Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of <em>Leishmania tarentolaeem> were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in <em>L. tarentolaeem> were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

A catalog of bird specimens associated with Prince Maximilian of Wied-Neuwied and potential type material in the natural history collection in Wiesbaden

Bird specimens collected by 19^th century explorer and ornithologist Prince Maximilian of Wied-Neuwied form one of the foundation collections of the American Museum of Natural History in New York. However, parts of his collection remained in Germany and came to the Museum Wiesbaden. Since Wied described numerous new species without designating types, some of these specimens might be type material. Here we present a catalog of the 30 Wiesbaden specimens associated with him and discuss their potential type status. We conclude that 17 individuals in 11 species are potential type specimens that should be considered in future taxonomic work.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Bilan nutritionnel au cours du développement de l'ectoparasite grégaire Dinarmus vagabundus et du solitaire Dinarmus basalis

R&eacute;sum&eacute; Nos m&eacute;thodes exp&eacute;rimentales permettent l'isolement d'une larve de sexe d&eacute;termin&eacute; par hôte de l'ectoparasite gr&eacute;gaire Dinarmus vagabundus et du solaitire, D. basalis. Des hôtes porteurs de 3 &agrave; 8 larves par hôte de D. vagabundus sont aussi isol&eacute;s. Dans ces conditions la quantit&eacute; de nourriture disponible est la m&ecirc;me pour toutes les densit&eacute;s larvaires &eacute;tudi&eacute;es.Les larves ent/m5734664812489gg/xxlarge9792.gif" alt="female" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9792.gif" alt="female" align="MIDDLE" BORDER="0"> &eacute;lev&eacute;es en solitaire des deux esp&egrave;ces assimilent une quantit&eacute; de nourriture significativement sup&eacute;rieure &agrave; celle assimil&eacute;e par les ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0">. Ceci conduit &agrave; des adultes ent/m5734664812489gg/xxlarge9792.gif" alt="female" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9792.gif" alt="female" align="MIDDLE" BORDER="0"> de poids moyen sup&eacute;rieur &agrave; celui des ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0">. Le poids moyen des ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> et des ent/m5734664812489gg/xxlarge9792.gif" alt="female" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9792.gif" alt="female" align="MIDDLE" BORDER="0"> de D. vagabundus diminue significativement aux fortes densit&eacute;s larvaires. L'intensit&eacute; de la liaison entre la quantit&eacute; de nourriture assimil&eacute;e et la biomasse produite s'affaiblit au fur et &agrave; mesure que la densit&eacute; larvaire par hôte augmente.Les ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> de D. vagabundus de poids moyen (0,42 mg) engendrent deux fois et demi plus de descendants que les ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> lilliputiennes (0,20 mg) &eacute;merg&eacute;es d'hôtes &agrave; forte densit&eacute; larvaire. Celles de D. basalis (0,65 g) sont moins prolifiques que les ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> ent/m5734664812489gg/xxlarge9794.gif" alt="male" align="MIDDLE" BORDER="0"> de D. vagabundus.e>     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from <em>Sulfolobus solfataricusem>, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.     

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of <em>Klebsiellaem> SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for <em>Klebsiellaem> serotype K8 capsular polysaccharide. e class="inline-figure">els-cdn.com/content/image/1-s2.0-0008621583850113-fx1.gif" height="95" alt="/>e>The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the <em>Klebsiellaem> K8 and SK1 polymers is akin to that found for <em>Klebsiellaem> polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.     

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.