A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.     

Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE*

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.     

Metabolic engineering of Escherichia coli to produce (2S, 3R, 4S)-4-hydroxyisoleucine

The stereo-specific l-isoleucine-4-hydroxylase (l-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to “shunt” destroyed TCA, thereby coupling l-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of l-isoleucine into 4-HIL with an 82% yield.     

Cross-reacting intraspecific antigens of Neisseria meningitidis. I. The isolation, immunochemical and serological characteristics of the cross-reacting antigens of N. meningitidis serogroup A

New data on the cross-reacting antigen of N. meningitidis, serogroup A, are presented. A complex of antigens has been isolated by treatment with tryptone X-100, ethanol precipitation and the subsequent treatment with trichloroacetic acid. The immunological analysis of the isolated preparation has shown that the proteinaceous part of the biopolymer contains 7 polypeptide fragments; of these, one fragment with a molecular weight of 31000 daltons has been found to constitute 49, 15% of all polypeptide fragments. The evaluation of the serological properties of this preparation in the precipitation test and the passive hemagglutination test has revealed that the preparation contains various cross-reactive antigenic determinants. Polyvalent erythrocyte diagnosticum obtained on the basis of this preparation permits the detection of antibodies to meningococci irrespective of their serogroup.     

The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free-living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.     

Regulators of Autophagosome Formation in Drosophila Muscles

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.