## 4 Discussion  
Since its inception, continuous culture has been used in microevolution studies [27]. More recently, the availability of high-throughput techniques has allowed us to study microevolution by following the adaptive changes of the transcriptomes (e.g., [7; 15; 28]), proteomes (e.g., [29; 30]), metabolomics (e.g., [31]), or genomes (e.g., [9; 10; 32]) of cell populations subject to continuous culture. It has been assumed that transcriptomic, proteomic, and metabolomic alterations reflect changes in the genomes of the microorganisms under study. In many cases, such an association has been directly proven (e.g., [8; 12]) confirming the generally accepted tenet that DNA is a carrier of heritable traits. The observation that became the topic of this work attracted our attention because it implied something different than is commonly believed. Our results revealed that slow, adaptive transcriptome changes in budding yeast cells subjected to microevolution may not always be the consequence of adaptive mutations of their genomes. The expression of over 400 genes was changed between the time point after 40 generations of growth (the beginning of the steady-state phase) and the time point after \begin{table}
\begin{tabular}{l l l l l l l} \hline \hline Wtm2p & 0.1 & 0 & 17 & 2.98 & 5.1 & Transcriptional modulator; involved in regulation of meiosis, silencing, and expression of RNR genes; involved in response to replication stress \\ \hline Rpi1p & 0.1 & 0 & 17 & 2.89 & 4.8 & Transcription factor, allelic differences between S28 SC and Sigma1278b; mediates fermentation stress tolerance by modulating cell wall integrity \\ \hline Com2p & 0.09 & 0.0001 & 16 & 2.94 & 4.3 & Transcription factor; _COM2_ transcription is regulated by Haa1p, Sok2p and Zap1p transcriptional activators; C. albicans homolog (MNLI) plays a role in adaptation to stress \\ \hline Sdd4p & 0.1 & 0.0002 & 12 & 2.93 & 3.5 & Putative transcription factor, induced in response to the DNA-damaging agent MMS \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Cont. 240 generations of growth (the adapted cell populations). As there were no common mutations that could be associated with these changes, they must have been encoded in some other way. The stability of continuous culture conditions in microevolution experiments is usually assured by growth limitation; the medium contains a limiting concentration of one of the nutrients, most often the carbon or nitrogen source [1]. Limitation assures the control of doubling time but at the same time results in starvation stress, which we intended to avoid in our experimental design. The dilution rate was kept at 0.17-0.18 \(\times\) h\({}^{-1}\), allowing the cell population to be maintained at a constant density equivalent to the late exponential growth phase. Moreover, to include as many genes as possible in our analysis, we used mineral, YNB-based growth medium with glycerol as a respiratory carbon source (see Materials and Methods). As documented by Figures 1 and 2, and Figure S3, the modulation of the _S. cerevisiae_ cell transcriptome in the course of continuous culture involves groups of genes belonging to distinct functional categories. Evidently, the observed changes in expression levels are specific and signify a shift in the biosynthesis/catabolism balance towards catabolism and towards diminishing the stress response. However, sometimes the cellular processes underpinning these changes are unclear. The observed transcriptome remodeling is interesting in several aspects. First, one might expect a change in gene expression in response to some variations in cell internal or external stimuli of any kind. However, we see the changes despite the absence of such variations. Moreover, changes in gene expression in response to stimuli are fast, measured in minutes or even in seconds, whereas the changes observed by us were slow, measured in days. To our knowledge, this phenomenon has not previously been reported in the literature.