Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli

Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.     

Hormone-sensitive stages in the sexual differentiation of laryngeal muscle fiber number in Xenopus laevis

The number of muscle fibers in the vocal organ of the adult male African clawed frog, Xenopus laevis, exceeds that of adult females. This sex difference is the result of rapid fiber addition in males between the end of metamorphosis, post-metamorphic stage 0 (PM0) and PM2. At PM0, male and female frogs have similar numbers of laryngeal muscle fibers. Males then add more muscle fibers than females and achieve an adult value that is 1.7 times the female number. Males castrated at PM0 have the same fiber number as females. Ovariectomy at PM0 does not alter muscle fiber addition in females. Gonadectomy at PM2 has no effect on fiber addition in either sex. Females attain masculine muscle fiber number if their ovaries are replaced with a testis at metamorphosis. Exogenous testosterone treatment at PM0 significantly increases fiber number in females but not in males. Exogenous testosterone given at PM2 has no effect on fiber number in females but decreases fiber number in males. We conclude that the testes are necessary for the marked addition of laryngeal muscle fibers seen in male X. laevis between PM0 and PM2. The masculine pattern of muscle fiber addition can be induced in females provided with a testis. Androgen secretion from the testes most probably accounts for masculinization of laryngeal muscle fiber number. After PM2, androgens are no longer necessary for muscle fiber addition and cannot increase fiber number in females.     

Alteration by heat shock and immunological characterization of Drosophila small nuclear ribonucleoproteins

Sera from human patients with systemic lupus erythematosus (SLE) have been shown to react with snRNP particles of both mammals and Drosophila (Mount, S. M. and J. A. Steitz. 1981. Nucleic Acids Res. 9:6351-6368). We have utilized fully characterized monospecific sera and specifically purified antibodies to carry out indirect immunofluorescence experiments with frozen sections of Drosophila embryos. Embryos subjected to severe heat shock before sectioning showed reduced binding of anti-Sm sera. Anti-nRNP sera reacted identically with antigens of heat shocked and non-heat-shocked sections. The reduction in anti-Sm fluorescence was restored by a brief salt wash. These results imply a noncovalent alteration in the conformation of Sm antigens with the administration of heat shock that can revert with exposure to salt. Drosophila antigens have been compared to mammalian standards, showing partial identity with bovine spleen extract (BSE) antigens when reacted with anti-Sm sera. The antigenic relatedness between affinity-purified heat-shocked and non-heat-shocked Drosophila antigens and their mammalian homologues was examined by quantitative ELISA methodology. In all cases, the Drosophila antigens from heat-shocked and non-heat-shocked embryos were identical. We theorize that the heat shock-induced alteration of Sm antigen reverst during extraction. Because the snRNP antigens have been shown to be involved in splicing, and because splicing is inhibited during heat shock (Yost, H. J., and S. Lindquist. 1986. Cell. 45:185-193), our results provide information on the nature and stability of a change in these antigens which may be a central element in control of the heat shock response.     

Mindestanforderungen an die artgerechte Haltung von Krokodilen in privaten Terrarien und zoologischen Einrichtungen

The minimum requirements for the keeping of crocodiles in private and public institutions are set up by a collective of authors. Important aspects such as the design of enclosures, secure handling and keeping, thermoregulation, protection of animals, feeding, behavioural enrichment and diseases and their prophylaxis are important topics. The authors critically deal with existing guidelines and give suggestions on how crocodiles can be kept appropriately and according to existing laws at the same time.Special attention is given to the aspects of current legislation. It is stressed that there should be given proper attention to the qualification of the keeping institution as to the biology of the animals as well as to security aspects.A table of species completes the text with a short array of important features to be considered in the keeping of crocodiles.     

The Validity of the Ussing Flux Ratio Equation in a Three-Dimensionally Inhomogeneous Membrane

Derivations of the Ussing flux ratio equation have, until now, required the membrane to be both bounded by parallel planes and homogeneous, except in the transmembrane direction. These constraints have been necessary for the theoretical demonstration that the equation is independent of membrane parameters in the absence of carriers, coupling, solvent drag, or “single-file” diffusion. In a new derivation, the flux ratio equation is shown to be valid in this kind of diffusion regime without regard to the three-dimensional structure of the membrane. Thus the constraints on both membrane homogeneity and membrane geometry are shown to be unnecessary. The general use of this equation to differentiate between simple, uncoupled diffusion and other membrane transport phenomena is thus placed on a firmer base. However, as in earlier derivations, it is necessary that isopotential, isobaric, constant concentration surfaces exist sufficiently close to the membrane on both of its sides.     

Apicoplast: keep it or leave it

Most Apicomplexans possess a relic plastid named apicoplast, originating from secondary endosymbiosis of a red algae. This non-photosynthetic organelle fulfils important metabolic functions and confers sensitivity to antibiotics. The tasks of this organelle is compared across the phylum of Apicomplexa, highlighting its role in metabolic adaptation to different intracellular niches     

Testing the monophyly of the New Zealand and Australian endemic family Conoesucidae Ross based on combined molecular and morphological data (Insecta: Trichoptera: Sericostomatoidea)

Conoesucidae (Trichoptera, Insecta) are restricted to SE Australia, Tasmania and New Zealand. The family includes 42 described species in 12 genera, and each genus is endemic to either New Zealand or Australia. Although monophyly has been previously assumed, no morphological characters have been proposed to represent synapomorphies for the group. We collected molecular data from two mitochondrial genes (16S and cytochrome oxidase I), one nuclear gene (elongation factor 1-α) (2237–2277 bp in total), and 12 morphological characters to produce the first phylogeny of the family. We combined the molecular and morphological characters and performed both a maximum parsimony analysis and a Bayesian analysis to test the monophyly of the family, and to hypothesize the phylogeny among its genera. The parsimony analysis revealed a single most parsimonious tree with Conoesucidae being a monophyletic taxon and sistergroup to the Calocidae. The Bayesian inference produced a distribution of trees, the consensus of which is supported with posterior probabilities of 100% for 15 out of 22 possible ingroup clades including the most basal branch of the family, indicating strong support for a monophyletic Conoesucidae. The most parsimonious tree and the tree from the Bayesian analysis were identical except that the ingroup genus Pycnocentria changed position by jumping to a neighbouring clade. Based on the assumption that the ancestral conoesucid species was present on both New Zealand and Australia, a biogeographical analysis using the dispersal-vicariance criteria demonstrated that one or two (depending on which of the two phylogenetic reconstructions were applied) sympatric speciation events took place on New Zealand prior to a single, late dispersal from New Zealand to Australia.