Immunolocalization of Na,K-ATPase in blowfly photoreceptor cells

The Na,K-ATPase (sodium pump) plays a central role in the physiology of arthropod photoreceptors as it re-establishes gradients for Na⁺ and K⁺ after light stimulation. We have mapped the distribution of the Na,K-ATPase in the photoreceptors of the blowfly (Calliphora erythrocephala) by immunofluorescent and immunogold cytochemistry, and demonstrate that the distribution pattern is more complex than previously presumed. High levels of sodium pumps have been detected consistently in all photoreceptors R1-8 on the nonreceptive surface, but no sodium pumps are found on the microvillar rhabdomere. Within the nonreceptive surface of the cells R1-6, however, the sodium pumps are confined to sites juxtaposed to neighboring photoreceptor or glial cells; no sodium pumps have been detected on the parts of the nonreceptive surface exposed to the intra-ommatidial space. In R7 and R8, the sodium pumps are found over the entire nonreceptive surface. The cytoskeletal protein spectrin colocalizes with the sodium pumps suggesting that linkage of the pump molecules to the spectrin-based submembrane cytoskeleton contributes to the maintenance of the complex pattern of pump distribution.     

Thiophene synthesis and distribution in young developing plants of Tagetes patula and Tagetes erecta

Thiophene synthesis and accumulation were investigated in organsof Tagetes patula and T. erecta. Thiophene accumulation startedrapidly in germinating seedlings of both species. Roots andhypocotyls were the major thiophene accumulating organs and5-(3-buten-1-ynyl)-2, 2-bithienyl (BBT) and 5-(4-acetoxy-1-butynyl)-2,2 -bithienyl (BBTOAc) were the major accumulated compounds.Higher thiophene concentrations were reached in Tagetes patulathan in T. erecta. Accumulation patterns for individual thiopheneswere different within organs, between organs and between bothspecies. Within hypocotyls of Tagetes patula, thiophene concentrationswere high in the epidermis and the vascular tissue and low inthe parenchymatic tissues of cortex and pith. Synthesis of thiopheneswas high in the roots and hypocotyls and very low in the leaves.Transport of thiophenes from the roots into the shoot occurred,but the rate of transport was too low to explain the high concentrationsin the hypocotyl. It is concluded that for the main part thiophenesare accumulated where they are synthesized. Key words: Tagetes, hiophenest, synthesis, accumulation, secondary metabolites     

Investigation of the dithiocarbamate-induced coupling of allylidene and carbonyl ligands on a tungsten center

Reaction of the allylidene tungsten complex [W(CPhCHCHMe)Br₂(CO)₂(4-picoline)] (1) with the dithiocarbamates MS₂CNR₂ (a: M=Na, R=Et; b: M=Na, R=Me; c: M=Li, R=Ph) in THF at 50 °C affords the vinylketene tungsten complexes [W(S₂CNR₂)₂(OCCPhCHCHMe)(CO)] (2a–c). At lower temperatures, four reaction intermediates (3–6) may be discerned. Spectroscopic studies indicate that these compounds contain η⁴-allyldithiocarbamate ligands which are generated by addition of dithiocarbamate across the metal-carbon double bond of the allylidene-tungsten unit in 1. The structures of [W(S₂CNEt₂)₂(OCCPhCHCHMe)(CO)] (2a) and of one intermediate, [W(η⁴-Et₂NCS₂CPhCHCHMe)(S₂CNEt₂)(CO)₂] (5a) were elucidated by X-ray crystallography.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity.     

Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

Light-stimulated proton transport into the vacuoles of leaf mesophyll cells does not require energization by the tonoplast pyrophosphatase

Photosynthetic characteristics of transgenic tobacco (Nicotiana tabacum L.) plants with a soluble pyrophosphatase in the cytosol of their leaf cells were compared to those of wild-type plants. Although the development of the transgenic plants was somewhat retarded compared to the wild type, as shown by stunted growth and delayed flowering, photosynthetic responses were comparable in transgenic and wild-type leaves of similar physiological age. In particular, light-dependent proton transport into the vacuoles of leaf mesophyll cells was not decreased in leaves of the transgenic plants, which did not contain pyrophosphate in the cytosol owing to the presence of a soluble pyrophosphase. This shows that light-stimulated proton pumping did not require the pumping activity of the tonoplast pyrophosphatase. Apparently, light-stimulated proton pumping can be based solely on the activity of the tonoplast ATPase.Abbreviation CDCF 5-(and 6-)arboxy-2,7-dichlorofluorescein This work was supported within the Sonderforschungsbereiche 176 and 251 of the University of Würzburg.     

BrdU incorporation reveals DNA replication in non dividing glial cells in the larval abdominal CNS ofDrosophila

Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) ofDrosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two otherDrosophila species,D. virilis andD. hydei.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.