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.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

Promoting regional sustainability by eco-province construction in China: A critical assessment

The regional level has been recognized as a good scale for implementing actions towards sustainable development (SD). An important pathway to improve regional sustainability is to locally carry out long-term ecological planning. In order to reduce environmental impacts from fast economic growth, several eastern provinces of China began Ecological Province Construction (EPC) at the end of 1990s. This paper presented a case for analyzing the progress of EPC in China via both qualitative and quantitative methods. We studied Shandong Eco-province Construction (SEPC) and evaluated its outcomes with its pre-set indicators in 2003–2010. A further evaluation with eco-efficiency and de-linking indices was conducted. The results indicated the pre-set indicators could not effectively reflect of the ecological province construction. It was recommended to introduce an evaluated framework combined with eco-efficiency and de-linking indices to indicate ecological planning at the regional scale.     

Plasmid-coded degradation of ethylbenzene and 1-phenylethanol in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens EB carries genes for the catabolism of ethylbenzene and 1-phenylethanol on a plasmid. The size of the plasmid as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 253–267 kb. By treatment with Mitomycin C, mutants of EB strain were obtained bearing a plasmid which had undergone an extensive deletion of about 80 kb. These mutants have lost the ability to grow on ethylbenzene and 1-phenylethanol as well as to synthesize meta-cleavage enzymes.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Chromosomal nodulation genes: Sym-plasmid containing Agrobacterium strains need chromosomal virulence genes (chvA and chvB) for nodulation

Summary The chromosomal genes chvA and chvB of Agrobacterium tumefaciens, which mediate attachment to plant cells, were found to be essential not only for tumour induction but also for the formation of root nodules on plants.     

Mechanism of the self-assembly of apoferritin from horse spleen

Apoferritin from horse spleen can be reversibly dissociated at pH 2 or in 7.2 M G-HCl (pH 3.5). Reconstitution of the native icositetramer in 0.1 M TEA buffer (pH 7.9) in the presence of 1 mM EDTA and 3 mM dithioerythritol leads to yields higher than 80%. To monitor the kinetic mechanism, intrinsic fluorescence, far-UV circular dichroism, and covalent cross-linking with glutaraldehyde were applied.The overall mechanism of assembly is characterized by a sequence of concentration-dependent association reactions involving structured monomers and a dimeric intermediate as the most prominent species, apart from trimers and dodecamers. The parallel decrease in monomers, dimers and trimers indicates that association equilibria precede the formation of the final assembly product.The assembly reaction is accompanied by characteristic changes in fluorescence emission and dichroic absorption. To a first approximation, renaturation and reassociation may be quantitatively described by one single rate-determining second-order process, subsequent to fast folding steps at the monomer level.Abbreviations CD circular dichroism - DTE dithioerythritol - G-HCl guanidinium chloride - SDS sodium dodecylsulfate - TEA triethanolamine - Tris tris hydroxymethylamino methane Dedicated to Professor Harold A. Scheraga on the occasion of his 65^th birthday