Continuous synthesis of the dnaA gene product of Escherichia coli in the cell cycle

Summary The dnaA gene product of Escherichia coli, identified as a weakly basic protein of about 48,000 daltons (Yuasa and Sakakibara 1980), can be separated from other celluar proteins by means of two-dimensional gel electrophoresis. Synthesis of the dnaA protein took place continuously during a cell growth cycle. The newly synthesized dnaA protein persisted stably for one generation. Thermosensitive dnaA protein produced by the dnaA167 mutant was stable at 30° C, but was disintegrated at 42° C. The amount of intact dnaA protein present in the mutant exposed to the high temperature for 60 min was less than a quarter of the amount at the time of the shift. The cells having the reduced amount of intact dnaA protein were capable of initiating a new round of chromosome replication at the low temperature without de novo synthesis of the dnaA protein. The potential of the mutant for initiation of DNA replication decreased with reduction in the amount of the thermoreversible dnaA protein. The mutations dnaA167 and dnaA46 had no significant effect on the syntheses of the dnaA mRNA and the protein product at the low and high temperatures.Abbreviations used SDS sodium dodecyl sulfate - kb kilobase pairs - TCA trichloroacetic acid     

Seven guaianolides from Eupatorium chinense

Two allyl hydroperoxy guaianolide sesquiterpene lactones (peroxyeupahakonin-A and -B) and five new guaianolides (eupahakonin-A and -B, eupahakonenin-A and -B, and eupahakonesin) were isolated from E. chinense and characterized. The allyl hydroperoxy sesquiterpene lactones were characterized by spectral and chemical methods. They were prepared chemically by photosensitized oxygenation of eupahakonin-A.     

Primary structure of mitochondrial glutamic oxaloacetic transaminase from rat liver: Comparison with that of the pig heart isozyme

The complete amino acid sequence of the mitochondrial glutamic oxaloacetic transaminase isozyme from rat liver is presented. The sequence contained 401 amino acid residues, 10 of which are methionine. Cyanogen bromide cleavage of mitochondrial glutamic oxaloacetic transaminase produced 12 peptides, one of which contained an internal homoserine residue resulting from incomplete cleavage by cyanogen bromide. The calculated molecular weight was 44,358. The sequence showed 94% homology with that of the corresponding isozyme from pig heart. These findings support the conclusion that the rate of evolution of the mitochondrial isozymes is lower than that of their cytosolic isozymes.     

Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.     

Differential Effects of Nitrate and Light on the Expression of Glutamine Synthetases and Ferredoxin-Dependent Glutamate Synthase in Maize

The effects of nitrate and light on the expression of genesfor glutamine synthetase (GS) isoproteins and ferredoxin-dependentglutamate synthase (Fd-GOGAT) were studied in different organsof maize seedlings by analyzing the levels of the respectivepolypeptides and mRNAs. In roots, the levels of plastidic GSand of a novel, root-specific GS molecule localized in the extraplastidiccompartment were increased markedly by nitrate, whereas Fd-GOGATand cytosolic GS remained at their initial levels. Ammonia wasnot effective in inducing the plastidic GS and Fd-GOGAT butit did induce the novel GS isoprotein. In leaves, cytosolicand plastidic GSs and Fd-GOGAT were present in both mesophyllcells (MC) and bundle sheath cells (BSC). Upon addition of nitrate,the level of plastidic GS increased preferentially in MC, andupon exposure of etiolated seedlings to light, the levels ofplastidic GS and Fd-GOGAT increased in BSC in a coordinatedmanner. The relationship between the expression of genes forGSs and Fd-GOGAT and the physiological role of the GS/GOGATcycle is discussed in terms of the characteristics of nitrogenmetabolism in roots, MC, and BSC. (Received August 11, 1992; Accepted September 21, 1992)     

Coordinate expression of Escherichia coli dnaA and dnaN genes

Summary The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lysogenic dnaN59 cells at the nonpermissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene fuction. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA ^- dnaN ⁺ phages remains weak upon simultaneous infection with dnaA ⁺ dnaN ^- phages. Thus the insertion and deletion in the dnaA gene influence in cis the expression of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter.     

Lignans of Larix leptolepis

Five lignans have been isolated from wood of Larix leptolepis. They are identified as 1-(4-hydroxy-3-methoxyphenyl)-2-4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy-propane- 1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-2-methoxy-4-[1-(E)-propen-3-ol]-phenoxy- propane-1,3-diol, 1-(4-hydroxy-3-methoxyphenyl)-2-(4-formyl-2-methoxyphenoxy)-propane-1,3-diol, 1,2-bis-(4-hydroxy-3-methoxyphenyl)-propane-1,3-diol and a trilignol, leptolepisol C.     

Strategy for the synthesis of large peptides: An application to the total synthesis of human parathyroid hormone [hPTH(1–84)]

A strategy suitable for the synthesis of larger peptides is proposed. It involves the following four considerations: (1) all of the side-chain functional groups are protected by benzyl-type protective groups; (2) a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, is used for the fragment-condensation reactions together with 1-hydroxybenzotriazole as the additive; (3) all the protective groups are cleaved simultaneously by the HF method in the final stage of the synthesis; and (4) side products formed are detected and removed by an efficient high-performance liquid chromatography procedure. The usefulness of these procedures is demonstrated taking the synthesis of human parathyroid hormone [hPTH(1–84)] as an example.