Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires σ factor that participates in both promoter recognition and DNA melting. RNAP lacking σ (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various σ factors. Using bubble templates containing the σD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of σ. This supports a model in which the UP element acts primarily through the α subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: σD restricts initiation to sites 8–9 nucleotides downstream of the conserved −10 element. Remarkably, σA also has a dramatic effect on start-site selection even though the σA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the σA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved −10 hexamer recognized by σA. Thus, σA appears to recognize the −10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, σ also localizes the transcription start site.

Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2–Cdc10, Controlling the Cell Cycle “Start”

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2+ gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2–Cdc10 complex. To identify the rep2+ function and molecularly define its domain organization, we reconstituted the Res2–Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2–Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.

PromEC: An updated database of Escherichia coli mRNA promoters with experimentally identified transcriptional start sites

PromEC is an updated compilation of Escherichia coli mRNA promoter sequences. It includes documentation on the location of experimentally identified mRNA transcriptional start sites on the E.coli chromosome, as well as the actual sequences in the promoter region. The database was updated as of July 2000 and includes 472 entries. PromEC is accessible at http://bioinfo.md.huji.ac.il/marg/promec

The Sda1 Protein Is Required for Passage through Start

We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis. Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo. Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size. Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start. Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins. The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start. Taken together, these findings reveal that Sda1 plays a critical role in G1 events. In addition, these findings suggest that Nap1 is likely to function during G1. Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2. In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton. Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.

The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start.

In yeast, commitment to cell division (Start) is catalyzed by activation of the Cdc28 protein kinase in late G1 phase by the Cln1, Cln2, and Cln3 G1 cyclins. The Clns are essential, rate-limiting activators of Start because cells lacking Cln function (referred to as cln-) arrest at Start and because CLN dosage modulates the timing of Start. At or shortly after Start, the development of B-type cyclin Clb-Cdc28 kinase activity and initiation of DNA replication requires the destruction of p40SIC1, a specific inhibitor of the Clb-Cdc28 kinases. I report here that cln cells are rendered viable by deletion of SIC1. Conversely, in cln1 cln2 cells, which have low CLN activity, modest increases in SIC1 gene dosage cause inviability. Deletion of SIC1 does not cause a general bypass of Start since (cln-)sic1 cells remain sensitive to mating pheromone-induced arrest. Far1, a pheromone-activated inhibitor of Cln-Cdc28 kinases, is dispensable for arrest of (cln-)sic1 cells by pheromone, implying the existence of an alternate Far1-independent arrest pathway. These observations define a pheromone-sensitive activity able to catalyze Start only in the absence of p40SIC1. The existence of this activity means that the B-type cyclin inhibitor p40SIC1 imposes the requirement for Cln function at Start.

How photons start vision.

Recent studies have elucidated how the absorption of a photon in a rod or cone cell leads to the generation of the amplified neural signal that is transmitted to higher-order visual neurons. Photoexcited visual pigment activates the GTP-binding protein transducin, which in turn stimulates cGMP phosphodiesterase. This enzyme hydrolyzes cGMP, allowing cGMP-gated cationic channels in the surface membrane to close, hyperpolarize the cell, and modulate transmitter release at the synaptic terminal. The kinetics of reactions in the cGMP cascade limit the temporal resolution of the visual system as a whole, while statistical fluctuations in the reactions limit the reliability of detection of dim light. Much interest now focuses on the processes that terminate the light response and dynamically regulate amplification in the cascade, causing the single photon response to be reproducible and allowing the cell to adapt in background light. A light-induced fall in the internal free Ca2+ concentration coordinates negative feedback control of amplification. The fall in Ca2+ stimulates resynthesis of cGMP, antagonizes rhodopsin's catalytic activity, and increases the affinity of the light-regulated cationic channel for cGMP. We are using physiological methods to study the molecular mechanisms that terminate the flash response and mediate adaptation. One approach is to observe transduction in truncated, dialyzed photoreceptor cells whose internal Ca2+ and nucleotide concentrations are under experimental control and to which exogenous proteins can be added. Another approach is to observe transduction in transgenic mouse rods in which specific proteins within the cascade are altered or deleted.

Dual transcriptional control by Ear3/COUP: negative regulation through the DR1 direct repeat and positive regulation through a sequence downstream of the transcriptional start site of the mouse mammary tumor virus promoter.

Ear3/COUP is an orphan member of the steroid/thyroid hormone receptor superfamily of transcription factors and binds most tightly to a direct repeat of AGGTCA with 1 nucleotide in between (DR1). Ear3/COUP also binds with a similar affinity to the palindromic thyroid hormone response element (TRE). This binding preference of Ear3/COUP is same as that of the retinoid X receptor (RXR), which is another member of the superfamily. In the present study, we identified a sequence responsible for Ear3/COUP-mediated transactivation in the region downstream of the transcription start site of the mouse mammary tumor virus promoter. This cis-acting sequence was unresponsive to RXR. When the DR1 or TRE sequence was added upstream of the promoter, transactivation by Ear3/COUP was completely abolished, whereas RXR enhanced transcription from the promoter. The mode of action of Ear3/COUP could be utilized to control complex gene expressions in morphogenesis, homeostasis, and development.