Reassociation of eukaryotic ribosomal subunits: dependence of reassociation on formation of a 40S initiation complex: effects of aurintricarboxylic acid and pactamycin.
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A new pactamycin analogue, 5"-fluoropactamycin, was prepared by directed biosynthesis. Supplementation of the fermentation medium of Streptomyces pactum, var. pactum with 3-amino-5-fluorobenzoic acid, an analogue of 3-aminobenzoic acid, an advanced precursor in pactamycin biosynthesis, resulted in co-production of pactamycin and the new pactamycin analogue. A similar feeding experiment with 3-amino-5-methylbenzoic acid did not result in formation of the corresponding methylated pactamycin analogue, but only in inhibition of pactamycin production. Comparison of antimicrobial and cytotoxic activities of pactamycin and 5"-fluoropactamycin showed no significant differences.
The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.
The translational orders of bovine leukemia virus (BLV) gag proteins and env proteins have been determined by the technique of pactamycin mapping. BLV-producing fetal lamb kidney (FLK) cells were pulse-labeled with 14C amino acids and [3H]glucosamine in the presence or absence of pactamycin which inhibits initiation of protein synthesis but not elongation. Viruses were harvested from the cell culture medium and viral proteins were analyzed by SDS-polyacrylamide gel electrophoresis. For each protein the " pactamycin ratio" was determined. The lower this ratio was for a given protein, the closer the protein would be to the amino terminus of the precursor polyprotein. From these experiments the following translational orders were derived: gag, NH2-p10-(p15(2) X p15(1)-p24-p12-COOH; and env, NH2- gp64 - gp30 -COOH. Investigation of isoelectric points of BLV proteins, a conserved property of retroviral proteins, supported the translational order of gag gene-coded proteins.
Target cells (P-815 mastocytoma cells) infected with ectromelia virus became susceptible to lysis by H-2 compatible specific effector T cells within one hour of exposure of the cells to virus. This is long before viral progeny are produced and shed from the cell. Polyacrylamide gel electrophoresis (PAGE) profiles of the plasma membranes from infected and uninfected P-815 cells pulsed with 35S-methionine for one or a few hours after infection with virus were very complex and showed no consistent differences. P-815 cells, infected with ectromelia virus in the presence of an inhibitor of protein synthesis, pactamycin, slowly became susceptible to cell mediate lysis when the pactamycin was removed. The number of polypeptide species synthesized under these conditions was reduced to only three, of molecular weights between 10,000-50,000 daltons. Specific, newly synthesized membrane components recognized by mouse convalescent sera were isolated by immune complexing and examined by PAGE. Six polypeptide bands were seen, the major one correlating with one observed in the pactamycin experiment. The results suggested that the convalescent serum recognized both viral and host cell coded antigens. The significance of these findings is discussed.