The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.     

Ligand-triggered conformational perturbations elicit changes at the single cysteinyl residue of spinach calmodulin

Following application of stoichiometric amounts of Ca2+ or specific partner peptides to spinach calmodulin, dynamic changes in the nanosecond range could be monitored at a strategically anchored fluorescence or spin probe. For these studies the single cysteinyl residue 26 of spinach calmodulin was labelled with a thiol-specific proxyl (i.e. 2,2,5,5-tetramethyl-1-pyrrolidinyl-oxyl) spin probe or with a bimane fluorescence probe. With Ca2+ and a specific ligand (mastoparan) present, fluorescence studies (anisotropy, lifetime) indicated that the rotational motion of the protein complex becomes slower relative to the motion of calmodulin in the absence of the specific ligand. The probe's attachment site 26 appears to reside in a fairly polar microenvironment as reported by a series of proxyl spin probes varying in label length. The rotational correlation time of the shortest spin probe markedly changed upon binding of a specific peptide to a calmodulin region distant from that of the monitoring spin probe. We interpret these observations as indicating that ligand-triggered conformational perturbations are eliciting specific responses at the cysteinyl residue 26 of spinach calmodulin.     

Morphology and biochemical genetics of Atlantic halibut, Hippoglossus hippoglossus (L.), from various spawning grounds

Potential phenotypic/genotypic differentiation of Atlantic halibut, Hippoglossus hippoglossus (L.), samples from two spawning grounds in north Norway (Malangen and Sørøysund) were studied. In addition, the genetic relationship between fish from north Norway, mid–Norway (Folia) and western Greenland (off Fredrikshåb) was investigated. Uni– and multivariate analyses of 15 morphometric measurements and six meristic counts revealed a considerable degree of homogeneity between Malangen and Sørøysund. No morphological characters were thus found suitable as diagnostic tools in the classification of potential halibut stocks. As determined from electrophoretic studies, the allelic distribution at the four polymorphic loci ADA, PGI–2, PGM–2 , and UTP (unidentified tissue protein) did not vary significantly when all four samples (Folia, Malangen, Sørøysund and Greenland) were considered together. However, when the Norwegian coast samples were split into a northern (Sørøysund and Malangen) and a middle (Folla) area, and each locus was regarded isolated, significant heterogeneity was detected at the PGM–2 locus, thus giving evidence for a potential heterogeneity along a north–south axis.     

Evidence for free and metabolically stable p53 protein in nuclear subfractions of simian virus 40-transformed cells.

To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren-transformed mouse (MethA) cells, and from various simian virus 40 (SV40)-transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40-transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissociation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T.     

Aluminum interaction with calmodulin. Evidence for altered structure and function from optical and enzymatic studies

The interaction of aluminum ions with bovine brain calmodulin has been examined by fluorescence spectroscopy, circular dichroic spectrophotometry and equilibrium dialysis, and by the calmodulin-dependent activation of 3',5'-cyclic nucleotide phosphodiesterase. These experiments show that aluminum binds stoichiometrically and cooperatively to calmodulin. Binding of aluminum at a molar ratio of 2:1 to calmodulin suffices to induce a major structural change. Estimates from spectroscopic data indicate that the binding affinity for the first mol of aluminum bound to the protein is about one order of magnitude stronger than that of calcium to its comparable site. These estimates agree with a dissociation constant of 0.4 microM derived from equilibrium dialysis experiments. Interaction of aluminum with calmodulin induces a helix-coil transition and enhances the hydrophobic surface area much more than calcium does. A molar ratio of 4:1 for [aluminum]/[calmodulin] is sufficient to block completely the activity of the calcium-calmodulin-dependent phosphodiesterase. Highly hydrated aluminum ions apparently promote solvent-rich, disordered polypeptide regions in calmodulin which, in turn, profoundly influence the protein's flexibility.     

Über markhaltige Nervenfasern und markhaltige Herringkörper in der Neurohypophyse des Elefanten

Zusammenfassung Der Tractus supraoptico-hypophyseus des Elefanten besteht aus markhaltigen Nervenfasern und zieht durch das Zwischenstück bis in die distale Neurohypophyse. In seinem Verlauf finden sich große Herringkörper, die teilweise Markscheiden besitzen. Der stark reduzierte Zwischenlappen liegt in Form von Zellnestern in dem Bindegewebe, das den distalen Teil umhüllt. Das neurosekretorische Material ist in üblicher Weise verteilt. Die Ergebnisse werden diskutiert.

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On myelinated nerve fibres and myelinated herring bodies in the neurohypophysis of the elephant

Summary The tractus supraoptico-hypophyseus of the elephant consists of myelinated fibres. It passes through the intermediate part towards the distal part. In its course there are Herring bodies. Some of them are provided with myelinated sheaths. The intermediate lobe is strongly reduced. It is situated in the connective tissue surrounding the distal part of the neurohypophysis and consists only of nests of epithelial cells. The localisation of the neurosecretory material corresponds to the usual distribution. The results are discussed.

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Die Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.

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Herrn Prof. Dr. Dr. E. Horstmann zum 60. Geburtstag gewidmet.     

Morphometrie der feinen Markfasern in der Sehrinde der Katze

Zusammenfassung In den Schichten I bis III der Sehrinde der Katze wurden die feinen markhaltigen Nervenfasern morphometrisch untersucht. Folgende Größen wurden bestimmt: 1. mit Hilfe des Punktzählverfahrens ein Volumenanteil von 7,1±1,6%. 2. Mit der Methode von Hennig (1958) in einem Raum von (0,1 mm)³ eine Oberfläche von 0,42±0,07 mm². 3. Mit Hilfe der Methode von Hennig (1963) in einem gleichen Raum eine Summenlänge der Markfasern von 152 mm. 4. Bei 3693 markhaltigen Nervenfasern ein Mittelwert aller Durchmesser von 0,51±0,08 . Die Größenverteilung der Durchmesser ist in der Abb. 2 enthalten. 5. Die Ergebnisse von 3. und 4. erlaubten eine Kontrollberechnung von Volumenanteil und Oberfläche. Die mit unterschiedlichen Methoden ermittelten Ergebnisse zeigen eine gute Übereinstimmung.

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Summary The small myelinated axons in layers I to III of the cat's visual cortex have been investigated by means of morphometric methods. It has been found that: 1. The myelinated fibres make up 7.1±1.6% of the total volume. 2. Within a volume of (0.1 mm)³ the myelinated fibres have a surface of 0.42±0.07 mm². 3. The compound length of the myelinated fibres within a volume of (0.1 mm)³ is 152 mm. 4. The diameters of 3,693 myelinated fibres have a mean value of 0.51±0.08 . The distribution of the diameters is shown in Fig. 2. 5. The results of 3. and 4. can be used to control the values given in 1. and 2. The values obtained by the two different methods are in good agreement.

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Die Untersuchungen wurden mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft ausgeführt.     

Triplex DNA in plasmids and chromosomes

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.     

Growing tobacco cells respond to rapid medium changes with a transient increase in localized Ca2+ activity

Summary A suspension of tobacco cells,Nicotiana tabacum L. BY-2, was subjected to a rapid change of medium, resulting in disturbance of growth. A subpopulation of growing cells responded to such a nutritional signal by establishing a transient, localized Ca²⁺ accumulation, as judged by chlorotetracycline fluorescence. Residing near or at the plasma membrane, this initial Ca²⁺ signal began to relax after 1 h to a value presumably corresponding to an equilibrium Ca²⁺ level. This response was susceptible to treatment with brefeldin A, an agent impacting vesicular traffic, as indicated by a further increase in fluorescence. By contrast, undisturbed growing and non-growing cells did not display a Ca²⁺ response, regardless of the presence of brefeldin A.