Identification and mapping of Epstein-Barr virus early antigens and demonstration of a viral gene activator that functions in trans.

The BamHI M DNA fragment of the Epstein-Barr virus (EBV) genome was inserted in two orientations into a simian virus 40-based expression vector, and the EBV-specific proteins produced in COS-7 monkey cells were examined. In one orientation, termed BamHI-M rightward reading frame 1 (BMRF1), a set of phosphoproteins ranging in size from 47,000 to 54,000 daltons was synthesized. These proteins reacted with monoclonal and polyclonal antisera, defining them as components of the EBV early antigen diffuse set of proteins (EA-D). The BamHI M DNA fragment in the opposite orientation, termed BamHI-M leftward reading frame 1 (BMLF1), directed the synthesis of a nuclear antigen detected by antibodies in serum from a patient with nasopharyngeal carcinoma. The BMLF1 antigen was not detected by monoclonal or polyclonal antibodies directed against the EA-D complex. A series of deletion mutants were constructed in the BamHI M DNA fragment, and the EA-D complex and BMLF1 antigen were mapped to discrete open reading frames in this DNA fragment. A test for several possible functions of these antigens showed that the BMLF1 antigen had the ability to activate or enhance, in trans, the level of expression of a gene under the control of the adenovirus early region 3 promoter or the simian virus 40 early promoter in the absence of its cis-acting enhancer. These experiments demonstrate a new gene function, encoded by EBV, that may be important in the positive regulation of viral or cellular genes.     

Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants

Rates of CO₂ assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO₂ in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25°C. Electron transport activity for CO₂ assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O₂, and between fluorescence and rates of CO₂ assimilation at 19 millibars but not 200 millibars O₂. The values of fluorescence measured at about 220 microbars intercellular CO₂ were not greatly affected by increasing O₂ from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO₂ partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO₂ partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO₂ partial pressures which reversed to a positive correlation at higher values of CO₂ partial pressures. Similar results were observed in the C₃ species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C₄ species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO₂ assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q_E) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

Activated N-ras gene induces neuronal differentiation of PC12 rat pheochromocytoma cells

Activated mouse N-ras gene transfected into PC12 rat pheochromocytoma cells suppressed proliferation and promoted neuronal differentiation. Normal mouse N-ras in a LTR-containing vector caused differentiation with a reduced efficiency, but normal N-ras in a vector lacking LTR sequences failed to alter the PC12 phenotype. Cultures of NGF-resistant PC12 variant subline U7 also showed outgrowth of neurites and cessation of cell division following transfection with the mutated ras gene. The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and suppressing proliferation, in contrast to their established oncogenic neoplasia-promoting activity in other cells.     

Ongoing hapten-specific delayed-type hypersensitivity prevents generation of cytolytic T lymphocytes to the same hapten

Cytolytic T lymphocytes (CTL) specific for trinitrophenyl (TNP)-altered self antigens can be generated in vivo through the simultaneous injection of TNP-modified syngeneic spleen cells and H-2-compatible, minor histocompatibility locus (Mls)-disparate auxiliary spleen cells into the footpads of mice. The latter stimulates host helper cells to produce differentiative and proliferative signals required for the generation of CTL. Advent of this protocol allowed investigation of the initiation of two different cell-mediated immune responses, delayed-type hypersensitivity (DTH) and the generation of CTL, in the same experimental animal. Mice presensitized for CTL were able to develop DTH as well as normal controls. However, when mice were first sensitized for DTH, they were thereafter incapable of generating CTL. This effect was hapten specific, relatively long lasting, and preventable by treating mice with cyclophosphamide before sensitizing for DTH. Adoptive transfer of lymphoid cells from DTH-immune mice conferred DTH reactivity upon naive recipients but not a suppressed CTL response. Therefore, cells mediating DTH were not responsible for suppression of CTL. The mechanism for suppression has been discussed from the viewpoint of the suppressor-T-cell circuits that are known to be generated when animals are sensitized for DTH and which are susceptible to treatment with cyclophosphamide.     

Cytokinin biosynthesis in plant tumour tissues

A range of endogenous cytokinins have been identified inDatura crown-gall tissue by GC-MS. Incorporation of [³H]adenine into zeatin riboside, zeatin and its nucleotide(s) is also shown. Metabolism studies usingcis- andtrans-isomers of zeatin riboside indicate that interconversion of the two isomers does not occur in this tissue. Data on the identity of major endogenous cytokinins in a genetic tumour line of tobacco is also provided.     

Mechanism of benzo(a)pyrene induction of alpha-human chorionic gonadotropin gene expression in human lung tumor cells

Human lung cells (ChaGo) derived from a bronchogenic carcinoma synthesize and secrete in the culture medium the alpha subunit of the glycoprotein hormone, human chorionic gonadotropin (alpha-hCG). The synthesis of alpha-hCG by ChaGo cells could be further stimulated by treatment with sublethal concentrations of the polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP), or dimethylbenzanthracene. The production of alpha-hCG could be correlated to the levels of alpha-hCG-specific mRNA sequences in control and PAH-treated cells. Further analysis of the RNA species (Northern blot) revealed that the level of the mature (approximately 1.0 kb) and the high molecular weight alpha-hCG specific nuclear RNA sequences (approximately 2.2 and 5 kb) were all greater in PAH-treated cells. Addition of [3H]BaP (0.25 microgram/ml) in the culture medium of ChaGo cells led to immediate uptake of the radioactive compound apparently by simple diffusion. SDS PAGE and subsequent fluorography revealed that the radioactive compound interacted and formed covalent complexes with cytoplasmic and nuclear proteins. This covalent interaction of the [3H]BaP molecule with cellular proteins could be significantly inhibited by either inhibiting the activity of the enzyme aryl hydrocarbon hydroxylase with 7,8-benzoflavone or by reducing the cellular concentration of the enzyme by simultaneous incubation with cycloheximide. These results suggested that in ChaGo cells, the observed covalent complexes were formed by the interaction of the BaP metabolites with cellular proteins. The concentrations at which 7,8-benzoflavone or cycloheximide inhibited formation of metabolites from [3H]BaP and their covalent interaction with cell protein did not affect the BaP-induced stimulation of alpha-hCG gene expression. However, the cytotoxic effects of BaP in ChaGo cells seemed to be exerted by the metabolism of the compounds. Results presented in this report suggest that BaP metabolism and the interaction of the metabolites with cell proteins were not essential for the BaP-induced modulation of alpha-hCG gene expression.     

Binding and internalization of heparin by vascular smooth muscle cells

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.     

Translocation of photoassimilate from leaves of two popyploid genotypes of tall fescue differing in photosynthetic rates

The photosynthetic rate of a decaploid genotype (1-16-2) of tall fescue ( Festuca arundinacea Schreb.) is about twice that of a common hexaploid genotype (V6-802) (Plant Physiol. 72: 16–21, 1983). Translocation of photosynthate out of the leaves is a possible means of regulating carbon assimilation. To evaluate this possibility, we have examined a) translocation velocity, b) time course of translocation from leaves, c) photoassimilate partitioning pattern into whole plants in pulse and chase experiments, and d) interveinal distances between two ploidy genotypes. Most of the ¹⁴C accumulated in sucrose, and the labelled carbon moved down the leaf blades at similar velocities (6 to 10 cm h⁻¹) in both genotypes. Recent ¹⁴C assimilate was rapidly translocated from the fed area of the leaf blade. For example, the decaploid and the common hexaploid had translocated 40 and 26% of the ¹⁴C, respectively, at 6 h, and 79 and 49% of the ¹⁴C, respectively, at 24 h. Partitioning of ¹⁴C among plant organs was considerably different between the genotypes after a 24 h chase. For example, out of the total ¹⁴C recovered from the whole plant, the decaploid had retained 40% in the labelled leaf with 10, 33 and 29% in other leaves, stem bases and roots, respectively; whereas the hexaploid had retained 91% in the labelled leaf with 4, 3 and 2% in other leaves, stem bases and roots, respectively. However, the higher rate of translocation was correlated with greater interveinal distances in the decaploid genotype. These results suggested that the higher translocation percentage in the decaploid than the hexaploid genotype was due to greater sink activity.