Formaldehyde fixation of cells does not greatly reduce the ability to amplify cellular DNA

Formaldehyde fixation of cells is routinely used to study DNA-protein interactions in vivo. In these studies, DNA is often analyzed using a polymerase chain reaction technique. Although it is known that formaldehyde can damage DNA, no studies have been performed so far to compare the efficiency of DNA amplification between normal and fixed cells. Here we show that formaldehyde fixation results in a 15% to 20% reduction in the ability to amplify cellular DNA. The loss of amplifiability is independent of the length of the amplification region and the degree to which DNA is compacted on packaging into chromatin.     

Using Principal Components and Factor Analysis in Animal Behaviour Research: Caveats and Guidelines

Principal component (PCA) and factor analysis (FA) are widely used in animal behaviour research. However, many authors automatically follow questionable practices implemented by default in general‐purpose statistical software. Worse still, the results of such analyses in research reports typically omit many crucial details which may hamper their evaluation. This article provides simple non‐technical guidelines for PCA and FA. A standard for reporting the results of these analyses is suggested. Studies using PCA and FA must report: (1) whether the correlation or covariance matrix was used; (2) sample size, preferably as a footnote to the table of factor loadings; (3) indices of sampling adequacy; (4) how the number of factors was assessed; (5) communalities when sample size is small; (6) details of factor rotation; (7) if factor scores are computed, present determinacy indices; (8) preferably they should publish the original correlation matrix.     

Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein

SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1α and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1α-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi⁺] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.     

The development of mouse spinal cord in tissue culture

Summary Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells and, finally, the cytodifferentiation of neurons were observed. A preliminary account of this work was given at the Tissue Culture Association Meeting in 1977, and the Canadian Federation of Biological Societies Meeting in 1977 (1,2). This work was supported by Grant MT 4235 from the Medical Research Council of Canada.     

Inhibition of biofouling by marine microorganisms and their metabolites

Development of microbial biofilms and the recruitment of propagules on the surfaces of man-made structures in the marine environment cause serious problems for the navies and for marine industries around the world. Current antifouling technology is based on the application of toxic substances that can be harmful to the natural environment. For this reason and the global ban of tributyl tin (TBT), there is a need for the development of "environmentally-friendly" antifoulants. Marine microbes are promising potential sources of non-toxic or less-toxic antifouling compounds as they can produce substances that inhibit not only the attachment and/or growth of microorganisms but also the settlement of invertebrate larvae and macroalgal spores. However, so far only few antilarval settlement compounds have been isolated and identified from bacteria. In this review knowledge about antifouling compounds produced by marine bacteria and diatoms are summarised and evaluated and future research directions are highlighted.     

DNA barcoding and minibarcoding as a powerful tool for feather mite studies

Feather mites (Astigmata: Analgoidea and Pterolichoidea) are among the most abundant and commonly occurring bird ectosymbionts. Basic questions on the ecology and evolution of feather mites remain unanswered because feather mite species identification is often only possible for adult males, and it is laborious even for specialized taxonomists, thus precluding large‐scale identifications. Here, we tested DNA barcoding as a useful molecular tool to identify feather mites from passerine birds. Three hundred and sixty‐one specimens of 72 species of feather mites from 68 species of European passerine birds from Russia and Spain were barcoded. The accuracy of barcoding and minibarcoding was tested. Moreover, threshold choice (a controversial issue in barcoding studies) was also explored in a new way, by calculating through simulations the effect of sampling effort (in species number and species composition) on threshold calculations. We found one 200‐bp minibarcode region that showed the same accuracy as the full‐length barcode (602 bp) and was surrounded by conserved regions potentially useful for group‐specific degenerate primers. Species identification accuracy was perfect (100%) but decreased when singletons or species of the Proctophyllodes pinnatus group were included. In fact, barcoding confirmed previous taxonomic issues within the P. pinnatus group. Following an integrative taxonomy approach, we compared our barcode study with previous taxonomic knowledge on feather mites, discovering three new putative cryptic species and validating three previous morphologically different (but still undescribed) new species.     

Continual assembly of desmosomes within stable intercellular contacts of epithelial A-431 cells

Subclones of human carcinoma-derived A-431 cell line stably producing fusion proteins consisting of the enhanced green fluorescent protein and either human desmoglein 2 (Dsg-GFP) or human plakoglobin (GFP-Pg) were used to examine the behavior of desmosomes in living cells. Immunofluorescence microscopy of the fixed cells showed that both fusion proteins, which were expressed in significantly lower levels relative to their endogenous counterparts, were efficiently recruited into desmosomes. Time-lapse confocal imaging of these cells reveals that such GFP-labeled desmosomes (GFP desmosomes) are stable structures which exhibit various dynamic and motile activities. The most notable are independent lateral mobility and fusion. Furthermore, the continual assembly of new nascent desmosomes is observed within stable contacts located at the middle of the epithelial sheet. A new GFP desmosome appears as a closely apposed group of fine patches which after a few minutes aggregate into a single structure. These three dynamic processes resulted in constant changes of desmosome distribution, numbers, and sizes. In addition, fluorescence recovery after photobleaching experiments showed that fine patches of desmosomal proteins may participate in desmosome maintenance. Such a diverse range of dynamic activities of desmosomes apparently produces flexible but tight cell-cell adhesion required for different morphogenetic events in epithelial structures.This work was supported by grant AR44016-04 from the National Institutes of Health     

A set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles,membranes and cytoskeletal elements in Medicago truncatula

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.