Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

IDENTIFICATION OF CYTOKININS IN SARGASSUM MUTICUM (PHAEOPHYTA) AND PORPHYRA PERFORATA (RHODOPHYTA)1

Combined gas chromatography-mass spectrometry (GCMS) was used to identify and quantify specific cytokinins from Porphyra perforate J. Ag. and Sargassum muticum (Yendo) Fensh. The level of isopentenyladenosine was estimated to be 0.6 μ·kg^?1 fresh weight in Porphyra and 0.9 μ·kg^?1 fresh weight in Sargassum. The level of cis-zeatin riboside was estimated to be 0.2 μ·kg^?1 fresh weight in Sargassum. This is the first definitive identification of a cytokinin from a red alga, and the second report from a brown alga.     

Application of otolith microchemistry to estimate the migratory history of Japanese eel Anguilla japonica on the Sanriku Coast of Japan

The age and migratory history of the Japanese eel, Anguilla japonica Temminck & Schlegel, collected in Miyako Bay along the Sanriku coast of Japan, was examined using the otolith microstructure and analysis of strontium (Sr) and calcium (Ca) concentrations conducted with wavelength dispersive X‐ray spectrometry by an electron microprobe. The line analysis of Sr : Ca ratios along the life history transect of each otolith showed a peak (ca. 15–17 × 10^?3) which corresponded with the period of their leptocephalus and early glass eel stages in the ocean. The mean Sr : Ca ratios from the elver mark to the otolith edge indicated that there were eels with several general categories of migratory history, including sea eels that never entered freshwater (average Sr : Ca ratios, ≥6.0 × 10^?3), and others that entered freshwater for brief periods but returned to the estuary or bay. This evidence of the occurrence of sea eels in this northern area indicates that Japanese eels of the Sanriku coast do not necessarily migrate into freshwater rivers during recruitment as do glass eels at the beginning of their growth phase; even those that do enter freshwater may later return to the marine environment. Thus, anguillid eel migrations into freshwater are clearly not an obligatory migratory pathway, but rather a facultative catadromy with seawater or estuarine residents as an ecophenotype.     

Purification and characterization of extracellular soluble and membrane-bound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis.

A membrane-bound insoluble alkaline phosphatase (APase) and an extracellular soluble APase were purified, respectively, from a membrane preparation of Bacillus subtilis 6160-BC6, which carries a mutation to produce APase constitutively, and from a culture fluid of a mutant strain. RAN 1, isolated from strain 6160-BC6, which produces an extracellular soluble APase. The two preparations were homogeneous, as judged by sodium dodecyl sulfate discontinuous gel electrophoresis and by gel electrophoreses in the presence of 8 M urea at pH 9.3 and 4.3. RAN 1 APase was crystallized. Both preparations possessed phosphatase and phosphodiesterase activities, and their pH optima were both at 9.5. They were competitively inhibited by phosphate or arsenate and were activated by the addition of Ca2+ but not by Zn2+. The APase and alkaline phosphodiesterase activities seemed to be contained in the same protein molecule. The molecular weight of 6160-BC6 APase was estimated to be 46,000 +/- 1,000, and that of RAN 1 APase was estimated to be 45,000 +/- 1,000. The largest difference between the 6160-BC6 and RAN 1 APase's was in solubility in low-ionic-strength solutions. Present results suggest that each enzyme is composed of a single polypeptide chain and that 6160-BC6 APase aggregates in solutions of low ionic strength.     

Calcium ion regulates the release of lipase of Fusarium oxysporum.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.     

Crystal structure of Streptomyces erythraeus trypsin at 2.7 A resolution.

The crystal structure of Streptomyces erythraeus trypsin (abbreviated as SET) has been determined in order to clarify the precise structure of the vicinity of the active site of serine protease and to understand its structure-function relationship. Crystals of SET were prepared at its active pH range (pH 5-10) without any inhibitors which might have affected the circumstances around the active sites. The structure model of SET was made based on the electron density map obtained by the multiple isomorphous replacement method at 3.5 A resolution, and refined by the restrained least-squares method. The current model yields a crystallographic R-factor of 0.272 for 4,968 reflections between 8 and 2.7 A resolution. Though the sequence homology among SET, Streptomyces griseus trypsin and bovine trypsin, 32-37%, is not so high, their overall structures are similar to each other. Comparison of the three molecular structures shows that: 1) the folding of the main chains of the three proteins is essentially the same though there are significant differences on the molecular surface; 2) the spatial arrangements of the catalytic triads in the three proteins are similar to each other; 3) in SET and S. griseus trypsin a short stretch of 3(10)-helix is found through Ala56 to Thr59; His57 in this segment is one important amino acid residue involved in the active sites.     

Comb cutting inRopalidia plebeiana: a new process of colony fission in social wasps (Hymenoptera: Vespidae)

Summary Females of an Australian polistine wasp,Ropalidia plebeiana, often use their mandibles to cut their nest-comb in spring, dividing it into two or more completely independent nests. Prior to the division, each of the major egg layers, often with some subordinates, tended to occupy a different part of a single comb. These females gnawed cells in the intermediate zone between such territories, and ultimately divided the comb. Many other females also built new nests near the nest aggregations, but addition of new nests by comb cutting represented 34.8 % of the increase in nest number. This method of colony fission is so far unknown in any eusocial Hymenoptera.     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.     

Nonradioactive detection of nucleic acid by the universal probe system

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.