Isolation of plasma membranes from Xenopus embryos

Summary Plasma membranes were isolated in high yield from Xenopus gastrulae by repeated sedimentation in discontinuous sucrose gradients. Most of the yolk was separated by lowspeed sedimentation before centrifugation on the discontinuous sucrose gradients. The isolation of plasma membranes was followed by covalent labelling of the surface of dissociated gastrula cells with diazoniobenzene sulphonate, by electron microscopy and the distribution of enzymatic markers. The isolated plasma membranes have a low neural inducing activity as compared to other cell constituents.     

Neural-inducing activity of nuclei and nuclear fractions from Xenopus laevis embryos

Summary From embryos (Xenopus laevis) of different developmental stages nuclei were isolated which exert neural inducing activity in the biological test. The active material could partly be extracted from the nuclei. Experiments for the isolation of nuclear ribonucleoprotein (RNP) particles have shown that the activity is localized at least in part in these particles. On the other hand, some neural inducer is not detached from chromatin and the nuclear matrix even with ionic detergents. Inducing activity was found in germinal vesicles and to a higher degree in the cytoplasm of oocytes, but in a masked, biologically inactive state.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Book reviews

Ohne Zusammenfassung

---

Book reviews

     

Gene synteny comparisons between different vertebrates provide new insights into breakage and fusion events during mammalian karyotype evolution

Background  

Genome comparisons have made possible the reconstruction of the eutherian ancestral karyotype but also have the potential to provide new insights into the evolutionary inter-relationship of the different eutherian orders within the mammalian phylogenetic tree. Such comparisons can additionally reveal (i) the nature of the DNA sequences present within the evolutionary breakpoint regions and (ii) whether or not the evolutionary breakpoints occur randomly across the genome. Gene synteny analysis (E-painting) not only greatly reduces the complexity of comparative genome sequence analysis but also extends its evolutionary reach.     

Polymorphic micro-inversions contribute to the genomic variability of humans and chimpanzees

A combination of inter- and intra-species genome comparisons is required to identify and classify the full spectrum of genetic changes, both subtle and gross, that have accompanied the evolutionary divergence of humans and other primates. In this study, gene order comparisons of 11,518 human and chimpanzee orthologous gene pairs were performed to detect regions of inverted gene order that are potentially indicative of small-scale rearrangements such as inversions. By these means, a total of 71 potential micro-rearrangements were detected, nine of which were considered to represent micro-inversions encompassing more than three genes. These putative inversions were then investigated by FISH and/or PCR analyses and the authenticity of five of the nine inversions, ranging in size from approximately 800 kb to approximately 4.4 Mb, was confirmed. These inversions mapped to 1p13.2-13.3, 7p22.1, 7p13-14.1, 18p11.21-11.22 and 19q13.12 and encompass 50, 14, 16, 7 and 16 known genes, respectively. Intriguingly, four of the confirmed inversions turned out to be polymorphic: three were polymorphic in the chimpanzee and one in humans. It is concluded that micro-inversions make a significant contribution to genomic variability in both humans and chimpanzees and inversion polymorphisms may be more frequent than previously realized.     

Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus

BACKGROUND: Viruses are being exploited as vectors to deliver therapeutic genetic information into target cells. The success of this approach will depend on the ability to overcome current limitations, especially in terms of safety and efficiency, through molecular engineering of the viral particles. METHODS: Here we show that in vitro directed evolution can be successfully performed to randomize the viral capsid by error prone PCR and to obtain mutants with improved phenotype. RESULTS: To demonstrate the potential of this technology we selected several adeno-associated virus (AAV) capsid variants that are less efficiently neutralized by human antibodies. These mutations can be used to generate novel vectors for the treatment of patients with pre-existing immunity to AAV. CONCLUSIONS: Our results demonstrate that combinatorial engineering overcomes the limitations of rational design approaches posed by incomplete understanding of the infectious process and at the same time offers a powerful tool to dissect basic viral biology by reverse genetics.     

Onset of significant pelagic carbonate accumulation after the Carnian Pluvial Event (CPE) in the western Tethys

Abundant calcispheres occur in Upper Carnian and Norian hemipelagic limestone successions of the Southern Apennines and Sicily. They exhibit a variety of morphologies that were investigated with optical and scanning electron microscopy (SEM). The most common morphology is that of a full solid sphere of radiaxial calcite crystals, 20–22 μm in diameter on average, with or without a minor hollow in the center. Smaller forms may be clusters of sub-micron crystals, only rarely disposed as to form a spherical test with a diameter of 10 μm or less. Larger forms are similar to small forms (clusters or spheres of sub-micron crystals) with an epitaxial calcite overgrowth. The taxonomic attribution of these calcispheres is uncertain, mostly because of their poor preservation, but a comparison is possible with some Mesozoic calcispheres attributed to calcareous dinocysts. The amount of epitaxial overgrowth is variable, but in most cases much larger than the original sphere. This prevents a significant evaluation of the contribution of calcispheres to carbonate pelagic sedimentation by point counting in thin-section. However, it can be shown that calcispheres become abundant only after a major climatic perturbation dated at the end of the Early Carnian, known as the Carnian Pluvial Event (CPE). This event involved a strong and prolonged enhancement of the hydrological cycle, with consequent supply of excess hydrogen carbonate to the oceans and increased seawater alkalinity. Although calcispheres of this type are known at least from the Middle Triassic, it is only shortly after the CPE that they become abundant, and the first common occurrence of calcareous nannoplankton in the western Tethys is thus Late Carnian in age.