We developed systems to rapidly express any yeast gene or to specifically degrade any protein, each with minimal untargeted disturbance of cell physiology. We illustrate applications of these new tools for elucidating the architecture and dynamics of genetic regulatory networks.

Despite rapid progress in characterizing the yeast metabolic cycle, its connection to the cell division cycle (CDC) has remained unclear. We discovered that a prototrophic batch culture of budding yeast, growing in a phosphate-limited ethanol medium, synchronizes spontaneously and goes through multiple metabolic cycles, whereas the fraction of cells in the G1/G0 phase of the CDC increases monotonically from 90 to 99%. This demonstrates that metabolic cycling does not require cell division cycling and that metabolic synchrony does not require carbon-source limitation. More than 3,000 genes, including most genes annotated to the CDC, were expressed periodically in our batch culture, albeit a mere 10% of the cells divided asynchronously; only a smaller subset of CDC genes correlated with cell division. These results suggest that the yeast metabolic cycle reflects a growth cycle during G1/G0 and explains our previous puzzling observation that genes annotated to the CDC increase in expression at slow growth.T wo kinds of periodic behavior have been characterized in slowly growing yeast cultures. The first, the classical cell division cycle (CDC), consists of four phases (G0/G1, S, G2, and M) that are easily distinguished by morphological criteria. When the growth rate of budding yeast is slowed by mutations or chemicals inhibiting growth, the duration of the G1/G0 phase increases relative to the durations of the S, G2, and M phases (1). Recently, we confirmed and quantified this CDC trend (Fig. 1A) in chemostat cultures whose steady-state growth rate was controlled by limiting natural nutrients (2, 3). The second kind of cycle, the yeast metabolic cycle (YMC), was first observed more than four decades ago (4) as periodic oscillations in the oxygen consumption of continuous, glucose-limited cultures growing in a chemostat. Like the CDC, the YMC can be divided phenomenologically into two phases: the low oxygen consumption phase (LOC), when the amount of oxygen in the medium is high because the cells consume little oxygen, and the high oxygen consumption phase (HOC), when the reverse holds (SI Appendix). We also reported previously (3) similar growth-rate changes in the relative durations of the phases of the YMC (Fig. 1B). As the growth rate increases, the relative duration of the LOC decreases whereas the relative duration of the HOC increases (Fig. 1B), similarly to the analogous changes in the CDC.These changes in the relative durations of the phases affect the composition of asynchronous cultures, because single cells from asynchronous cultures cycle metabolically (5, 6) and thus the fraction of cells in a particular phase is proportional to the duration of that phase relative to the entire cycle period. The increase in the relative duration of a phase results in the increase in the fraction of cells in that phase, and thus an increase in the population-average expression levels of genes peaking during that phase. Consider, for example, a ribosomal gene that peaks in expression during the HOC phase; at slow growt...

Purpose: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients.Experimental Design: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response.Results: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases.Conclusions: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis.

Supplementary Table 1 from Prospective Gene Signature Study Using microRNA to Identify the Tissue of Origin in Patients with Carcinoma of Unknown Primary