The roles of the two highly unstable components F and P involved in the bioluminescence of euphausiid shrimps

Bioluminescence of euphausiids takes place when a fluorescent tetrapyrrole F and a highly unstable protein P react in the presence of oxygen. A previous study on the euphausiid Meganyctiphanes norvegica indicated that F acts as a catalyst and P is consumed in the luminescence reaction, differing from the luminescence system of dinoflagellates in which a tetrapyrrole luciferin, nearly identical to F, is enzymatically oxidized in the presence of dinoflagellate luciferase. In the present study, P was extracted from Euphausia pacifica as well as from M. norvegica, then purified separately by affinity chromatography on a column of biliverdin–Sepharose 4B, completing the whole process in less than 5h. The samples of P obtained from both species had a molecular weight of 600,000, a purity of about 80%, and a specific activity 50–100 times greater than that previously found. The activity of P rapidly decreased in solutions, even at 0°C, and the inactivation of P derived from M. norvegica was more than four times faster than that derived from E. pacifica. The kinetics of the luminescence reaction was investigated with F and P whose concentrations were systematically varied. The reaction was characteristically slow and involved two different reaction rates; the turnover number at 0°C was 30/h for the initial 20 min and 20/h after the initial 1 h. The total light emitted in a 50-h period indicated that the bioluminescence quantum yield of F was about 0.6 at 0°C, and P recycled many times in the luminescence reaction. Thus, the present results conclusively show that F is a luciferin and P is a luciferase of an unusually slow-working type, contrary to early report.     

Substrate Preference in a Strain of Megasphaera elsdenii, a Ruminal Bacterium, and Its Implications in Propionate Production and Growth Competition

The NIAH 1102 strain of Megasphaera elsdenii utilized lactate in preference to glucose when the two substrates were present. Even when lactate was supplied to cells fermenting glucose, the cells switched substrate utilization from glucose to lactate and did not utilize glucose until lactate decreased to a low concentration (1 to 2 mM). Since substrate utilization was shifted gradually without intermittence, typical diauxic growth was not seen. The cyclic AMP content did not rise markedly with the shift in substrate utilization, suggesting that this nucleotide is not involved in the regulation of the shift. It was unlikely that propionate was produced from glucose, which was explicable by the fact that lactate racemase activity dropped rapidly with the exhaustion of lactate and cells actively fermenting glucose did not possess this enzyme. A coculture experiment indicated that M. elsdenii NIAH 1102 is overcome by Streptococcus bovis JB1 in the competition for glucose, mainly because M. elsdenii NIAH 1102 is obliged to utilize lactate produced by S. bovis JB1; i.e., glucose utilization by M. elsdenii NIAH 1102 is suppressed by the coexistence of S. bovis JB1.     

A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.     

Flower-Inducing Activity of Exogenous Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Rich Medium

Flower-inducing activity of lysine was examined in Lemna paucicostata151, a weakly responsive short-day plant, cultured on nitrogen-richmedium under long-day conditions (continuous light). Lemna paucicostata151 was homogenized in a solution of lysine and the homogenatewas centrifuged. The supernatant (lysine-containing extract)was added to nitrogen-rich medium after passage through a membranefilter to give various concentrations of lysine in the medium.Flowering was induced in plants grown for six days on mediumthat contained lysine at concentrations above 0.25 µM.In plants grown on medium that contained 1 µM lysine,a significant flowering response was observed on the fourthday of culture. However, the flower-inducing activity of lysinedisappeared when the lysine-containing extract was added tothe medium and the medium was then autoclaved, suggesting thatthe active principle is unstable to autoclaving. Among derivativesof lysine tested, lysine hydroxamate had the highest flower-inducingactivity and lysyl lysine had almost same activity as that oflysine. When added to the medium without homogenization withplant material, lysine and lysyl lysine had flower-inducingactivity but lysine hydroxamate did not induce flowering. (Received April 26, 1993; Accepted November 8, 1993)     

Spatial distribution of root activity and nitrogen fixation in sorghum/pigeonpea intercropping on an Indian Alfisol

A medium-duration pigeonpea cultivar (ICP 1–6) and a hybrid sorghum (CSH 5) were grown on a shallow Alfisol in monocropping and intercropping systems. Using a monolith method, spatial distribution of nodulation, acetylene reduction activity (ARA) and root respiration were measured.The number, mass and ARA of nodules decreased exponentially with distance from the plant base except at the late reproductive stage. Nodulation and ARA tended to be higher in the intercrop than in the monocrop.Respiration rate of roots increased with distance from the plant base and reached a maximum value at about 20–30 cm. The rate was higher in pigeonpea than in sorghum and also higher in intercrop than in monocrop.This study suggests that pigeonpea roots are physiologically more active than sorghum roots, implying that pigeonpea may become a strong competitor for nutrients in the soil when intercropped. The nitrogen-fixing ability of pigeonpea may be enhanced by intercropping because the sorghum rapidly absorbed inorganic N which would otherwise inhibit N₂ fixation.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

cis-Elements and Protein Factors Related to Regulation of Transcription of Arylsulfatase Gene during Sea Urchin Development

Eight restriction fragments (I–VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus , and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between −2.8 kb and −2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis -acting elements that interact with nuclear proteins in a sequence-specific manner. Among them, two sequences, a G-string and a GATCTCCCC, were determined by DNA footprinting as sites of protein-DNA interaction. The DNA-binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA-binding proteins through their modification is discussed.