Beginning in the 1990s, Kuriyama, Klevecz, Murray and colleagues pioneered the study of short-period, 40-min oscillations observed during continuous culture of an industrial fermentation strain of S. cerevisiae
]. By sampling populations of cycling cells at frequent intervals, low-amplitude, genome-wide fluctuations in transcription and numerous metabolic parameters were detected during these short-period oscillations [7• •
]. Periodic changes in gene expression were subsequently observed during the longer-period, 4–5 h oscillations [8• •
]. Importantly, both the short-period (~40 min) and long-period (~4–5 h) cycles revealed that the majority of yeast genes appeared to be cyclically regulated as a function of the oscillations in oxygen consumption [7• •
]. However, there was little correlation between the two datasets with respect to the phases during which particular classes of transcripts peaked [12
]. This suggested that the long-period and short-period cycles are quite different, at least by the criteria of periodic transcription and gene expression.
The short-period cycles suggested that the temporal separation between the oxidative (oxygen-consuming) and reductive phases is propagated through the yeast transcriptome [7• •
]. The temporal segregation of biological processes was more apparent in the long-period cycles, where over half the yeast genome showed high-amplitude, periodic expression, with different genes being expressed at their highest levels at completely different times during these oscillations [8• •
]. Furthermore, the genes that were highly overrepresented in the set of periodic genes were predominantly involved in metabolism and protein synthesis, with gene products that localize to the mitochondria also significantly overrepresented [8• •
]. These gene expression studies from the long-period cycles (hereon referred to as the Yeast Metabolic Cycle, or YMC) also suggested why the genes that peak in the oxygen-consuming phase (ribosomal proteins, translation initiation factors, genes involved in amino acid biosynthesis, etc.) may be significantly upregulated in this phase: these processes are energetically demanding, and their expression correlates perfectly with a burst of mitochondrial oxidative phosphorylation [8• •
The data from these studies suggested an overall logic underlying the long-period YMC, where cellular processes are not just separated by subcellular spatial compartmentalization of metabolic enzymes, but are also tightly regulated in time [8• •
]. The oscillating transcripts of the YMC fall within three distinct, temporally separated phases organized about the cycles of oxygen consumption [8• •
]. These were designated as the Oxidative phase (OX), the Reductive-Building phase (RB), and the Reductive-Charging phase (RC) [8• •
]. In the OX phase, cells rapidly consume molecular oxygen in the form of a burst of mitochondrial respiration. This phase is characterized by the sudden transcriptional upregulation of genes involved in ribosomal biogenesis, the translational machinery, amino acid biosynthesis, sulfur metabolism and numerous other genes involved in growth. In the RB phase, cells enter S phase and complete the cell division process. This activity correlates with an increase in transcripts of genes involved in the cellular building machinery, including DNA replication, spindle pole components, and other genes involved in the cell cycle. The majority of gene products encoding mitochondrial proteins also peak in this phase. In the RC phase, transcripts of many genes associated with starvation, stress, and cell survival increase before the cells prepare to enter another OX phase. Thus, the OX phase can be likened to ‘growth’, the RB phase to ‘division’, and the RC phase to ‘survival/quiescence’. Cells continuously alternate among these three metabolic phases, each associated with particular transcriptional programs, during continuous growth. One of the most interesting and significant examples of temporal compartmentalization is the finding that cell division is gated to the RB phase during which the rate of oxygen consumption decreases [8• •
]. Such gating of cell division was also reportedly observed during short-period 40 min oscillations, despite the lack of a corresponding upregulation of cell cycle genes [7• •
A separate series of comprehensive gene expression studies was performed on yeast cells grown in various nutrient-limited chemostat cultures under changing growth rates [14
]. These studies aimed to determine groups of genes that are either positively or negatively correlated with growth rate. Strikingly, there was a substantial overlap in the genes that are positively correlated with growth rate and those that peak during the OX, growth, phase of the long-period YMC [16• •
]. Moreover, many genes that were negatively correlated with increasing growth rate in this study peaked during the quiescent-like RC phase of the YMC [16• •
]. Thus, through two independent experimental means, similar groups of genes were found to be co-regulated with respect to growth and cellular metabolic state. Collectively, these data predict that cells might undergo such metabolic cycles outside of a steady-state glucose-limited growth environment, and that the chemostat merely facilitates the establishment of a unique synchrony that enables the macroscopic observation of these oscillations and their systematic dissection. illustrates the organization of the different phases of the long-period YMC, with different functionally related biological processes temporally separated in these phases.
Figure 1 The Yeast Metabolic Cycle (YMC). A schematic illustration of the long-period cycles of oxygen consumption during continuous growth that have been termed the Yeast Metabolic Cycle. Cell growth, which is accompanied by the increased expression of growth (more ...)
The periodic gene expression data collected over three consecutive long-period metabolic cycles was used to investigate the tightly controlled sequence of transcriptional events that span the budding yeast cell cycle [17•
]. Eukaryotic cell division is a highly complex and precisely orchestrated process involving specific gene transcription, translation, protein localization and turnover. Using deconvolution of the unique profile shapes of cell cycle genes, the peaks of expression of cell cycle regulated genes were timed at an unprecedented resolution of ~2–3 min [17•
]. Such precise timing was perhaps enabled by the slower growth of cells in the YMC (compared to cells growing in high-glucose stationary cultures) as well as the extraordinary synchrony of cells undergoing such metabolic oscillations, which often persists for weeks. These data revealed a previously unanticipated wave of gene transcription around the G1 to S transition of the cell cycle, and a just-in-time transcription of genes involved in the initiation of DNA replication [17•
]. These studies suggest that the YMC may be an excellent system to investigate the temporal sequence of events that comprise the budding yeast cell cycle program because it imposes an unusually synchronous progression of cells through the cell cycle.