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

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae 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 L. tarentolae 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.     

Correlations in one-dimensional fully developed chaos

Correlations in the baker map and the tent map as examples of one-dimensional, fully developed chaos are considered. It is shown, utilizing symbolic dynamical systems derived from these maps, that the vanishing second-order correlation function is not sufficient to guarantee uncorrelatedness. Importance of the higher-order, especially third-order, correlation functions is emphasized for chaotic systems. In search of the quantities that grasp correlational behaviors as a whole in chaotic systems, it is proposed to use the fixed-separation correlation integral, which is a modified quantity of the usual correlation integral devised to calculate the fractal dimension of strange attractors, for these maps. It is shown that the new quantity contains all the even-number orders of autocorrelation function that are commonly considered.     

凋亡而不出现DNA梯形带一例

用普通琼脂糖凝胶电泳UVB照射后分别培养24、36小时的NIH3T3细胞DNA,均未出现梯形带,但从细胞形态上看,大部分细胞发生凋亡并出现凋亡小体。电泳UVB照射后培养24小时的昆明小鼠胸腺细胞DNA,出现典型的凋亡梯形带。说明细胞在发生凋亡时DNA并不总是从核小体之间断裂的,不能把DNA梯形带作为判断细胞凋亡的唯一标准。     

The use of environmental DNA of fishes as an efficient method of determining habitat connectivity

This study demonstrated the use of environmental DNA (eDNA) to determine habitat connectivity for migration of fishes between the sea and river. Environmental DNA is DNA fragments released by fishes in water, which can be used as a species-specific marker of the presence/absence of the target species. A year-round water sampling regime at 15 sites on the Yodo River, Japan, was conducted to determine whether three major man-made barriers on the river inhibited the migration of fishes using species-specific detection of DNA fragments from three target migrant species, temperate seabass, Lateolabrax japonicus, flathead grey mullet, Mugil cephalus, and ayu, Plecoglossus altivelis altivelis. The presence/absence of eDNA from target species was consistent with known patterns of species’ seasonal migration. The detection of the DNA of temperate seabass and flathead grey mullet at sites upstream of the dam closest to the river mouth indicated successful upstream migration of these species via a fish ladder bypassing the dam. On the other hand, DNA of these two species was not detected from the upstream side of the two remaining dams, which are not equipped with fish ladders. Ayu is the only species among the three target species with a land-locked population in Lake Biwa located at the headwater of Yodo River. Ayu DNA was detected at most of the sites in the freshwater area during the warm months; however, in the coldest month of February, eDNA was only detected in the uppermost site of Yodo River at the southern tip of Lake Biwa. The eDNA we detected at this site suggests that it was derived from juvenile ayu spending their winter months in the lake. These results suggest that the eDNA analysis presented here can accurately track the seasonal migration of fishes in a river, demonstrating its application as an indicator of habitat connectivity for fishes in association with man-made barriers in a river. The sampling of eDNA involves merely scooping a tank full of water; therefore, it is a simple, rapid, and cost-effective method for long-term monitoring of habitat connectivity associated with the construction of barriers in a river.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex

The interaction of a structurally characterized Sr–Fe nitrosyl complex with DNA has been studied by UV–vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6 × 10⁴ M⁻¹. Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H₂O₂.     

Local spin dynamics in magnetic molecular chains studied by NMR and μSR

We present NMR and μ⁺SR study of spin dynamics in one-dimensional and quasi-one-dimensional molecular magnets of recent synthesis. In particular, we focus on the so called Gd(hfac)₃NIT-R and CoPhOMe magnetic chains families. For Gd-R helimagnets we show some differences between “weakly frustrated systems” and “fully frustrated systems”. The different behaviour is due to the different radical inserted in the chains (R = Me, Ph for “weakly frustrated systems” and R = iPr, Et for “fully frustrated systems”). The existence of different phase transitions, particularly to 3D long-range magnetic order in Gd-Ph and to chiral order in Gd-iPr, is remarked together with a comparison between results obtained from macroscopic and microscopic investigating techniques. As regards CoPhOMe slowly relaxing chain, the ¹H NMR measurements confirm the freezing of the spins at low temperature, which prevents the 3D long-range order, and display the presence of two relaxation mechanisms related to distinct contributions to the local spin relaxation.     

Nanoprocessability of a one-dimensional oxalato-bridged cobalt(II) complex with 1,2,4-triazole

The polymer {[Co(ox)(Htr)₂] · 2H₂O}_n (ox = oxalate dianion; Htr = 1,2,4-triazole) (1) has been synthesized and characterized by FT-IR spectroscopy, thermal analysis, variable-temperature magnetic measurements and X-ray diffraction methods. The physical analysis allows us to propose a one-dimensional structure in which [Co(Htr)₂]²⁺ units are bridged by bis-bidentate oxalato ligands. Magnetic measurements at variable temperature show an overall antiferromagnetic behavior of the compound. Isolated chains of this polymer have been obtained by sonication of 1 in water and deposition on mica or on mica treated with poly-l-lysine. Circular molecules and nano-fibres have been isolated on Highly Oriented Pyrolitic Graphite (HOPG) by casting deposition of sonicated solutions of 1 in ethanol. The direct reaction on HOPG surface between Co^II, H₂ox and Htr has proved a useful route to isolate one-dimensional systems on surfaces. The development of new strategies to characterize these types of polymers on surfaces opens the possibility to perform nano-scale studies on their properties and their potential use as nano-materials.     

Coordination polymers of Cu(II) and Cd(II) with bifunctional chelates of the type dipicolylamino-alkylcarboxylate, (NC5H4CH2)2N(CH2)nCO2H (n = 1, 2, 3 and 4)

A series of bifunctional chelates of the type dipicolylamino-alkylcarboxylate (NC₅H₄CH₂)₂N(CH₂)_nCO₂H (n = 1-4; HL¹-HL⁴, respectively) has been prepared. Reactions of the ligands in aqueous methanol/N,N-dimethylformamide with the appropriate Cu(II) salts yielded the compounds [CuL¹](NO₃)·H₂O (1·H₂O), [CuL²(H₂O)]BF₄·H₂O (2·H₂O), [Cu(HL³)(SO₄)]₂ (3) and [CuL⁴(NO₃)]·MeOH (4·MeOH). While compounds 1, 2 and 4 are one-dimensional, the detailed connectivities within the chains are quite distinct, depending on factors such as alkyl chain length and ligation of aqua ligands or anionic components. In contrast to 1, 2 and 4, the structure of 3 is molecular, a binuclear assembly of edge-sharing Cu(II) ‘4+2’ distorted octahedra. The Cd(II) species, [{CdL²}₂(SO₄)]·4H₂O (5·4H₂O), prepared from HL² and CdSO₄·nH₂O in aqueous methanol/N,N-dimethylformamide, is two-dimensional, with a network constructed from binuclear units of seven coordinate Cd(II), , linked through bridging SO₄²⁻ groups to produce an assembly of linked hexagonal rings [{CdL²}₂(SO₄)]₆.     

Tau Protein Diffuses along the Microtubule Lattice

Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.