BIOCHEMISTRY: NIRENBERG AND MATTHAEI PROC. N. A. S.template RNA. Other explanations, however, are fully plausible, and it is not possible at this state to rule out alternative interpretations. In the following paper, further experiments on amino acid incorporation using the system described here are presented. It will be shown that in addition to the usual requirements, the system is stimulated by template RNA. Summary.-Cell-free E. coli extracts have been obtained which actively incorporate amino acids into protein. Methods were devised whereby these extracts could be dialyzed and stored for long periods of time at -150 without undue loss of activity. The characteristics of amino acid incorporation by such stored extracts were strongly suggestive of de novo protein synthesis, for incorporation required both ribosomes and 105,000 X g supernatant fractions, ATP and an ATPgenerating system, was stimulated by a mixture of other L-amino acids, and was markedly inhibited by puromycin, chloramphenicol, and RNAase. The initial rate of amino acid incorporation was not inhibited by DNAase; subsequent incorporation was greatly inhibited. The possible relationship of the DNAase inhibition of amino acid incorporation into protein to the synthesis of "messenger" RNA was briefly discussed.* Supported by a NATO Postdoctoral Research Fellowship.1 Tissikres, A., D. Schlessinger, and F. Gros, these PROCEEDINGS, 46, 1450. 2 Matthaei, J. H., and M. W. Nirenberg, Fed. Proc., 20, 391 (1961).3 Kameyama, T., and G. D. Novelli, Biochem. Biophys. Res. Comm., 2, 393 (1960). 4 Kirsch, J. F., P. Siekevitz, and G. E. Palade, J. Biol. Chem., 235, 1419 (1960). 5 Mora, P. T., E. Merler, and P. Maury, J. Am. Chem. Soc., 81, 5449 (1959). 6 Anson, M. L., J. Gen. Physiol., 22, 79 (1938). 7 Sevag, M. G., D. B. Lackmann, and J. Smolens, J. Biol. Chem., 124, 425 (1938). 8 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, ibid., 193, 265 (1951). 9 Allfrey, V. G., and A. E. Mirsky, these PROCEEDINGS, 44, 981 (1958).10 Hurwitz, J., A. Bresler, and R. Diringer, Biochem. Biophys. Res. Comm., 3, 15 (1960).11 Stevens, A., ibid., 3, 92 (1960).12 Weiss, S. B., and T. Nakamoto, J. Biol. Chem., 236, PC18 (1961). 13 Siekevitz, P., ibid., 195, 549 (1952
Fusion of spleen cells from a mouse immunized with chicken embryo retina cells with clonal mouse myeloma cells yielded a lymphocyte hybrid cell line that produced antibody that bound to neural tissue such as retina, brain, spinal cord, and dorsal root ganglia but not to other tissues tested. The antigen was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies in chicken retina, but little or no antigen was detected on axons or dendrites, Muller cells, or retina pigment cells. The activity of antigen A2B5 is relatively stable at 100°C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside, and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition at 0.2 AM) or by N-acetylneuraminic acid (half-maximal inhibition at 5000 MM) but not by other purified gangliosides tested. These results suggest that the antigen is a complex ganglioside in plasma membranes of retina neuron cell bodies but not axons or dendrites. Chicken embryo retina cells have been used to study biological processes that require interactions between neurons, such as cell aggregation (1, 2), adhesiveness (3), and synapse formation (4-7). To define surface molecules of retina neurons, we have used the technique, introduced by Milstein and coworkers (8-10), of antibody production by hybrid cells formed by fusion of mouse myeloma cells with spleen cells from mice immunized with chicken embryo retina cells. This approach is useful because lymphocyte hybridomas can synthesize large quantities of monoclonal antibody with specificity for a single antigen determinant. Hybridoma cell lines have been reported which synthesize antibodies specific for cell surface antigens as diverse as the Forsmann antigen (11), HLA determinants (12), tumor-specific antigens (13), and an antigen detected on human neuroblastoma cells and fetal brain (14) (also see ref. 15).Other techniques also have been used to produce antisera against nervous system antigens. These include xenogenic immunization followed by extensive absorption with non-neuronal tissues (16,17) and immunization with neural cell lines (18-20), partially purified synaptosomes, plasma membranes, and protein fractions (21,22).In this report we describe the characterization of a monospecific antibody synthesized by lymphocyte hybrid A2B5 cells that recognizes a surface antigen restricted to cell bodies of most, or all, retina neurons. Flow General, Hamden, CT). The cell suspension was incubated for 30 min at 37°C; cells then were sedimented at 1000 X g; the supernatant solution was harvested by using Titertek harvesting filters (Flow General, and the radioactivity in the supernatant solutions that was absorbed by the filters was determined. Saturating concentrations of antibody A2B5 in the presence of complement released 50-60% of radioactive material from cells compared to that released by 0.3% Triton X-...
Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.
Narcotics affect adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.8.1.1J in two opposing ways, both mediated by the opiate receptor. The first process is the readily reversible inhibition of the enzyme by narcotics; the second is a compensatory increase in enzyme activity which is delayed in onset and relatively stable. Late positive regulation of the enzyme counteracts the inhibitory influence of morphine and is responsible for narcotic dependence and tolerance. The coupled inhibitory and positive regulatory mechanisms for adenylate cyclase provide a means of activating and deactivating neural circuits hours after the initial event and thus may play a role in a memory process. Recent observations with neuroblastoma X glioma hybrid cells indicate that the binding of morphine and other opiates to narcotic receptors results in an inhibition of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (1-3) and a decrease in cAMP levels in intact cells (1, 2, 4, 5). Similar observations have been made with brain (6, 9) but the heterogeneity of cell types present and apparent lability of the brain enzyme may be responsible for conflicting observations (8)(9)(10). Both dependence upon opiates and tolerance to these compounds were hypothesized to result from either an increase in the number of molecules of adenylate cyclase or a long-lived factor which affects the rates of adenylate cyclase activity or turnover (1). In this report, we describe the results of experiments which were designed to test the hypothesis illustrated diagramatically in Fig. 1. The addition of morphine results in the rapid inhibition of adenylate cyclase activity and a resultant decrease in intracellular cAMP levels. Further incubation reveals a second regulatory process which involves a compensatory increase in adenylate cyclase activity termed late positive regulation. The increase in adenylate cyclase activity counteracts the inhibition of enzyme activity by morphine and cAMP levels are restored to the normal value. Cells now are tolerant to morphine and are also dependent upon the narcotic, since withdrawal of the drug or the addition of a specific narcotic antagonist will raise cAMP levels to abnormally high values and secondarily produce a gradual return to the normal level of adenylate cyclase activity.In this communication we report data which demonstrate a rapid inhibition and a late positive regulation of adenylate cyclase which are dependent upon narcotics and account for the phenomena of narcotic dependence and tolerance. METHODS AND MATERIALSThe source of each chemical and the medium and growth conditions for neuroblastoma X glioma hybrid NG108-15 were described previously (1).Assay of cAMP in Intact Cells and Medium. Growth medium was changed 12 hr before assay. Narcotic was added to the growth medium [Dulbecco's modification of Eagle's medium (DMEM), hypoxanthine-aminopterinthymidine (HAT), and 10% fetal bovine serum] and plates were incubated in a humidified atmosphere of 90% air-10% C02. A...
A rapid, sensitive method is described for measuring C(14)-aminoacyl-sRNA interactions with ribosomes which are specifically induced by the appropriate RNA codewords prior to peptide-bond formation. Properties of the codeword recognition process and the minimum oligonucleotide chain length required to induce such interactions are presented. The trinucleotides, pUpUpU, pApApA, and pCpCpC, but not dinucleotides, specifically direct the binding to ribosomes of phenylalanine-, lysine-, and proline-sRNA, respectively. Since 5'-terminal, 3'-terminal, and internal codewords differ in chemical structure, three corresponding classes of codewords are proposed. The recognition of each class in this system is described. The template efficiency of trinucleotide codewords is modified greatly by terminal phosphate. Triplets with 5'-terminal phosphate are more active as templates than triplets without terminal phosphate. Triplets with 3'- or 3' (2')-terminal phosphate are markedly less active as templates. These findings are discussed in relation to the probable functions of terminal codewords. The modification of RNA and DNA codewords, converting sense into missense or nonsense codewords, is suggested as a possible regulatory mechanism in protein synthesis.
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