Thymidylate synthase gene of herpesvirus ateles.

The putative thymidylate synthase (TS) gene of herpesvirus ateles, a T-lymphotropic tumor virus of New World primates, has a single large open reading frame encoding a polypeptide of 32.9 kilodaltons. The gene is transcribed into an unspliced 2.4-kilobase mRNA that is abundantly expressed late in virus replication. The AT-rich 5' untranslated leader sequence of TS mRNA in herpesvirus ateles-infected cells is remarkable in length (1,184 nucleotides), containing 29 minicistrons; this may indicate a role in translation regulation.     

Diurnal Lhc gene expression is present in many but not all species of the plant kingdom

The diurnal and circadian expression of light-harvesting genes (Lhc) is well documented for many plant species of the Angiospermae division. Here we present the diurnal mRNA levels of species of the Gymnospermae, Pteridophyta, Bryophyta and Phycophyta divisions. Except for four Coniferophytina species, diurnal Lhc mRNA accumulation is detected in fern, moss and algae, supporting the idea that the concept of circadian clock-controlled gene expression is an ancient process. Possible reasons why plants need the circadian clock control mechanism are discussed.Dedicated to Dr H. W. Heldt on the occasion of his 60th birthday.     

Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

Phosphatidylcholine synthesis in platelets is stimulated by diacylglycerol but not by phorbol ester

The biosynthesis of phosphatidylcholine (PC) in platelets was followed by measuring the incorporation of 32Pi. Incorporation into PC was stimulated by treatment with Clostridium perfringens phospholipase C or with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. However, neither the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate or thrombin stimulated 32Pi incorporation into PC. We conclude that phorbol ester does not stimulate the hydrolysis of PC to diacylglycerol in platelets.     

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.     

Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

Epidermal melanocytes (MC) synthesise melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melano-genesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E₁, E₂, D₂, F_2α, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradliated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P<0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanognesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C.     

Nitric oxide differentially regulates the gene expression of caspase genes but not some autophagic genes.

Nitric oxide (NO) is fundamentally important molecule which produces a wide range of cellular effects with the most poorly understood one being alteration in the sensitivity to cell death. The objective of this study was to test the hypothesis that NO would differentially affect caspase or autophagy gene expression in a manner that might account for the disparate actions of NO to either enhance or protect against cell death. Neonatal mouse cardiomyocytes in culture were treated with the NO donor SIN-1 (3-morpholinosydnonimine hydrochloride) for up to 20 h. RNA was collected, after either 2, 4 or 20 h, labeled and hybridized to cDNA microarray slides The concentration of SIN-1 was selected after concentration response studies of SIN-1 on cell viability, assessed by the MTT assay. The cDNA microarrays were used that contained the mouse genome version 2.0 with genes for enzymes crucial to apoptosis, namely caspases-1, -2, -3, -6, -7, -8, -9, -11, -12 and -14, as well as for enzymes crucial to autophagy namely beclin-1, Apg5l and Apg12l. Considering the entire 20 h period, treatment with SIN-1 was associated with significant (p<0.05) changes in five caspases. In contrast, there were no changes in the three separate genes involved in autophagy. Time course experiments showed a consistent increase in caspase-8, -11 and -14, and a consistent decrease in caspase-1 and -6. Notably, caspase-1 showed a persistent and marked reduction so that after 20 h of treatment, caspase-1 was dramatically reduced, almost ten fold, to 0.14+/-0.11 of control. In conclusion, these results suggest that: (i) NO regulates the expression of genes involved in apoptotic but not some involved in autophagic cell death; (ii) the more recently discovered caspase-14 may have a role in the heart; (iii) NO-induced alteration of different caspases may explain the ability of NO to either enhance or protect against cell death depending on whether associated factors involve, respectively caspases-8, -11, and -14 or -1 and -6.     

Uptake of thallous ions by mitochondria is stimulated by nonactin but not by respiration alone

Nonrespiring rat-liver mitochondria swell in media containing high concentrations of thallous nitrate, indicating passive penetration of Tl+. This swelling could be further stimulated by 10 nM or more nonactin while even 1 microM valinomycin was without effect. Nonactin was also much more potent than valinomycin in stimulating swelling of respiring mitochondria in the presence of thallous acetate. It is evident that nonactin acts as a potent ionophore of Tl+ able to promote both the passive and energized uptake of Tl+ in mitochondria. The distribution of Tl+, present in trace concentrations below 1 mM, was measured during energisation by respiration both in the presence and absence of ionophores. Respiration induced net uptake of Tl+ only in the presence of ionophores, though Tl+ as a permeant cation was expected to sense respiration-induced changes in the membrane potential. The data may be interpreted as indicating that no transmembrane potential is formed upon energisation, but localized fields, which are able to interact with the lipophilic ionophore complexes of Tl+, but not with the hydrophilic cation Tl+. This interpretation is valid only if thermodynamic equilibrium has been reached.     

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.     

Control of endogenous gene expression timing by introns

Comparison of gene expression from transgenes and endogenous genes with or without introns reveals a time-regulating role of introns in natural biological systems.