Local thermodynamic stability scores are well represented by a non-central student's t distribution

Local folding in mRNAs is closely associated w ith biological functions. In this study, we reveal the whole distribution of local thermodynamic stability in the complete genome of the poliovirus P3/Leon/37 and the single-stranded RNA sequences that corresponds to the nucleotide sequence of the complete genome sequence (1 667 867 bp) of Helicobacter pylori (H. pylori) strain 26695. Local thermodynamic stability in the RNA sequences is measured by two standard z -scores, significance score and stability score. To estimate the distribution of thermodynamic stability, a model based on the non-central Student's t distribution has been developed. Significant patterns of extremes that are either much more stable or unstable than expected by chance are detected. Our results indicate that the highly stable and statistically more significant folding regions are predominantly in non-coding sequences in the two genome sequences. Moreover, the highly unstable folding regions, on the contrary, are predominantly in the protein coding sequences of H. pylori. The observed differences across the complete genomic sequences are statistically very significant by a chi2-test. These extreme patterns may be useful in searching for target sequences for long-chain antisense RNA and for locating potential RNA functional elements involved in the regulation of gene expression including translation, mRNA localization and metabolism.     

“Rule of Six”: How Does the Sendai Virus RNA Polymerase Keep Count?

The "rule of six" stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6n + 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3'-OH end (3'-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3'-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.     

Nef-induced CD4 downregulation: a diacidic sequence in human immunodeficiency virus type 1 Nef does not function as a protein sorting motif through direct binding to beta-COP

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the beta subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63-73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with beta-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to beta-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to beta-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.     

Novel chromosomal insertional translocation in chicken uncovered by double color FISH

A novel insertion 78,ZZ or 78,ZW, ins(3;1)(q25q27;undetermined) was revealed in chicken by double color fluorescent in situ hybridization (FISH). A fragment of chromosome 1 spanning either from q13-14 to q34-35, or from q14-21 to q36-41 bands, had been translocated to chromosome 3 at a site located between q25 to q27 bands. This has resulted in the generation of an interstitial deletion in chromosome 1 and an insertional translocation in chromosome 3. Chickens with this balanced insertional translocation are asymptomatic carriers and their fertility is not affected, but embryo mortality increases. Greater than 50% occurrence of unbalanced gametes are observed. However, progeny sex ratio is not affected.     

Molecular phylogeny of the Ceratosolen species pollinating Ficus of the subgenus Sycomorus sensu stricto: biogeographical history and origins of the species-specificity breakdown cases

The 14 species of Ficus of the subgenus Sycomorus (Moraceae) are invariably pollinated by Ceratosolen species (Hym. Chalcidoidea), which in turn reproduce in the fig florets. They are distributed mostly in continental Africa, Madagascar, and the Mascarene and Comoro Islands, but 1 species extends its geographical range all over the Oriental region. Fig-pollinator relationships are usually strictly species specific, but exceptions to the 'one-to-one' rule occur within the group we studied. In order to understand both the biogeographical history of the Ceratosolen species associated with Ficus of the subgenus Sycomorus and the origins of the specificity breakdown cases, we have used cytochrome b sequences to reconstruct a phylogeny of the fig wasps. The results show that the pollinators from the Malagasy region and those from continental Africa form two distinct clades, which probably diverged after the crossing of the Mozambique Channel by an ancestral population. The Oriental wasp species show strong affinities with the African species. The two species-specificity exceptions are due to different evolutionary events. The occurrence of the two West African pollinators associated with F. sur can be explained by successive speciation events of the mutualistic partner without plant radiation. In contrast, we hypothesize that C. galili shifted by horizontal transfer from an unknown, presumably extinct, Ficus species to F. sycomorus after this native Malagasy fig species colonized Africa.     

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Distribution of glycogen phosphorylase and cytochrome oxidase in the central nervous system of the turtle Trachemys dorbigni

Glycogen phosphorylase (GP) and cytochrome oxidase (CO) activities were mapped histochemically in the brain of the turtle Trachemys dorbigni. In the telencephalon, both activities occurred in the olfactory bulb, in all cortical areas, in the dorsal ventricular ridge, striatum, primordium hippocampi and olfactory tubercle. In the diencephalon, they were identified in some areas of the hypothalamus, and in rotundus and geniculate nuclei. Both reactions were detected in the oculomotor, trochlear, mesencephalic trigeminal nuclei, the nucleus of the posterior commissure, torus semicircularis, substantia nigra and ruber and isthmic nuclei of the mesencephalon. In all layers of the optic tectum GP activity was found, but CO only labelled the stratum griseum centrale. In the medulla oblonga both enzymes appear in the reticular, raphe and vestibular nuclei, locus coeruleus and nuclei of cranial nerves. In the cerebellum, the granular and molecular layers, and the deep cerebellar nuclei were positive for both enzymes. The Purkinje cells were only reactive for CO. In the spinal cord, motor and commissural neurones exhibited a positive reaction for the two enzymes. However, CO also occurred in the marginal nucleus and in the lateral funiculus. These results may be useful as a basis for subsequent studies on turtle brain metabolism.     

Conus venoms: a source of toxins which interact with membrane- potential-dependent sodium channels

Marine snails of the genus Conus, as they are carnivorous predators, have a venom apparatus used to capture their prey. The toxins contained in the venoms of Conidae, called conotoxins, are of a particular high degree of diversity and represent powerful tools in the neuroscience field. Indeed, these toxins specifically bind with a high affinity to receptors and ionic channels. Therefore, they provide original pharmacological tools which receive increasing investigation both to identify and study some functions of the nervous systems and to characterize new types and closely related subtypes of receptors or ionic channels. The voltage-gated sodium channel, because of its fundamental role in cell membrane excitability, is the specific target of a large number of animal and vegetal toxins. Actually, at least seven toxin receptor sites have been identified on this channel-protein. These toxins, and in particular conotoxins, are used to precise the role of different types and/or closely related subtypes of sodium channels in the peripheral and central nervous systems. The focus of the present review is to summarize our current knowledge of the consequences of physiological interactions between different conotoxin families and sodium channels.     

Word-processor macro for restriction endonuclease analysis

This paper describes a Microsoft Word 97 macro designed for restriction endonuclease analysis. Selected DNA fragments in the active Word document can be analyzed through a dynamic dialog box that formats the enzyme restriction lists for further analysis. The results can be obtained in a new Word document with the name of the enzymes, number of cuts and positions. This macro has several advantages: the results can be printed in a format suitable for record keeping, no additional programs are required and it is simple to use.