Elsevier

Gene

Volume 236, Issue 1, 5 August 1999, Pages 33-42
Gene

Monitoring dynamics of gene expression in yeast during stationary phase

https://doi.org/10.1016/S0378-1119(99)00264-4Get rights and content

Abstract

The commonly used genetic approaches in yeast are designed to identify defects in cell/colony growth. In order to identify genes which control molecular mechanisms during quiescence (‘stationary phase’), different tactics are required. We describe the development of a new genetic approach based on the previous observations that gene expression in quiescent Saccharomyces cerevisiae cells is largely repressed. For studying the mechanism controlling the repression of gene expression in stationary phase, we use UBI4-lacZ as a reporter gene. The product of this fusion gene was shown previously to encode an unstable protein in dividing cells. We show here that it is also unstable in stationary cells. We demonstrate that the relatively short half-life of this reporter protein can be utilized to monitor the dynamics of the repression of gene expression during stationary phase in liquid culture, using ACT1 or SSA3 promoters as the model promoters. By adapting a colony color test, we show that the reporter gene can also be used to monitor gene expression in quiescent colonies, thus serving as a tool to screen for defects in the regulation of this process during growth arrest. The utility of the approach was demonstrated by confirming the defects of top1Δ and bcy1Δ cells to appropriately express the ACT1p-UBI4-lacZ in stationary phase. The mutant colonies were easily discernible from wild-type colonies by our color test. Finally, using SSA3p-UBI4-lacZ as the reporter gene, we found that the 5′-untranslated region of SSA3 mRNA is sufficient to repress translation of the reporter mRNA after entry of the cells into stationary phase. The possibility that the short length of the SSA3 5′-untranslated region is a major determinant of the inefficient translation of SSA3p-UBI4-lacZ in stationary phase is discussed.

Introduction

In nature, unicellular organisms only sporadically experience conditions that support rapid growth. More frequently they have to cope with periods of starvation. Nevertheless, much more is known about the molecular mechanisms that govern growth than those playing a role during starvation. Some of the reasons for this gap are the lack of adequate reporters for studying the dynamics of gene expression, and the lack of genetic approaches for identifying genes involved in coping with starvation. The commonly used genetic approaches are usually aimed at identifying genes whose defects, directly or indirectly, affect growth. These include genes whose mutations cause either a simple or synthetic lethality, slow growth, a defect in cell division, or the inability to grow at a non-permissive temperature. The commonly accepted view is ‘growth oriented’ to the extent that essential genes are usually defined as those essential for growth. Genes whose functions are essential for maintaining viability during quiescence but not during growth (e.g., RPB4, BCY1) are usually regarded as ‘non-essential’ genes.

Most of our experience of stationary phase (SP) has been gained by studying cells in liquid cultures. Following the transition of Saccharomyces cerevisiae cells from logarithmic growth to starvation-induced SP, a decrease in the overall metabolism is detected. Low metabolism during starvation has probably evolved to preserve energy, thus enabling cells to survive long periods of starvation. Various molecular processes, such as transcription (Choder, 1991), translation (Boucherie, 1985, Fuge et al., 1994) and protein turnover (Fuge et al., 1994), show relatively low activity during SP. The mechanisms underlying the inhibition of gene expression in SP and the signaling pathways that control the repression processes are little understood. Inhibition of transcription is governed by a mechanism involving topoisomerase I, encoded by TOP1. Thus, cells lacking TOP1, which grow and transcribe their genome normally during logarithmic growth, are defective in the transcription repression that is normally induced in SP, and continue to transcribe genes relatively efficiently following entry into SP (Choder, 1991). In spite of the substantial decline in the global gene expression in SP, the cellular protein level is decreased only about two- to three-fold following the shift from logarithmic growth to SP, and remains relatively unchanged during long periods of time in later stages (Fuge et al., 1994, Werner-Washburne et al., 1996, Werner-Washburne et al., 1993; Choder, unpublished observation). It is reasonable to assume, therefore, that in addition to the low expression of genes, protein turnover is also substantially decreased in SP (Fuge et al., 1994). The detailed mechanisms underlying repression in transcription, translation and protein turnover remain to be determined.

Whereas the vast majority of the studies on the yeast molecular biology were performed with cells in liquid cultures, genetic approaches are based on features associated with colony growth and/or maturation. Until recently, it was not clear whether the quiescence state that colony cells enter at the end of their growth can serve as a good model for studying SP in liquid culture. We have recently examined the growth and maturation of yeast colonies (Meunier and Choder, 1999). We found that, if colonies are dense enough (≥200 colonies per 9 cm plate), an exponential growth phase is followed by a sharp transition into a quiescence phase in which practically all the cells cease to divide. During the colony growth phase, cells exhibit morphological and biochemical features that characterize growing cells in liquid culture. During the colony quiescence phase, cells exhibit morphological and biochemical features that characterize stationary cells in liquid culture. For example, changes in gene expression during the shift from growth to quiescence in colony cells are similar to those observed during the shift from growth to SP in liquid culture (Meunier and Choder, 1999). Thus, it seems that a dense population of colonies that naturally cease to divide due to the depletion of nutrients and/or as a result of signaling molecules that colonies secrete that induce their growth arrest (Palkova et al., 1997) can serve as a model system for studying control mechanisms operating during SP in liquid culture.

Escherichia coli lacZ is a popular reporter gene which has been extensively exploited to identify and delineate regulatory sequences in the 5′ non-coding regions of yeast genes (Guarente, 1983, Myers et al., 1896, Rose and Botstein, 1983). Nevertheless, this reporter has a serious limitation as its product, β-galactosidase (βgal), is very stable. Therefore, this reporter is not suitable for monitoring decrease in the tested activity (e.g., transcription, translation), as any reduction in its expression does not result in a parallel reduction in the level of its stable product. lacZ has also been used as a reporter gene in many genetic screens. In most of these screens, the chromogenic βgal substrate, 5-bromo-4-chloro-indolyl-beta-d-galacto-pyranoside (Xgal), is added to the plates and the color develops during the colony growth (Guarente, 1983) and thus reflects the expression of the reporter gene during growth. Following entry of the colony cells into SP, the color remains largely unchanged due to the stability of both the colored compound and βgal. In some other screens, Xgal is added to mature colonies (Duttweiler, 1996, Vojtek et al., 1993). In these cases, however, it is not possible to distinguish between the true expression status of the reporter gene at the time of assay and its expression that had occurred before the assay was perfomed.

Here we describe an approach for analyzing repression in gene expression which occurs in SP. We show that the fusion genes UBI4-lacZ, containing an isoleucine codon at the junction between the two genes and previously found to give rise to an unstable βgal (Bachmair et al., 1986), is also sufficiently unstable in SP. We demonstrate that this reporter is suitable for monitoring the dynamics of gene expression during SP. We used the reporter to develop a method for identifying mutants defective in the control of gene expression during SP.

Section snippets

ACT1p-lacZ

ACT1 promoter was amplified by PCR (30 cycles of 94°C for 20 s, 50°C for 20 s, 72°C for 30 s, followed by 3 min at 72°C), using OMC57 (5′-CGGGATCCAAAACCCTTAAAAACATATG-3′) and OMC43 (5′- AAACTGCAGAGCATTGTTAATTCAGTAAATTTTC-3′) as the forward and reverse primers, respectively. The reverse primer includes the translation start site (bold). BamHI and PstI sites (underlined) were introduced into the forward and reverse primers, respectively. The promoter fragment, bounded by the positions +1 to −472

ACT1 transcriptional repression can be monitored accurately by the ACT1p-UBI4-lacZ reporter gene

ACT1, encoding actin, is a representative gene whose transcription is typically repressed following the shift from logarithmic growth to SP (Choder, 1991, Choder and Young, 1993, Paz et al., 1999). To monitor the kinetics of ACT1 transcriptional repression, we searched for a reliable reporter gene whose product has a short half-life. To this end, we took advantage of the existence of lacZ fusions encoding unstable βgal molecules. Bachmair et al. (1986) have demonstrated that the half-life of

Acknowledgements

We thank A. Varshavsky for pUB23, G. Simchen and R. Sternglanz for yeast strains, and A. Krauskoff for critically reading the manuscript. This work was supported by the Israel Science Foundation to M.C., and by a post-doctoral fellowship supported by the French Embassy in Israel to J.R.M.

References (33)

  • A. Bachmair et al.

    In vivo half-life of a protein is a function of its amino-terminal residue

    Science

    (1986)
  • W.R. Boorstein et al.

    Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae

    Mol. Cell. Biol.

    (1990)
  • H. Boucherie

    Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae

    J. Bacteriol.

    (1985)
  • J.F. Cannon et al.

    Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase

    Mol. Cell. Biol.

    (1987)
  • M. Choder

    A general topoisomerase I-dependent transcriptional repression in the stationary phase in yeast

    Genes Dev.

    (1991)
  • M. Choder

    A growth rate-limiting process in the last growth phase of the yeast life cycle involves RPB4, a subunit of RNA polymerase II

    J. Bacteriol.

    (1993)
  • Cited by (0)

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    Present address: Loreal, 1, avenue E. Schueller, F-93600 Aulnay Sous Bois, France.

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