Gel retardation analysis of E. coli M1 RNA-tRNA complexes.

We have analyzed complexes between tRNA and E. coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels. The RNA subunit of E. coli RNase P formed a specific complex with mature tRNA molecules. A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function. Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion. Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist. Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation. At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed. This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.     

Immunophenotyping of mitotic cells from long-term cultures of chorionic villi

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.     

Testing the success of palaeontological methods in the delimitation of clam shrimp (Crustacea,Branchiopoda) on extant species

Fossil spinicaudatan taxonomy heavily relies on carapace features (size, shape, ornamentation) and palaeontologists have greatly refined methods to study and describe carapace variability. Whether carapace features alone are sufficient for distinguishing between species of a single genus has remained untested. In our study, we tested common palaeontological methods on 481 individuals of the extant Australian genus Ozestheria that have been previously assigned to ten species based on genetic analysis. All species are morphologically distinct based on geometric morphometrics (p ≤ 0.001), but they occupy overlapping regions in Ozestheria morphospace. Linear discriminant analysis of Fourier shape coefficients reaches a mean model performance of 93.8% correctly classified individuals over all possible 45 pairwise species comparisons. This can be further increased by combining the size and shape datasets. Nine of the ten examined species are clearly sexually dimorphic but male and female morphologies strongly overlap within species with little influence on model performance. Ornamentation is commonly species-diagnostic; seven ornamentation types are distinguished of which six are species-specific while one is shared by four species. A transformation of main ornamental features (e.g. from punctate to smooth) can occur among closely related species suggesting short evolutionary timescales. Our overall results support the taxonomic value of carapace features, which should also receive greater attention in the taxonomy of extant species. The extensive variation in carapace shape and ornamentation is noteworthy and several species would probably have been assigned to different genera or families if these had been fossils, bearing implications for the systematics of fossil Spinicaudata.     

Die Feinstruktur der Schweineaorta beim experimentellen Lathyrismus

Zusammenfassung Die Veränderungen der Aortenfeinstruktur, welche eine Vergiftung mit-Amino-Propio-Nitril (Lathyrusfaktor) bei jungen Schweinen hervorruft, werden beschrieben.Der Benninghoffsche Spannapparat der Media wird frühzeitig durch eine Lösung der muskulo-elastischen Kontinuität gelockert. Im Vordergrunde der morphologischen Veränderungen stehen dann Zerfallserscheinungen der elastischen Substanz, welche zu einer Desintegration der elastischen Lamellen sowie der elastischen interlamellären Schrägverbinder führen und endlich eine völlige Strukturauflösung der ganzen Mediaorganisation mit herdförmigen Totalnekrosen zur Folge haben. Das mit dem Mediafasersystem strukturell und funktioneil eng verbundene kollagen-elastische Fasergitter der Intima zerfällt; die subendotheliale Intima wird dabei hochgradig aufgetrieben durch eine Einlagerung von Ödemflüssigkeit und wahrscheinlich auch Blutserum. Die feinnetzig-zweiphasige Normaldarstellung der Grundsubstanz geht verloren. Die Feinstruktur der Kollagenfibrillen bleibt unverändert; es wird darauf hingewiesen, daß trotzdem eine schwere Schädigung auch des Kollagens anzunehmen ist. Die Mediamuskelzellen wandeln sich weitgehend in Fibroblasten um; die Langhans-Zellen der Intima werden in gleicher Weise aktiviert. Zellnekrosen treten in der Media nur dann auf, wenn sekundär die subakute Aortenwandverdickung eine Mangelsituation für den Zelleigenstoffwechsel hervorruft.In der Umgebung der Vasa vasorum kommt es zu kleinen Blutungen, die mit Fortschreiten der Mediazerstörung in große, dissezierende Blutungen übergehen können. Der wahrscheinlich durch die Elastizitätsverminderung der Media eingeleitete Mechanismus der Gefäßrupturierung wird beschrieben und mit dem vonGöre und vonDoerr vermuteten Mechanismus der Bildung von Aneurysmata dissecantia der menschlichen Aorta in Parallele gesetzt.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. — FräuleinElke CarStensen schulden wir Dank für unermüdliche, gewissenhafte und geschickte technische Mitarbeit.     

Contribution of the different planktonic microbial assemblages to ETS activity in the Ligurian frontal area: north-west Mediterranean Sea

Spatial and size distribution of micro-organisms and their ETSactivity has been investigated in Ligurian Sea surface watersalong the Nice-Calvi transect across frontal areas from 18 to37 km offshore (TOMOFRONT 1 and 2 cruises, April 1988 and April-May1989 respectively). Aplastidic and plastidic nanoflagellatesand aplastidic picoflagellates were present in numbers closeto 0.25 x 10⁴ cells ml^–1, whereas plastidic picoflagellatesaccounted for about half this number. Correlations have beenevidenced between plastidic and aplastidic micro-organisms withinthe same size group, suggesting that they belong to a well-definedecosystem. The highest correlation between total ETS activityand abundance of the considered size groups was observed fornanoflagellates (r = 0.94, n = 22, and r = 0.90, n = 22 foraplastidic and plastidic cells respectively). The importanceof the role of nanoflagellates in surface waters, with respectto the overall ETS activity, was supported by results from sizefractionation which assigned to the 3–10 µm sizerange a 73.3% contribution to overall ETS activity. Resultsemphasize analysing global ETS activity of natural samples inorder to derive relationships between the different populationspresent in the sampled water. It is suggested that couplingflow cytometry to the ETS approach should be very helpful inthat respect.     

Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O

H₂–forming N ⁵,N ¹⁰ –methylenetetrahydromethanopterin dehydrogenase is a novel type of hydrogenase that contains neither nickel nor iron-sulfur clusters. Evidence has been presented that the reaction mechanism catalyzed by the enzyme is very similar to that of the formation of carbocations and H₂ from alkanes under superacidic conditions. We present here further results in support of this mechanism. It was found that the purified enzyme per se did not catalyze the conversion of para H₂ to ortho H₂, a reaction catalyzed by all other hydrogenases known to date. However, it catalyzed the conversion in the presence of the substrate N ⁵,N ¹⁰ –methenyltetrahydromethanopterin (CH≡H₄MPT⁺), indicating that for heterolytic cleavage of H₂ the enzyme-CH≡H₄MPT⁺ complex is required. In D₂O, the formation of HD and D₂ from H₂ rather than a para–ortho H₂ conversion was observed, indicating that after heterolytic cleavage of H₂ the dissociation of the proton from the enzyme-substrate complex is fast relative to the re-formation of free H₂.     

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex.     

Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole     

Recent approaches to probe functional groups in ribonuclease P RNA by modification interference

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt nucleotide(s) - PAGE polyacrylamide gel electrophoresis     

The ant’s path integration system: a neural architecture

 A model is developed by which path integration as observed in many animal species could be implemented neurobiologically. The proposed architecture is able to describe the navigation behaviour of Cataglyphis ants, and that of other social insects, at the level of interacting neurons. The basic idea of this architecture is the concept of activity patterns travelling along neural chains. Although experimental evidence has yet to be provided, this concept seems biologically plausible and not limited to the navigation problem. Neural chains are able to represent variables by activity patterns with high accuracy and temporal stability. Moreover, they are able to integrate incremental signals with high precision. Cyclical chains of neurons show superior performance as soon as cyclical variables are to be represented and integrated. Finally, representation of cyclical variables by travelling activity peaks allows simple approximations of goniometric functions as they are used in path integration systems. Received: 15 November 1994/Accepted in revised form: 30 May 1995