Figure 1.

An engineered network with delayed autoinhibition exhibits pulses of protein expression. (A) Schematic diagram of the reporter used for the study of intron delays in a gene network with delayed autoinhibition (sequences and assembly strategy are detailed in Supplemental Table 1). The mouse β-actin promoter (Pactin) that includes its first intron drives the expression of the reporter. Fluorescence detection is possible because of YFP, the Venus fast-maturing variant of yellow fluorescent protein. TetR is the tetracycline repressor that, when delivered to the nucleus because of a fusion with a nuclear localization signal (NLS), binds to operator sequences (o) that have been engineered into the promoter to drive repression by steric hindrance and promoter looping-out. The PEST sequence (rich in proline, glutamic acid, serine, and threonine) is a protein degradation element originating in the mouse ornithine decarboxylase gene. AU-rich elements present in the 3′-untranslated region of our gene were included to reduce the stability of the mRNA. Destabilization of both the protein and mRNA increases the responsiveness of the negative feedback loop. (B) Fluorescence micrographs of YFP signal and phase contrast micrographs from a 3T3 cell of a clone that contains a Flp-In, 3-kb version of the gene depicted in A. Images were taken every 6 min, but only images for every 10th exposure are presented. (C) Time-lapse trajectory of the quantified average fluorescence intensity in arbitrary units (a.u.) from the cell in B. The data presented were acquired in a 28-h window that encompasses a single cell division.