d) MPL1-HA-YFP accumulation within macronuclei of live T. thermophila cells expressing an allelic series of fusion proteins—WT, (AAA)6-12, and (AAG)12—was visualized by epifluorescence microscopy. Different exposures times are indicated on the right to demonstrate the relative accumulation of each variant. (e) Western blot analysis was performed with whole cell lysates made from T. thermophila cells expressing the MLP-HA-YFP fusion proteins. Protein from equivalent cell numbers was loaded in each lane and detected using YFP specific antibody (top panel) and normalized to the nuclear histone species, histone H3 trimethyl-lysine 4 (H3K4m) (bottom panel). Positions of the full-length fusion protein (YFP), normalization control (H3k4m), and sizes of molecular weight markers (MWM) are indicated. Degradation of excess fusion protein is readily apparent as faster migrating species below the full-length MLP1-HA-YFP. (f) Steady state levels of fusion gene constructs measured by qRT-PCR. Relative levels of the mRNA for (AAG)12 and (AAA)6–12 are presented as percentage of the wild type (WT) construct mRNA levels. Error bars represent mean±s.d. values (n=3).
