A heat-shock promoter fusion to the Ac transposase gene drives inducible transposition of a Ds element during Arabidopsis embryo development

A heat-shock promoter fusion to the Ac transposase gene (hs::TPase) was constructed and introduced into Arabidopsis. In five transformants containing the fusion the abundance of transposase mRNA increased approximately 120-fold on exposure to high temperatures. Hybrid plants containing hs::TPase and a Ds element inserted in a streptomycin resistance gene (Ds::SPT) were made and these plants were self-fertilized either after heat shocking at different stages in development or without exposure to high temperature. The progeny of these plants were sown on streptomycin-containing medium and the frequency with which variegated or streptomycin-resistant (strep^R) seedlings occurred was used as an indication of the frequency of Ds excision. Very few of the progeny of plants not exposed to heat shock or of those heat shocked only during vegetative development were variegated or strep^R. However, plants that were heat shocked after the appearance of flower buds and during seed development produced high frequencies (approaching 100%) of variegated, but very few strep^R, progeny. Furthermore, when variegated seedlings were grown to maturity and self-fertilized without further exposure to heat shock then strep^R seedlings often occurred at high frequency among their progeny. Southern analysis indicated that the majority of these strep^R plants contained a transposed Ds at a new location. These data indicate that in response to heat shock Ds excision frequently occurs in embryonic cells which ultimately give rise to the gametes, as well as in cells of the developing cotyledons. The importance of an inducible transposon system for transposon tagging is discussed.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

Patterns of trace metal accumulation in Swedish marine nemerteans

Total body burdens of Zn, Pb, Al and Cu were measured in Swedish marine nemerteans sampled from an area close to a major source of pollution. Accumulation patterns varied both seasonally and spatially. The results suggest that nemerteans may be useful bioindicator species in environmental monitoring programmes.     

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.     

Effect of prostaglandin E1 on chemiluminescence response and adherence of human polymorphonuclear leukocytes to human cultured endothelial cells of prostaglandin E1 treated polytraumatized patients

We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen-derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group. Production of oxygen-derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p < 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post-trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p < 0.01) within the first 6 d post-trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen-derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium tumefaciens T-DNA Indoleacetic Acid-Biosynthetic Genes

A key regulator of cambial growth is the plant hormone indoleacetic acid (IAA). Here we report on altered wood characteristics and growth patterns in transgenic hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) expressing Agrobacterium tumefaciens T-DNA IAA-biosynthetic iaaM and iaaH genes. Eighteen lines simultaneously expressing both genes were regenerated. Of these, four lines, verified to be transgenic by northern blot analysis, were selected and raised under controlled growth conditions. All four lines were affected in their growth patterns, including alterations in height and stem diameter growth, internode elongation, leaf enlargement, and degree of apical dominance. Two transgenic lines, showing the most distinct phenotypic deviation from the wild type, were characterized in more detail for free and conjugated IAA levels and for wood characteristics. Both lines showed an altered IAA balance, particularly in mature leaves and roots where IAA levels were elevated. They also exhibited changes in wood anatomy, most notably a reduction in vessel size, an increase in vessel density, and changes in ray development. Thus, the recent development of techniques for gene transfer to forest trees enabled us to investigate the influence of an altered IAA balance on xylem development in an intact experimental system. In addition, the results demonstrate the possibility of manipulating wood properties in a forest tree through controlled changes of IAA concentration and distribution.     

Effects of fish predation and habitat type on stream benthic communities

The influence of habitat on interactions between a fish predator (brown trout Salmo trutta) and a benthic invertebrate community was studied in nine field enclosures (8 ×3 m) in a creek in southern Sweden. Three habitat treatments were tested, a shallow sandy habitat, a deep habitat containing a mixture of large and small cobbles and a moderately deep habitat with large cobbles. The one month-long experiment showed that there were no major differences in the abundance and biomass of the benthic macroinvertebrate fauna among these habitats as no functional groups of invertebrates and only a few taxa differed between treatments. Invertebrate drift rates decreased over time, which was probably related to seasonal changes in invertebrate life cycles or to effects of predation independent of habitat type, as there was no difference between treatments.     

IN VIVO MEASUREMENT OF ACTIVE OXYGEN PRODUCTION IN THE BROWN ALGA FUCUS EVANESCENS USING 2′,7′-DICHLOROHYDROFLUORESCEIN DIACETATE1

Intracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) to the fluorescent dye 2′,7′-dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were incubated in seawater containing DCFH-DA under a range of conditions, and it was also possible to load algae with DCFH-DA and then follow subsequent DCF production in emersed tissue. DCF formation was linear for at least 2 h in both darkness and light, with the rate of formation increasing with the light level. DCF formation was temperature dependent. It also increased when algae were treated with H₂O₂ or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O^?₂ production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentrations of H₂O₂. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH-DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds subjected to environmental stresses.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.