Response to Gibberellin Structural Variants Shows that Ability to Inhibit Flowering Correlates with Effectiveness for Promoting Stem Elongation of Some Plant Species

The flowering response of three plant species Fuchsia hybrida, Pharbitis nil, and Spathiphyllum "Petite" has been examined after treatment with synthetic and natural gibberellins (GAs) including GA1, GA3, GA5, 2,2-dimethyl GA4, various of their 3a-hydroxyl epimers, the methyl ester of GA3, a 15b-hydroxyl GA3, and some 16,17-dihydro derivatives. Of necessity, application techniques differed between species and dose responses cannot be compared. However, comparisons across species were possible on the basis of their differential responses to the various GAs. Flowering was inhibited and, in an inverse way, stem elongation promoted when GA3 or GA5 was applied to Fuchsia or, at high doses, to Pharbitis. The increased stem growth was apparently responsible for inhibition of flowering because epimerization of the hydroxyl at C-3 of GA3 or modification of GA5 to 16,17-dihydro GA5 created compounds that were growth inactive and that no longer inhibited flowering. However, the response of Pharbitis was more complex because both these GAs and their derivatives promoted flowering at doses subthreshold for growth. For Spathiphyllum, only promotion of flowering was evident with any GA, and the structural variants were slightly active or inactive. Thus, there are at least two antagonistic actions of GA on flowering and for Pharbitis either inhibition or promotion can be shown. The possibility is discussed that the inhibitory action of GA on flowering involves diversion of assimilate away from the shoot apex and into the elongating stem.     

NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using (19)F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K(d), of isoflurane binding to nAChR at 15 degrees C was found to be 0.36 +/- 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X(b), determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T(2)) of (19)F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k(-1), is 1.7 x 10(4) s(-1). The on rate, k(+1), can thus be calculated to be approximately 4.8 x 10(7) M(-1) s(-1), suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k(+1) and k(-1) are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.     

Cloning of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from terpenoid antibiotic-producing Streptomyces strains

We have isolated a mutant lacking 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity from a terpenoid antibiotic (terpentecin) producer, Streptomyces griseolosporeus MF730-N6, which uses both the mevalonate and nonmevalonate pathways for the formation of isopentenyl diphosphate, by screening terpentecin non-producing mutants. Terpentecin is known to be synthesized via the mevalonate pathway. The gene encoding HMG-CoA reductase (hmgg) was cloned and identified by complementation of the mutant, using a self-cloning system developed in this study for strain MF730-N6. The corresponding hmgs gene for HMG-CoA reductase was also cloned from Streptomyces sp. KO-3988, which produces the terpenoid antibiotic furaquinocin. Sequence analysis of hmgg and hmgs showed that both genes encode polypeptides of 353 amino acids which are 84% identical to each other. A search of protein sequence databases revealed that both gene products were also similar to HMG-CoA reductases from a variety of other organisms, including Streptomyces sp. CL190 (hmgg is 89% and hmgs 85% identical to its CL190 homolog), sea urchin (40.3 and 40.5%), German cockroach (37.6 and 38.4%), and Camptotheca acuminata (39.7 and 40.8%). Received: 17 May 1999 / Accepted: 10 September 1999     

Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis

The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The beta-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.     

Cell Reproduction and Morphological Changes in Mycoplasma capricolum

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.Mycoplasmas are parasitic bacteria that have extremely low G+C contents and small genomes (9). Their morphology is irregular because of the lack of a peptidoglycan layer.In Escherichia coli, initiation of chromosomal DNA replication occurs once during the cell’s replicative cycle, and the nucleoids partition before cell division (13). The chromosomal replication of E. coli initiates in a small region and proceeds in both directions. It is mainly controlled by the timing and frequency of initiation, while the velocity of replication is constant.In mycoplasmas, chromosome replication also starts at a fixed site, followed by bidirectional progression (19–21, 25, 40). As in many eubacteria (36), the dnaA gene is expressed and plays important roles in the initiation of replication (35). These observations suggest that the outline of chromosome replication of mycoplasmas is similar to that of E. coli. However, the process of mycoplasma cell reproduction has not been clarified. Moreover, the cell division cycle of E. coli cannot be simply applied to mycoplasmas because of their irregular cell morphology (4). A model has been suggested for the cell cycle of Mycoplasma mycoides (6, 30, 31), which is closely related to Mycoplasma capricolum (39). According to this model, an elementary rounded body grows into a filamentous form and then new elementary rounded bodies are developed within this filament and released, but this model has not been adequately substantiated.In this study, we analyzed the process of DNA replication, cell morphology, and viability under various conditions of M. capricolum and proposed a model of cellular reproduction for this bacterium.