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At least 19 recordsLinked to original sources
PubMed1990

Resistance to pactamycin in clones of Streptomyces lividans containing DNA from pactamycin-producing Streptomyces pactum.

A pactamycin (Pc)-resistance determinant (pct) from Streptomyces pactum has been isolated on a 4.9-kb KpnI fragment. The original construct involving plasmid pIJ702 was highly unstable in Streptomyces lividans, leading to deletion of the pct gene from the vector. Subcloning of pct into an alternative vector (pOJ160) led to the generation of a more stable clone which possessed Pc-resistant ribosomes, and reconstitution analysis established that 16S rRNA was responsible for such resistance. Post-transcriptional modification of rRNA is probably the mechanism of resistance since the cloned DNA fragment did not appear to encode 16S rRNA.

Cloning, Molecular↗
PubMed1977

Protein synthesis and amnesia: studies with emetine and pactamycin.

Two antibiotic inhibitors of protein synthesis, emetine and pactamycin, have been tested for their effects on cerebral and peripheral protein synthesis and amnesia. Peripherally administered emetine but not pactamycin inhibited cerebral protein synthesis, although this inhibition was lower than that observed with cycloheximide or anisomycin. Pactamycin had a lesser effect on adrenal protein synthesis than emetine. This was reflected in the ability of emetine but not pactamycin to block ACTH-induced corticosteroidogenesis. Anisomycin and cycloheximide caused amnesia in a passive avoidance task, whereas pactamycin and emetine did not. These results are inconsistent with the amnesia being due to inhibition of protein synthesis in a peripheral organ. They are also inconsistent with the amnesia being due to the suppression of an adrenocortical response as previously suggested. No obvious correlation between amnesia and the mechanism of protein synthesis was observed. The most parsimonious explanation is that inhibition of cerebral protein synthesis is necessary for amnesia.

Adrenal Glands↗
PubMed1986

Photolabeling of protein components in the pactamycin binding site of rat liver ribosomes.

The antitumoral and antibacterial drug pactamycin can be radioactively labeled by iodination without loss of biological activity. Using the labeled pactamycin, the ribosomal binding site of the drug on rat liver ribosomes has been studied by affinity labeling techniques taking advantage of the photoreactive acetophenone group present in the molecule. When 40 S ribosomal subunits are labeled, one major spot of radioactivity is found associated to protein S25. In addition, weaker spots related to proteins S14/15, S10, S17 and S7 can also be detected in the autoradiogram of the two-dimensional gel slab. Since pactamycin inhibits protein synthesis initiation, the proteins forming its binding site must be related to some step of this process. By comparison with results from pactamycin affinity labeling of Escherichia coli ribosomes (Tejedor, F., Amils, R. and Ballesta, J.P.G. (1985) Biochemistry 24, 3667-3672) these proteins could lie in the mRNA and initiation factors binding region of the rat liver ribosome.

Animals↗
PubMed1978

Proteins of Rous-associated virus 61, an avian retrovirus: common precursor for glycoproteins gp85 and gp35 and use of pactamycin to map translational order of proteins in the gag, pol, and env genes.

Cells infected by Rous-associated virus 61 (RAV-61) contained a precursor-like protein, pr90, that was specifically precipitated by antiserum directed against envelope glycoproteins, gp85 and gp35. Tryptic peptide mapping showed that pr90 contained tryptic sequences of both gp85 and gp35. Pactamycin mapping experiments indicated that the two glycoproteins are translated from the env-mRNA in the order (5') gp85--gp35. The pactamycin mapping experiments also indicated a translational order of p10--(p27, p12)--p15 for the gag proteins; this agreement with the order previously reported from tryptic mapping studies on precursor pr76 of avian myeloblastosis virus implied that the stoichiometry of the core proteins was unchanged when virions were assembled in the presence of pactamycin. The reverse transcriptase proteins, unlike those of the env and gag genes, fell on the right side of the pactamycin map. This result is in accord with the idea that most, if not all, of the reverse transcriptase protein is translated by read-through of the gag(pol) message rather than by translation of a hypothetical pol-mRNA devoted solely to synthesis of that protein.

Avian Leukosis Virus↗
PubMed1985

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes.

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.

Antibiotics, Antineoplastic↗
PubMed1991

Site-specific methylation of 16S rRNA caused by pct, a pactamycin resistance determinant from the producing organism, Streptomyces pactum.

Ribosomal resistance to pactamycin in clones of Streptomyces lividans containing DNA (pct) from Streptomyces pactum, the pactamycin producer, involves methylation of 16S RNA. The modified residue A-941 in S. lividans 16S rRNA (A-964 in the homologous Escherichia coli sequence) is converted to 1-methyladenosine, and the ribosomal ability to bind pactamycin is reduced or abolished.

Base Sequence↗
PubMed1972

Gene order of encephalomyocarditis virus as determined by studies with pactamycin.

Previous work has shown that translation of the encephalomyocarditis (EMC) viral ribonucleic acid (RNA) generates at least three primary products, polypeptides A, F, and C. The A and C polypeptides then undergo post-translational cleavages to complete the production of the stable viral polypeptides (delta, beta, gamma, alpha, G, I, F, H, and E). In this communication we show that A, F, and C are produced in equimolar amounts giving further support to the theory that the RNA of picornaviruses has only a single site for the initiation of protein synthesis. The biosynthesis of viral proteins in EMC virus-infected HeLa cells was studied in the presence of pactamycin at concentrations which preferentially inhibit the initiation of protein synthesis. The amount of each polypeptide formed during the residual period of protein synthesis observed after the addition of pactamycin was used as a criterion for ordering the genes on the viral RNA. The results obtained indicate that the primary gene products are ordered on the EMC viral RNA 5' --> 3' A-F-C and that the stable products are ordered delta-beta-gamma-alpha-G-I-F-H-E. Moreover, the intermediate chains B and epsilon map in the capsid region, whereas the intermediate chain D maps in the E region. This order is largely consistent with previously established relationships of the viral polypeptides and thus indicates that pactamycin is a valid tool for "genetic" mapping of polycistronic RNA molecules with single initiation sites.

Amino Acids↗
PubMedDate not supplied

Binding sites of the antibiotics pactamycin and celesticetin on ribosomal RNAs.

The binding sites of the antibiotics pactamycin and celesticetin on the rRNAs of Escherichia coli ribosomes were investigated by a chemical footprinting procedure. Pactamycin protected residues G-693 and C-795 in 16S RNA which are located in an important functional region of the 30S subunit participating in initiation complex formation and ribosomal subunit interaction. Celesticetin altered the reactivities of 5 residues A-2058, A-2059, A-2062, A-2451 and G-2505 within the central loop of domain V of 23S RNA which has been implicated in peptidyltransferase activity. Inferences are drawn concerning the mode of action of the antibiotics.

Anti-Bacterial Agents↗
PubMed1976

Analysis of the two steps in polypeptide chain initiation inhibited by pactamycin.

Earlier work has shown that the inhibition by pactamycin (PM) of polypeptide chain initiation in reticulocyte extracts is associated with (1) a defect in the joining of the 60S subunit to the smaller initiation complex to form an 80S complex ("joining reaction") (Kappen, L. S., Suzuki, H., and Goldberg, I. H. (1973), Proc. Natl. Acad. Sci. U.S.A. 70, 22) and (2) a block after the synthesis of the initial dipeptide (Kappen, L. S., and Goldberg, I. H. (1973), Biochem. Biophys. Res. Commun. 54, 1083). The relative contributions of these two effects to the action of PM and their relationship to one another were evaluated in a system employing sparsomycin that permits both initiation at a certain number of initiation sites and limited oligopeptide formation without termination and release. The degree to which PM blocks the "joining reaction" and leads to the accumulation of 48S initiation complexes that either remain free or are bound to polysomes without the corresponding 60S subunit ("half-mers") was estimated by treatment of polysomes with RNase. Met-tRNAfMet binding factors are required to stabilize the RNase-generated 48S complexes. Under conditions where the initiation factor required for the "joining reaction" functions catalytically, presumably by cycling on and off initiation complexes, PM usually inhibits 80S complex formation 50-70%. Where "joining" is not limiting (presence of at least stoichiometric amounts of joining factor or high Mg2+ concentration) PM leads to the maximal accumulation of the initial dipeptide, Met-Val, in the P-site on the ribosome, indicating a block in a subsequent step in elongation. Binding studies with [3H]PM and the inability of PM to inhibit elongation of preformed Met-Val indicate that PM must interact with the ribosomes at an early stage of initiation. Taken together these data are compatible with the suggestion that PM does not interfere with the ribosomal "joining reaction" per se, but prevents the release and reuse of the joining factor, and in so doing blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome.

Animals↗
PubMed1971

Determination of the gene sequence of poliovirus with pactamycin.

By examination of the virus-specific polypeptides formed after the addition of pactamycin, an inhibitor of protein chain initiation, to infected cells, it has been possible to tentatively locate the virus coat proteins at the amino terminus of the large, virus-specific protein precursor, and, therefore, to assign the coat protein cistron to the 5' end of the RNA.

Amino Acid Sequence↗
PubMed1971

Effect of pactamycin on synthesis of poliovirus proteins: a method for genetic mapping.

We have studied the effect of the drug pactamycin on protein synthesis in poliovirus-infected HeLa cells. At a concentration which primarily inhibits initiation of protein synthesis, the spectrum of poliovirus proteins synthesized is markedly changed. The amount of NCVP 1, the capsid precursor, is greatly reduced relative to NCVP 2 and the amount of NCVP X is slightly reduced. Since it is believed that there is only one major site for the initiation of protein synthesis on the poliovirus genome, we interpret this differential effect on the poliovirus proteins to be an indication of their relative distance from the initiation site. On this basis, we propose a gene order for the poliovirus genome (5' --> 3') of NCVP 1, NCVP X, NCVP 2.

Antibiotics, Antineoplastic↗
PubMed1986

PD 113,618 and PD 118,309, new pactamycin analogs.

Two antibiotics were isolated from culture broths of a Streptomyces sp. and identified as 8"-hydroxypactamycin and 7-deoxypactamycin. The latter antibiotic was shown to be identical to cranomycin. An additional compound, 8"-hydroxypactamycate, was also isolated.

Antibiotics, Antineoplastic↗
PubMed1992

The role of protein synthesis in the decay of phosphoenolpyruvate carboxykinase messenger RNA.

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.

Animals↗
PubMed1976

Cytotoxic T cells recognize very early, minor changes in ectromelia virus-infected target cells.

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.

Animals↗