Selection for mutants with low nitrate uptake ability in rice (Oryza sativa)

Screening for mutants deficient in the high affinity system of nitrate uptake was performed using mutagenized M₂ population of rice ( Oryza sativa , cv. Nipponbare or Kinmaze). For selecting mutants, M₂ seedlings were transferred individually to 10 ml solution containing 250 μ M potassium nitrate and 500 μ M calcium sulphate at 20 or 28°C. After 6 or 24 h, nitrate concentration of the solution was determined with a nitrate selective electrode and the seedlings showing impaired nitrate uptake were selected as nitrate uptake deficient variants. Of 74 variants, three were confirmed to be mutants with low nitrate uptake ability in the M₃ generation. Potassium uptake ability also decreased in the mutants. Three mutants were divided into two groups based on the analysis of nitrate reductase (NR, EC 1.6.6.1) activity and chlorate resistance. Two, NUE13 and NUE36 , had a lower level of NR activity than the original cultivar and were not resistant to chlorate, while the seedlings of NUE50 had the same level of NR activity as the original cultivar and were more resistant to chlorate than the original cultivar. All mutants were resistant to cesium, a toxic ion analogue for potassium, suggesting that the decreased levels of both nitrate and potassium uptake were coupled to the change of plasma membrane H⁺-ATPase activity.     

Molecular Cloning and Characterization of the cDNA for Discoidin II of Dictyostelium discoideum

By using monoclonal antibodies directed against discoidin II,we have isolated cDNA clones from axenically grown Ax-2 cells.On cDNA clone (D2) condtained a 1.2-k.b insert encoding theentire discoidin II protein, which is conposed of 257 aminoacid residuces and has a calculated molecular mass of 28,574.The amino acid sequences, determined by Edman degradation ofsix tryptic peptides of discoidin II, were identical to thosededuced from the cDNA sequences. The protein bears no resemblanceto any proteins in the data banks, except that its sequenceis 49% identical with the amino acid sequence of discoidin I.Discoidin II shares with discoidin I both a carbohydratebindingsite and an Arg-Gly-Asp (RGD) sequence, which has been foundin fibronectin in mammalian cells. With the onset of aggregation(8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate.A similar pattern of regulation occurs at the protein level. ¹Present address: MRC Laboratory for Molecular Cell Biology,University College London, Gower Street, London, WC1E 6BT UnitedKingdom     

Dimer Formation of a Cell-Cell Adhesion Protein, gp64 of the Cellular Slime Mold, Polysphondylium pallidum

The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistanttypes of cell-cell adhesion. The major component of them hasbeen identified as a glycoprotein with a molecular mass of 64kDa on SDS-PAGE (referred to as gp64). We found that a substantialamount of the gp64 run as dimer, when gp64 was dissolved inSDS-sample buffer without 2-mercaptoethanol and then subjectedto electrophoresis. The occurrence of a homophilic dimer wasdemonstrated by analyzing the dimer-like band on a gel for itsamino acid sequence and amino acid composition. The dimer-likeband also was analyzed by three sorts of monoclonal antibodies,two of which recognize respectively a conforniational epitopeand a denatured epitope of the protein moiety of gp64. The dataindicate that the native conformation of gp64 is necessary fordimer formation. ²Present address: Institute of Immunological Science, HokkaidoUniversity, Sapporo, 060 Japan     

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

A new process for the simultaneous enzymic synthesis and purification of N-(benzyloxycarbonyl)- -aspartyl- -phenylalanine methyl ester (ZAPM), a precursor of aspartame, has been developed. The enzymic reaction between N-(benzyloxycarbonyl)- -aspartic acid (ZA) and -phenylalanine methyl ester (PM) was carried out in a biphasic hollow-fibre rector with an aqueous phase an a butyl acetate phase. The reaction took place in the aqueous phase and by maintaining the pH at 5, the product (ZAPM) was extracted into the organic phase. Product purity was greater than 90% and reasonable productivity could be achieved with this system.     

Interannual fluctuations in recruitment and growth of the sardine,Sardinops melanostictus,in the sea of Japan and adjacent waters

Recruitment and growth of the sardineSardinops melanostictus fluctuated markedly in the Sea of Japan and adjacent waters between 1978 and 1993. Stock size was calculated using Virtual Population Analysis and average body length in each age class was determined by the number of annual rings on the scales. There is an inverse correlation between average water temperature at a depth of 50 m in the coastal area of the mainland of Japan in winter (January to March) and recruitmentR defined as the number of individuals at 1 year old. There is also an inverse correlation between spawning stock sizeE and reproductive success in (R/E). A multiple regression model using spawning stock size and water temperature in winter as independent variables can explain 73% of variance in reproductive success. It suggests that both density-dependent and density-independent factors perform important roles determining reproductive success. There is an inverse correlation between body length and stock size and this suggests that there is a density-dependent effect on the growth of the sardine.     

Exercise-induced splitting of the inorganic phosphate peak: investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy

To investigate the splitting of the inorganic phosphate (Pi) peak during exercise and recovery, a time-resolved ³¹phosphorus nuclear magnetic resonance spectroscopy (³¹P-MRS) technique was used. Seven healthy young sedentary male subjects performed knee flexion exercise in the prone position inside a 2.1-T magnet, with the surface coil for ³¹P-MRS being placed on the biceps femoris muscle. After a 1-min warm-up without loading, the exercise intensity was increased by 0.41 W at 15-s intervals until exhaustion, followed by a 5-min recovery period. The ³¹P-MRS were recorded every 5 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of the Pi and phosphocreatine peaks were performed. Five of the seven subjects showed two distinct Pi peaks during exercise, suggesting two different pH distributions in exercising muscle (high pH and low pH region). In these five subjects, the high-pH increased rapidly just after the onset of exercise, while the low-pH peak increased gradually approximately 60 s after the onset of exercise. During recovery, the disappearance of the high-pH peak was more rapid than that of the low-pH peak. These findings suggest that our method ³¹P-MRS provides a simple approach for studying the kinetics of the Pi peak and intramuscular pH during exercise and recovery.     

Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 M, whereas the IC₅₀ value was 15 M for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly Ser) in the subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.