35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Tree killing by herbicide producing ants for the establishment of pureTococa occidentalis populations in the Peruvian Amazon

Populations ofTococa occidentalis (Melastomataceae) and the inhabiting ants (Myrmelachista sp.) were observed for more than eight months in the Peruvian Amazon (Sira mountains). They represent a complex coevolutionary system: the plants offer shelter (leaf domatia, hollow stems) and food (leaf glands), whereas the ants kill all surrounding plants, including large trees up to 10 m, by chemical weapons. Experiments with exposed plants revealed a highly specialized way to attack meristematic tissue and leaf nervature, which leads to a quick decay of the plant individuals. The clearing of the vegetation by the ants allows theTococa population to expand mostly by vegetative shoots to large monocultures (up to 30 m in diameter) free from any other plant species. Artificially introduced plant individuals, from differentT. occidentalis populations, are regarded as a foreign species by the ants.The succession of such aTococa-Myrmelachista system begins with one or a few founder plants on a light place in the midst of the vegetation.Myrmelachista soon inhabits their host plants which otherwise would not survive and begin to clear the place from all foreign plant species.Tococa expands quickly, forming circle shaped populations. Distantly situated canopy trees shade theTococa population after a number of years and cause their decay. The whole place appears contaminated for years and no other plant can establish itself. Some of the consequences of these open places are erosion and a severe influence on the regeneration of the forest.     

Observations on egg production rates and seasonal changes in the internal morphology of Mediterranean populations of Acartia clausi and Centropages typicus

Egg production rates in wild populations of Acartia clausi and Centropages typicus, sampled biweekly in the Gulf of Naples from October 1985 to July 1987, showed marked seasonal fluctuations with maximum values in early spring that proceeded the annual maxima for adult female densities in summer. A positive correlation between chlorophyll a concentrations and egg production was evident only during the early spring phytoplankton bloom. A strong diminution in egg deposition occurred later in spring and continued throughout the summer notwithstanding high chlorophyll concentrations. In winter, when population abundances for adult females were lowest, egg production rates were always higher than in summer. Differences in egg production rates coincided with pronounced morphological changes between summer and winter populations of both species. The most striking of these changes consisted, in winter, in the presence of a dark brown fluid-like mass of granular material that seemed to freely bathe the gonads. The presence of this substance only during periods of elevated egg production suggests that it may enhance egg production rates when the adult population reaches minimum annual levels. Such a mechanism of self-regulation may operate to dampen the effects of environmental variability thereby contributing to maintain a conservative structure in coastal copepod communities.     

Cell motility measurements with an automated microscope system

The motility of 3T3 cells has been studied using a newly developed automated microscope system which is capable of recognizing live unstained cells growing in tissue culture. A large number of individual cells can be rapidly identified and characterized and their precise positions recorded. All cells can be revisited automatically every few minutes, and the new cell positions can be determined. Quantitative data from up to 1 000 cells can then be obtained, and cell movement parameters like cell speed, distance travelled, direction of movement, etc., can be measured for individual cells and for the whole cell population. In addition, for any number of chosen cells, high-resolution digitized images can be taken for further morphological studies, including acquisition of images of individual cells.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

Toxigenic Potential of Some Fusarium Isolates from Southeast Asia

The isolation and characterization of 10 isolates of six Fusarium spp. from plant and soil samples collected in Southeast Asia is reported. The ability of these isolates to produce trichothecenes both in liquid cultures (CZ, GYEP, and MYRO) and on rice was assessed, and their toxigenic potential was examined by skin assay and gavage studies with culture filtrates. Although culture filtrates of all the isolates caused minor damage to test animals, only that of F. equiseti DAOM 189762 produced trichothecenes.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10^-8 s up to 10^-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 10³, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.Abbreviations used are ESR electron spin resonance - St-ESR saturation transfer electron spin resonance - GDGT glyceroldialkyl-glycerol-tetracther - GDNT glycerol-dialkyl-nonitoltetraether - 5 SASL 12SASL and 16SASL, stearic acid spin labels, N-oxyl-4,4-dimethyloxazolidine derivatives of 5-ketostearic acid, 12-ketostearic acid and 16-ketostearic acid, respectively - DSC differential scanning calorimetry