Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

Formation of a stable and catalytically active complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26

Formation of a stable complex of the two essential components of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26, which represents the catalytically active state of this enzyme, is observed in the presence of a relatively high concentrations of inorganic pyrophosphate or one of the substrates, isopentenyl diphosphate or farnesyl diphosphate. The apparent molecular mass of the complex is estimated to be about 50 kDa by gel filtration with Superose 12.     

Facilitation of electrofusion of mouse lymphoma cells by the proteolytic action of proteases

Cell fusion of mouse lymphoma (L5178Y) was achieved by applying electrical pulses under dielectrophoresis. The presence of dispase, pronase or trypsin facilitated the electric pulse-induced cell fusion. Heat-inactivated pronase was no longer effective. Protease inhibitors (aprotinin and p-tosyl-L-lysine chloromethylketone) suppressed the effect of trypsin. Even in the absence of proteases, the cells pretreated with dispase or pronase underwent fusion with high probabilities, as far as free calcium ions were present in the external solution. It is concluded that facilitation of electrofusion by proteases is due to their proteolytic activities.     

Undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26: essential factors for the enzymatic activity

An undecaprenyl diphosphate synthase fraction, which was free of other prenyltransferases and was active without the addition of detergent or phospholipid, was obtained by Sephadex G-100 chromatography of cell-free extracts of Micrococcus luteus B-P 26 cells. The addition of small amounts of Triton X-100 to this fraction caused a marked loss of the enzyme activity, but the activity was gradually restored as further detergent was added. When the enzyme fraction was chromatographed on DEAE-cellulose, the synthase was partially purified, but the activity was not detected unless assayed with addition of the detergent or a lipid fraction of this bacterium. Among the three phospholipids isolated from this bacterium, cardiolipin and phosphatidylglycerol had a marked effect in activating lipid-depleted undecaprenyl diphosphate synthase, but O-lysylphosphatidylglycerol, which occurs prominently in this bacterium, had little effect.     

Membrane potential changes associated with differentiation of enterocytes in the rat intestinal villi in culture

Fragmented villi of the small intestine isolated from newborn rats were maintained in culture for periods of up to 4 weeks. In culture, floating globular villi and adherent villi were distinguished. Intracellular recordings were made from both types of villi. The membrane potential was highest (about ?70 mV) in the cells at the tip of the villus and was lowest (about ?18 mV) in the cells near the crypt region, showing a continuous gradation according to the cell location. The membrane potential in cultured mature enterocytes was due to the contribution of the electrogenic Na⁺ pump as well as to the Ca²⁺-activated K⁺ conductance. Both the components were temperature and metabolic energy dependent. Based on these data, it is suggested that the Ca²⁺ transport mechanism and the electrogenic Na⁺ pump are involved in the process of cell differentiation or maturation of the intestinal epithelia.     

Biochemical Studies on Fatty Liver

The relation of the fatty liver with some enzyme activities concerning the amino acid metabolism was investigated. Fatty livers were produced by an amino acid imbalanced diet containing 8% of casein supplemented with 0.3% of DL-methionine (threonine deficient), and serine dehydrase ( = threonine dehydrase = cystathionine synthetase), homoserine dehydrase ( = cystine splitting enzyme = cystathionase), and threonine aldolase activities were determined.

Under this condition, the threonine aldolase activity was hardly altered, but the serine dehydrase and the homoserine dehydrase activities were fairly variable. However, the variation of these enzyme activities did not seem to have appreciable relation with the fatty liver, but rather had a connexion with the dietary protein level or calory content.     

Enzymatic Synthesis of Trehalose from Maltose

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.

Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.     

Structure of Malonogalactan: Carbon-13 Nuclear Magnetic Resonance Spectra of Malonogalactan

Six kinds of arachin subunits were isolated by isoelectric focusing in sucrose density gradient in the presence of 6 m urea and 0.2 m β-mercaptoethanol. These subunits (S₁ to S₆) had different isoelectric points (pI 5.8, 6.0, 6.3, 7.1, 7.4 and 8.3) and they were found in parent arachin in the weight ratio of 2.5 : 2.2 : 2.6 : 1.6 : 1.1 : 1.0. The molecular weights and N-terminal amino acids of the isolated subunits were 35,500 and valine (S₁), 37,500 and isoleucine (S₂), 40,500 and isoleucine (S₃) and 19,500 and glycine (S₄, S₅ and S₆). While, the parent arachin contained approximately one mole of valine, two moles of isoleucine and three moles of glycine as the N-terminal amino acids per 180,000 g. It is, therefore, concluded that arachin consists of six different subunits to form the molecular weight 180,000. No disulfide bond takes part in the subunit association of arachin, because the dissociation in a urea solution occurred in the absence of β-mercaptoethanol.     

The Structure of Leucanicidin,a Novel Insecticidal Macrolide Produced by Streptomyces halstedii

( – )-Invictolide [(3R,5R,6S,1′R)-3,5-dimethyl-6-(1′-methylbutyl)-tetrahydro-2H-pyran-2-one] was synthesized in 16 steps from 2-methylpentanal.