Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

The endopolyphosphatase gene: Essential in Saccharomyces cerevisiae

Endopolyphosphatases (Ppn1) from yeast and animal cells hydrolyze inorganic polyphosphate (poly P) chains of many hundreds of phosphate residues into shorter lengths. The limit digest consists predominantly of chains of 60 (P60) and 3 (P3) Pi residues. Ppn1 of Saccharomyces cerevisiae, a homodimer of 35-kDa subunits (about 352-aa) is of vacuolar origin and requires the protease activation of a 75-kDa (674-aa) precursor polypeptide. The Ppn1 gene (PPN1) now has been cloned, sequenced, overexpressed, and deleted. That PPN1 encodes Ppn1 was verified by a 25-fold increase in Ppn1 when overexpressed under a GAL promoter and also by several peptide sequences that match exactly with sequences in a yeast genome ORF, the mutation of which abolishes Ppn1 activity. Null mutants in Ppn1 accumulate long-chain poly P and are defective in growth in minimal media. A double mutant of PPN1 and PPX1 (the gene encoding a potent exopolyphosphatase) loses viability rapidly in stationary phase. Whether this loss is a result of the excess of long-chain poly P or to the lack of shorter chains (i.e., poly P60 and P3) is unknown. Overexpression of the processed form of Ppn1 should provide a unique and powerful reagent to analyze poly P when the chain termini are unavailable to the actions of polyPase and poly P kinase.

Cell cycling and patterned cell proliferation in the Drosophila wing during metamorphosis.

In metamorphosing wing discs, progression through the cell cycle takes place, as in larval discs, in nonclonally derived clusters of cells synchronized in the same cell cycle stage. Contrary to early discs, there are temporal and spatial heterogeneities in cell proliferation associated with wing margin, vein, intervein, and middle intervein territories. Within these territories, there are no indications of a wave progression of the cell cycle. Mitotic orientations are, as in early discs, at random but there is a preferential allocation of postmitotic cells along the proximodistal axis, thus explaining the elongated shape of the resulting clones along this axis. Shapes of clones in mature discs and in evaginated wings are similar, thus excluding major morphogenetic movements during evagination. After the proliferative period, all the cells are arrested in G1 phase. The final number of cells of the wing is fixed independently of experimental perturbations that alter the cell division schedule. These results are discussed in the context of a model of wing morphogenesis.

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.

G1 arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637.

The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.

Cell cycle-dependent modulation of telomerase activity in tumor cells.

Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. We tested this hypothesis by arresting human tumor cell lines at different stages of the cell cycle. Induction of quiescence by serum deprivation did not affect telomerase activity. Cells arrested at the G1/S phase of the cell cycle showed similar levels of telomerase to asynchronous cultures; progression through the S phase was associated with increased telomerase activity. The highest level of telomerase activity was detected in S-phase cells. In contrast, cells arrested at G2/M phase of the cell cycle were almost devoid of telomerase activity. Diverse cell cycle blockers, including transforming growth factor beta1 and cytotoxic agents, also caused inhibition of telomerase activity. These results establish a direct link between telomerase activity and progression through the cell cycle.

Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.

The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

Identification of two flavivirus-like genomes in the GB hepatitis agent.

A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.