Martí Aldea

A SET OF VECTORS WITH A TETRACYCLINE-REGULATABLE PROMOTER SYSTEM FOR MODULATED GENE EXPRESSION IN SACCHAROMYCES CEREVISIAE

A set of Saccharomyces cerevisiae expression vectors has been developed in which transcription is driven by a hybrid tetO‐CYC1 promoter through the action of a tetR‐VP16 (tTA) activator. Expression from the promoter is regulated by tetracycline or derivatives. Various modalities of promoter and activator are used in order to achieve different levels of maximal expression. In the presence of antibiotic in the growth medium at concentrations that do not affect cell growth, expression from the tetO promoter is negligible, and upon antibiotic removal induction ratios of up to 1000‐fold are observed with a lacZ reporter system. With the strongest system, overexpression levels comparable with those observed with GAL1‐driven promoters are reached. For each particular promoter/tTA combination, expression can be modulated by changing the tetracycline concentration in the growth medium. These vectors may be useful for the study of the function of essential genes in yeast, as well as for phenotypic analysis of genes in overexpression conditions, without restrictions imposed by growth medium composition. © 1997 by John Wiley & Sons, Ltd.

Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters.

The cell division ftsQAZ cluster and the ftsZ‐dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their −10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

The Cln3 cyclin is down-regulated by translational repression and degradation during the G1 arrest caused by nitrogen deprivation in budding yeast

Nutrients are among the most important trophic factors in all organisms. When deprived of essential nutrients, yeast cells use accumulated reserves to complete the current cycle and arrest in the following G1 phase. We show here that the Cln3 cyclin, which has a key role in the timely activation of SBF (Swi4–Swi6)‐ and MBF (Mbp1–Swi6)‐dependent promoters in late G1, is down‐regulated rapidly at a post‐transcriptional level in cells deprived of the nitrogen source. In addition to the fact that Cln3 is degraded faster by ubiquitin‐dependent mechanisms, we have found that translation of the CLN3 mRNA is repressed ∼8‐fold under nitrogen deprivation conditions. As a consequence, both SBF‐ and MBF‐dependent expression is strongly down‐regulated. Mainly because of their transcriptional dependence on SBF, and perhaps with the contribution of similar post‐transcriptional mechanisms to those found for Cln3, the G1 cyclins Cln1 and 2 become undetectable in starved cells. The complete loss of Cln cyclins and the sustained presence of the Clb–cyclin kinase inhibitor Sic1 in starved cells may provide the molecular basis for the G1 arrest caused by nitrogen deprivation.

Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity

Control of cell cycle progression by stress‐activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7‐docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1–Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.

Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene.

The bolA gene, which is involved in the morphogenetic pathways of Escherichia coli, was sequenced and two potential promoters were identified. Expression from promoter P1, proximal to the bolA structural gene is specifically induced during the transition to the stationary phase of growth. This promoter contains an unusual‐‐10 region (CGGCTAGTA), which defines a new class of E. coli promoters necessary for the dramatic increase in the rate of synthesis of a large set of proteins during the cessation of logarithmic growth. This conclusion was confirmed by identifying two additional E. coli promoters and one plasmid promoter, which also were induced during the transition to the stationary phase of growth. Analysis of proteins produced during the exponential and stationary phases of growth in a bolA null mutant suggest a possible role for the BolA protein in the induction of the expression of penicillin‐binding protein 6 (PBP6) in the transition to the stationary phase. Supporting this hypothesis is the presence of a putative DNA‐binding domain within the bolA coding sequence.

Transcription of ftsZ oscillates during the cell cycle of Escherichia coli

The FtsZ protein is a key element controlling cell division in Escherichia coli. A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells. The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates. This cell cycle dependency is specifically due to the two proximal ftsZ promoters. A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer. In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate‐limiting role of this protein in cell division. Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated. This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles.

The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

High‐affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase‐FTR1 permease complex. Expression of the high‐affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non‐induced levels of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi‐Iwai et al., EMBO J. 14: 1231–1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high‐affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest at the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high‐affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirements for cell growth.

Basic helix-loop-helix proteins bind to TrkB and p21(Cip1) promoters linking differentiation and cell cycle arrest in neuroblastoma cells.

ABSTRACT Differentiation of precursor into specialized cells involves an increasing restriction in proliferative capacity, culminating in cell cycle exit. In this report we used a human neuroblastoma cell line to study the molecular mechanisms that coordinate cell cycle arrest and neuronal differentiation. Exposure to retinoic acid (RA), a differentiation agent in many cell types, causes an upregulation of neurotrophin receptor TrkB and the cyclin kinase inhibitor p21Cip1 at a transcriptional level. Full transcriptional activation of these two genes requires canonical E-box sequences found in the TrkB and p21Cip1 promoters. As reported for other E-box-regulated promoters, ectopic expression of E47 and E12 basic helix-loop-helix (bHLH) proteins enhances RA-dependent expression of TrkB and p21Cip1 , whereas the inhibitory HLH Id2 exerts opposite effects. In addition, ectopic expression of E47 and NeuroD, a neuronal bHLH protein, is able to activate TrkB transcription in the absence of RA. More importantly, we show that E47 and NeuroD proteins bind the TrkB and p21Cip1 promoter sequences in vivo. Since they establish a direct transcriptional link between a cell cycle inhibitor, p21Cip1 , and a neurotrophic receptor, TrkB, bHLH proteins would play an important role in coordinating key events of cell cycle arrest and neuronal differentiation.

G1 cyclins block the Ime1 pathway to make mitosis and meiosis incompatible in budding yeast.

Diploid yeast cells switch from mitosis to meiosis when starved of essential nutrients. While G1 cyclins play a key role in initiating the mitotic cell cycle, entry into meiosis depends on Ime1, a transcriptional activator regulated by both nutritional and cell‐type signals. We show here that G1 cyclins downregulate IME1 transcription and prevent the accumulation of the Ime1 protein within the nucleus, which results in repression of early‐meiotic gene expression. As G1‐cyclin deficient cells do not require nutrient starvation to undergo meiosis, G1 cyclin would exert its role by transmitting essential nutritional signals to Ime1 function. The existence of a negative cross‐talk mechanism between mitosis and meiosis may help explain why these two developmental options are incompatible in budding yeast.

Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae.

Moderate hyperosmotic stress on Saccharomyces cerevisiae cells produces a temporary delay at the G1 stage of the cell cycle. This is accompanied by transitory downregulation of CLN1, CLN2 and CLB5 transcript levels, although not of CLN3, which codes for the most upstream activator of the G1/S transition. Osmotic shock to cells synchronized in early G1, when Cln3 is the only cyclin present, causes a delay in cell cycle resumption. This points to Cln3 as being a key cell cycle target for osmotic stress. We have observed that osmotic shock causes downregulation of the kinase activity of Cln3–Cdc28 complexes. This is concomitant with a temporary accumulation of Cln3 protein as a result of increased stability. The effects of the osmotic stress in G1 are not suppressed in CLN3‐1 cells with increased kinase activity, as the Cln3–Cdc28 activity in this mutant is still affected by the shock. Although Hog1 is not required for the observed cell cycle arrest in hyperosmotic conditions, it is necessary to resume the cell cycle at KCl concentrations higher than 0.4 M.