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B M Olivera

Publications and source records attributed to B M Olivera.

At least 145 records · Page 8Linked to original sources
PubMed1984

Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives.

The 13 amino acid toxic peptide from the marine snail Conus geographus, conotoxin GI, blocks the acetylcholine receptor at the neuromuscular junction. In this report, we describe a method for analyzing disulfide bonding in nanomole amounts of small cystine-rich peptides. The procedure involves partial reduction and a double-label alkylation of cysteine residues. Using this method, we show that the natural conotoxin GI has a (2-7, 3-13) disulfide configuration. The structure of conotoxin GI has been confirmed by chemical synthesis. The preparation and purification of molecularly homogeneous, iodinated derivatives of this toxin are also described. All derivatives, including the [diiodohistidine,diiodotyrosine]conotoxin GI, retained at least half of the biological activity of unmodified toxin. Since the tetraiodinated toxin, which is greater than 25% by weight iodine, retains considerable toxicity, unmodified histidine and tyrosine residues in conotoxin GI are not crucial for biological activity.

Amino Acid Sequence↗
PubMed1984

Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus.

We demonstrate that the activity of the major DNA topoisomerase I from calf thymus is severely inhibited after modification by purified poly(ADP-ribose) synthetase. Polymeric chains of poly(ADP-ribose) are covalently attached to DNA topoisomerase I. These observations with highly purified enzymes suggest that poly(ADP-ribosylation) may be a cellular mechanism for modulating DNA topoisomerase I activity in response to the state of DNA in the nucleus. Although extensive poly(ADP-ribosylation) of the Mr = 100,000 DNA topoisomerase I from calf thymus resulted in greater than 90% enzyme inhibition, exogenous poly(ADP-ribose) does not, by itself, inhibit topoisomerase activity. After modification, the apparent molecular weight of both the topoisomerase enzyme protein and of the topoisomerase enzyme activity was increased. In vitro, the extent of modification of DNA topoisomerase I could be controlled either by changing the ratio of topoisomerase to the synthetase or by varying the reaction time. More than 40 residues of ADP ribose per topoisomerase molecule could be added by the synthetase. Analysis of a poly(ADP-ribosylated) topoisomerase preparation that was about 50% inhibited revealed an average polymer chain length of 7.4, with 1-2 chains per enzyme molecule.

Animals↗
PubMed1984

Poly (ADP-ribosylation) and DNA topoisomerase I in different cell lines.

The modification of DNA topoisomerase I activity by poly (ADP-ribosylation) is a potentially important control mechanism for this ubiquitous enzyme which is widely believed to play a role in replication (2, 3). Our studies of the phenomenon so far suggest that DNA topoisomerase I activity may be regulated in response to the state of the DNA in the nucleus since the modification enzyme, poly (ADP-ribose) synthetase, is sensitive to interruptions in the normal double helical structure of DNA (10). Although this phenomenon has been most extensively characterized for the purified enzymes from calf thymus (5), preliminary evidence for poly (ADP-ribosylation) of topoisomerase I is presented in a nonthymic system, the mammalian cell line CHO EM9. Attempts to detect poly (ADP-ribosylation) in yeast cells have so far proven to be unsuccessful.

Animals↗
PubMed1984

Analysis of mu DNA replicated on cellophane discs.

We have presented restriction analyses which indicate that the first transposition event after induction can be carried out and analyzed in vitro using cellophane disc lysates. This would be a first step in describing in molecular terms a "wiring diagram" of replicative transposition.

Bacteriophage mu↗
PubMed1984

Mu DNA replication in vitro: criteria for initiation.

An in vitro system for investigating Mu replication nd transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates. Two conditions reported to specifically decrease Mu DNA replication in vivo ( Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.

Bacteriophage mu↗
PubMed1984

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions.

In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.

Anaerobiosis↗
PubMed1983

Conotoxin MI. Disulfide bonding and conformational states.

The toxic peptide from Conus magus venom (conotoxin MI) is a 14-amino acid peptide (McIntosh, M., Cruz, L. J., Hunkapiller, M. W., Gray, W. R., and Olivera, B. M. (1982) Arch. Biochem. Biophys. 218, 329-334) which inhibits the acetylcholine receptor. In this work we have confirmed the primary structure and established the disulfide bonding configuration (Cys 3-Cys 8; Cys 4-Cys 14) by direct chemical synthesis of the toxin with specific disulfide bridges. Natural and synthetic toxins were compared by several methods. Fast atom bombardment mass spectroscopy confirmed that the synthetic product had the expected molecular mass and number of exchangeable hydrogens. Ultraviolet CD spectra were closely comparable in shape and magnitude for the two materials, which were also identical in biological activity and chromatographic behavior. We have also established that, although the peptide is highly cross-linked with two disulfide bridges, it can slowly equilibrate between two conformations. A simulation analysis suggests that the conformers have half-lives of approximately 12 and approximately 72 min at 0 degrees C, decreasing approximately 2-fold for every 10 degrees C increase in temperature.

Amino Acid Sequence↗
PubMed1983

Flexibility in RNA priming of Okazaki pieces at the E. coli replication fork.

We present results which suggest considerable flexibility in the RNA priming of Okazaki pieces at the E. coli replication fork. Using film lysates on cellophane discs, we have identified RNA at the 5' ends of Okazaki pieces. All four ribonucleotides are found to be present at the RNA-DNA junction if all four ribonucleoside triphosphates are used. However, if only ATP, or ATP and GTP are used, then only 2' (3')AMP, or 2' (3')AMP and 2' (3')GMP are found at the RNA-DNA junction. A nearest neighbor analysis of RNA associated with Okazaki pieces using alpha 32P-CTP as a probe shows a similar dependence of nearest neighbor composition on the ribonucleoside triphosphate composition of the incubation mixture. Thus, the nucleotide composition of the RNA primers at the ends of Okazaki pieces varies as a function of the ribonucleoside triphosphates available.

DNA↗
PubMed1983

Poly(ADP-ribosylation) of a DNA topoisomerase.

A DNA topoisomerase activity, copurifying with poly(ADP-ribose) synthetase from calf thymus, is greater than 95% inhibited if extensive poly(ADP-ribosylation) is allowed to occur. The inhibited DNA topoisomerase, which has drastically different elution properties on hydroxylapatite, can be reactivated by mild alkaline treatment. These results are consistent with a poly(ADP-ribosylation) of the DNA topoisomerase and covalent attachment of the poly(ADP-ribose) moieties to the topoisomerase by alkali-labile bonds.

Animals↗
PubMed1983

Bacteriophage Mu DNA replication in vitro.

An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.

Coliphages↗
PubMed1983

Bacteriophage Mu: a transposing replicon.

Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP. If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions. The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA. This and previous data are consistent with replication occurring within Mu boundaries. Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs. These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro.

Bacteriophage mu↗
PubMed1983

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium.

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.

6-Aminonicotinamide↗
PubMed1983

An indispensable gene for NAD biosynthesis in Salmonella typhimurium.

We have located the nadD locus between lip and leuS at 14 min on the Salmonella typhimurium chromosome, and we have shown it to be the structural gene for nicotinic acid mononucleotide adenylyltransferase. This is the first indispensable gene of pyridine nucleotide metabolism that has been identified. Mutants altered at this locus, isolated by their 6-aminonicotinamide resistance phenotype, accumulate abnormally large pools of nicotinic acid mononucleotide in vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assays reveal markedly lower transferase activity in mutant extracts than in nadD+ extracts. The partial dominance of nadD mutants when placed in a nadD+/nadD diploid suggests that nicotinic acid mononucleotide adenylyltransferase is a multimeric enzyme.

Amide Synthases↗
PubMed1982

Transient generation of displaced single-stranded DNA during nick translation.

We show that displaced single-stranded overhangs are transiently generated and destroyed during nick translation by E. coli DNA polymerase I. Evidence that hyper-rec mutants have an increased frequency of such overhang structures is discussed. The transient generation of overhangs may be significant for general recombination. The 5' leads to 3' exonuclease activity of polymerase I specifically hydrolyzes such overhangs to yield a nick. Overhangs are generated by polymerization, but after every polymerization step, either polymerase or exonuclease can act--55% of the time, polymerization occurred first. At this frequency overhangs of greater than or equal to 12 nucleotides are generated every 1300 nucleotides polymerized. We suggest that many DNA strand discontinuities are displaced single-stranded overhangs, rather than gaps or simple nicks.

Bacteriophage phi X 174↗
PubMed1982

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium.

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.

NAD↗