Biosynthesis of eideine:: II. Localization of edeine synthetase within Bacillus brevis Vm4

Edeine-synthesizing polyenzymes, associated with a complex of cytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from cells intensively synthesizing edeines (18–20 h culture) contained edeine B. Edeine B was found to be bound covalently to the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1–0.3 μmol/mg protein, depending on the age of cells.Detachment of edeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH₄)₂SO₄ at 45–55% saturation or by DEAE-cellulose colum fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to protein fraction of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16–22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine b of specific activity: 80 units/μmol was released.The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associatd with this complex did not effect the DNA-synthesizing activity.     

Inactive form of edeine in the edeine-producing Bacillus brevis Vm 4 cells.

1. Exogenous edeine inhibits the synthesis of DNA and protein, but not that of RNA, in extracts of edeine-producing Bacillus brevis Vm 4 cells. This is analogous to the effect of edeine on extracts obtained from edeine-sensitive cells. 2. Producer cells, in contrast to sensitive ones, are not permeable to exogenous edeine. DNA synthesis in producer cells rendered permeable by toluene treatment becomes sensitive to edeine. 3. No free edeine could be detected in post-log producer cells during maximal synthesis of edeine. Nascent edeine exists in the cell in a biologically inactive form, bound to a fast-sedimenting fraction. Edeine B, identical to the antibiotic present in the medium, is released from this fraction by mild treatment with alkali.     

Stability of protein synthesis initiation complexes in the presence of edeine

The antibiotic inhibitor edeine has been used to study various aspects of the initiation of protein synthesis in a rabbit reticulocyte cell-free system. Edeine prevents assembly of the 80S initiation complex while allowing accumulation of a 44S initiation intermediate. The complete 80S initiation complex, once formed, is stable in the presence of edeine. The functioning of the initiation complex, as judged by release of methionyl-puromycin, is only partially inhibited by a concentration of edeine which fully inhibits formation of the initiation complex. The above effects of edeine on a eukaryotic system differ from the effects edeine has been found to have in a prokaryotic system.     

GTP hydrolysis during methionyl-tRNAf binding to 40 S ribosomal subunits and the site of edeine inhibition.

Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.     

The uptake and subcellular localization of the peptide antibiotic edeine a in HeLa cells in suspension culture

The uptake of [¹⁴C]thymidine, [¹⁴C]uridine and [¹⁴C]leucine by HeLa cells incubated in the presence of 1.52 μg/ml edeine A is inhibited by 7.5, 0 and 4%, respectively. Though edeine A has no gross cytopathic effect on HeLa cells, the peptide antibiotic enters the cells and h after addition to cell cultures is found in the nuclei. After 6 h of incubation, the highest intracellular concentration of edeine is located in the nuclear fraction, but, after 12 h, a higher proportion is in the post-mitochondrial supernatant fraction where it is associated with protein components in the range of molecular weights of 20 000 and 9 500 D. In the nucleus most of the [¹⁴C]edeine is bound to the chromatin fraction after 2 h of incubation. Exhaustive deoxyribonuclease digestion of the chromatin fraction releases all the radioactivity into one ultraviolet absorbing peak, which sediments to a density of 20% sucrose. Exhaustive ribonuclease digestion of the chromatin fraction releases all the radioactivity into two ultraviolet absorbing peaks which sediment to a density of 20 and 40%, respectively; subsequent proteolytic digestion of the RNAse-treated chromatin fraction frees about 70% of the edeine A from the ultraviolet absorbing peaks. This suggests that intranuclear edeine A associates with proteins in the chromatin. The radioactivity was recovered from the enzymatically digested chromatin fractions and characterized as biologically active edeine A.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

Purification of bacteriophage T7 DNA-membrane complex and its application to the in vitro recombination reaction

In order to construct an in vitro recombination system of T7 DNA, the reaction products of which resemble those in vivo in structure, T7 DNA-membrane complex which is free from concomitant DNase activity was purified from T7 phage-infected cells. T7-infected cells were lysed with T4 lysozyme/Brij58, and T7 DNA-membrane complex was purified through three successive density gradient centrifugations. The properties of the complex on exposure to defined nucleases and observation of the complex by electron microscopy revealed that in T7 DNA-membrane complex, both ends of a linear T7 DNA are bound with membrane components. A mixture of 32P-labeled T7 DNA-membrane complex and BU-labeled T7 DNA-membrane complex was incubated with T7 exonuclease and T7 DNA-binding protein, and the reaction products with intermediate density were purified. Most of the products were found to have structures similar to that of the recombination intermediate found in T7-infected cells upon electron microscopic examination.     

The synthesis of DNA by DNA-membrane complexes from irradiated E. coli B/r and Bs-1: the role of the membrane.

DNA-membrane complexes were isolated from lysed E. coli B/r and Bs-1, either by low g forces from a low salt solution, or by high g forces through a discontinuous sucrose gradient. The latter method was more gentle. Irradiation of the intact bacteria had no effect on the membrane macromolecules or on RNA components of these complexes. DNA loss was not significant after irradiation under anoxic conditions but complexes isolated from from Bs-1 irradiated in air showed an appreciable decrease in DNA content. In the presence of the appropriate nucleotide mixture, both 'free' DNA, found in the supernatant fractions, and rapidly sedimented membrane-associated DNA were able to synthesize DNA in the absence of added polymerase. DNA synthesis associated with 'free' DNA was more sensitive to radiation than that associated with DNA bound to the membrane, which appeared to moderate the effects of radiation on new DNA synthesis. It is concluded that the depression of DNA synthesis is primarily a result of irradiation-induced changes on genome-DNA. The interpretation of earlier work from our laboratories that DNA-membrane complexes contained the macromolecular structure which responded to radiation with a high o.e.r. is not supported by the evidence in this work.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine.

N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.     

Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus.

Analysis of duck hepatitis B viral DNA by gel electrophoresis, Southern blotting, and binding to benzoylated naphthoylated DEAE-cellulose showed that a protein is bound to the minus-strand virion DNA as well as to the full-length single strand, minus-strand species, and minus-strand DNA intermediates isolated from replicating complexes present in infected duck liver. By utilizing a modified dideoxynucleotidyl sequencing method, it was shown that the protein is covalently bound to the smallest detectable growing strands (ca. 30 bases) and that minus-strand synthesis begins at a unique site. These results support the notion that the protein may function as a primer for synthesis of the minus-strand DNA.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Amplification and cloning of the Chinese hamster glutamine synthetase gene.

A Chinese hamster ovary cell line, KG1MS which is resistant to 5 mM methionine sulphoximine overproduces glutamine synthetase. Overproduction of this 42 000 mol. wt. polypeptide is not seen in either parental KG1 or revertant KG1MSC4-0 cell lines which are resistant to 3 microM and 8 microM methionine sulphoximine, respectively. Restriction endonuclease analysis of DNA from KG1MS cells produces a pattern of amplified DNA fragments not seen in parental KG1 or revertant KG1MSC4-0 digests. Hybridization of cDNA probes complementary to KG1MS poly(A) mRNA against Southern blots of KG1MS restriction digests identifies a specific subset within these amplified sequences which is not detected by cDNA probes made from parental KG1 poly(A) mRNA. One 8.2-kb BglII fragment of KG1MS DNA identified by cDNA hybridization has been cloned to produce recombinant pGS-1. mRNA hybrid selected by pGS-1 translates to a 42 000 mol. wt. polypeptide which co-migrates in polyacrylamide gels with the over-produced protein in KG1MS cells and purified glutamine synthetase. pGS-1 also hybridizes to several mRNA species abundant in KG1MSC4-M, but not KG1, poly(A) mRNA extracts. The high level of resistance to methionine sulphoximine shown by KG1MS cells is due to amplification of a DNA sequence of at least 50 kb which contains the coding region for the enzyme glutamine synthetase.     

Site of nuclear DNA replication in embryonic cells of the sea urchin Strongylocentrotus intermedius]

In the sea urchin embryonic cells, all newly synthesized nuclear DNA (n-DNA) pulse-labeled by 3H-thymidine was found within DNA-membrane complex (DNA-mc) isolated by centrifugation of lysates of nuclei after their treatment with Sarkosyl, Brij-35, or sodium dodecylsulfate through neutral sucrose (10--30%) gradients. This attachment has been shown not to be an artifact due to the unspecific effect of the detergents or the destabilization of the secondary structure of n-DNA because the association of the exogenous 14C-DNA with nuclear membrane and chromatin did not occur during the isolation of the DNA-mc. n-DNA was not replaced from DNA-mc when the latter was isolated in the excess of unlabeled denatured DNA. n-DNA associated with DNA-mc behaved as a precursor of chromosomal DNA. It is suggested that in sea urchin embryonic cells the synthesis of nuclear DNA is carried out by the replicative complex attached to the nuclear membrane.     

Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA.

A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.     

Enzyme-bound intermediates in the biosynthesis of bacitracin.

1. Bacitracin synthetase, a three-component enzyme complex which catalyzes synthesis of the dodecapeptide bacitracin A, has been prepared from Bacillus licheniformis strains ATCC 10716, AL and SB 319. During synthesis of bacitracin, the amino acids (smaller amounts) and peptides are covalently bound to the enzyme complex. The nature of the bindings suggest that the amino acids and peptides are thioester linked. 2. The peptides, identified by thin-layer chromatography after performic acid liberation were Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu-Ile, Ile-Cys-Leu-Glu-Ile-Lys-Orn, Ile-Cys-Leu-Glu-Ile-Ile-Orn-Ile, Ile-Cys-L-EU-Glu-Ile-Lys-Orn-Ile-Phe, Ile-Cys-Leu-Glu-Ile-L-YS-Orn-Ile-Phe-His-Phe-His and Ile-Cys-Leu-Glu-Ile-Lys-Orn-Ile-Phe-His-Asp. 3. The labelled peptides covalently bound to bacitracin synthetase were intermediates in bacitracin synthesis. 4. Chain growth is initiated on one enzyme component (A) by the addition of isoleucine and cysteine. The sequential addition of the other amino acids proceeds in the C-terminal direction until the pentapeptide is formed. Further addition of amino acids and production of bacitracin are obtained by adding the other enzyme components (B and C) to the incubation mixture.     

The fractionation of calf thymus DNA on histone KAP - Sepharose 4B columns

It was found that fractionation of calf thymus DNA on homologous histtone KAP covalently bound to CNBr activated Sepharose 4B depends on the molecular weight of DNA. The maximum of elution of high molecular DNA (m. wt. above 5 x 10(6)) was observed at 0.56 M NaCl and that of degraded DNA (m. wt. 0.8 x 10(6)) at 0.52 M NaCl. Significant differences in melting temperatures and melting intervals were observed among fractions obtained from low molecular DNA as a result of enrichment of some fractions in satellite DNAs. These differences were very small in DNA of m. wt. above 5 x 10(6). The results are discussed in terms of specific areas which may exist on calf thymus DNA molecules playing the role of loci, where lysine-rich histone KAP is preferentially bound.     

Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding

The crystal structures of the universal translation-initiation inhibitors edeine and pactamycin bound to ribosomal 30S subunit have revealed that edeine induces base pairing of G693:C795, residues that constitute the pactamycin binding site. Here, we show that base pair formation by addition of edeine inhibits tRNA binding to the P site by preventing codon-anticodon interaction and that addition of pactamycin, which rebreaks the base pair, can relieve this inhibition. In addition, edeine induces translational misreading in the A site, at levels comparable to those induced by the classic misreading antibiotic streptomycin. Binding of pactamycin between residues G693 and C795 strongly inhibits translocation with a surprising tRNA specificity but has no effect on translation initiation, suggesting that reclassification of this antibiotic is necessary. Collectively, these results suggest that the universally conserved G693:C795 residues regulate tRNA binding at the P site of the ribosome and influence translocation efficiency.     

On the mechanism for formation of RNA . DNA complexes from lymphocytes.

Early intermediates in DNA synthesis by human lymphocytes were studied for the possible association of RNA with nascent DNA. Nucleic acid extracts from cells pulse-labeled with [3H] uridine contain RNA that is associated with DNA in Cs2SO4 equilibrium density gradients. The amount of RNA bound to DNA was greatly reduced by repeated denaturation and equilibrium centrifugation. An apparently similar complex between RNA and DNA was formed in reconstruction experiments in which purified [3H] uridine-labeled RNA was mixed with purified DNA. The association between RNA and DNA could be eliminated in the reconstruction experiments and greatly reduced in extracts from pulse-labeled cells by denaturation and equilibrium centrifugation in the presence of formaldehyde. These studies demonstrate that noncovalent bonding between RNA and DNA can account for most, and possibly all, of the RNA with density close to DNA in Cs2SO4 gradients of nascent DNA preparations. In addition, the results indicate that ribonucleotide, demonstrated by other methods to be covalently bound to nascent DNA, must constitute less than 1/5 of the total nucleotide in the molecule.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.