PDA1mRNA: a standard for quantitation of mRNA inSaccharomyces cerevisiaesuperior toACT1mRNA

The expression of many genes is regulated at the transcriptional level. Information about transcriptional regulation of a gene may provide insight into its function and mechanism by which transcription is regulated. A straightforward technique to analyze the transcription level of a gene is to determine its mRNA concentration in cells grown under the conditions of interest by Northern analysis (1). This technique usually involves separation of RNA molecules by gel electrophoresis, blotting onto membrane filters and subsequent hybridization to a suitable probe. However, due to practical problems, the amount of mRNA detected is often not representative for the actual mRNA concentration in the cell. This problem is partially overcome by determining the amount of the mRNA of interest relative to an internal standard, usually mRNA from a gene that is constitutively transcribed. Actin mRNA encoded by the ACT1 gene in S.cerevisiae is commonly used as such a standard (2). ACT1 is constitutively transcribed during most conditions tested. We found however, that the concentration of ACT1 mRNA decreased when cultures of S.cerevisiae reached or proceeded into stationary phase (Fig. 1). Hence, under these conditions the level of ACT1 mRNA is not constant thus jeopardizing its function as an internal standard Since actin is a component of the cytoskeleton which is mainly required during cell division, it might be questioned if transcription ACT1 is constitutive at other than the logarithmic growth stage. This could be a serious hindrance in experiments with batch cultures where the growth stage is usually inadequately defined or when samples from stationary phase cultures have to be analyzed We have investigated the transcriptional regulation of the PDA! gene which encodes the E la subunit of the pyruvate dehydrogenase complex from S.cerevisiae (3). PDA1 mRNA of about 1300 nt is present in approximately one third of the concentration of ACT1 mRNA in the exponential growth phase as determined by dot-blot hybridizations and easily detected in total RNA samples from S.cerevisiae (4). The level of PDAl mRNA is almost constant under all conditions tested. These include various growth stages in batch cultures on glucose. We also compared mRNA levels during growth on glucose, galactose, pyruvate and ethanol in rich media in batch cultures and during growth on glucose and ethanol and under anaerobic conditions in continuous cultures in defined media. In all cases the same amount of PDA1 mRNA relative to the total amount of RNA is found (5). In contrast to ACT], 1 2 3 4 5 6 7 8 9 10