The physical map ofMus musculus chromosome 11 reveals evolutionary relationship with different syntenic groups of genes inHomo sapiens

Summary The physical localization of sequences homologous to three cloned genes was determined by in situ hybridization to metaphase chromosomes. Previous work had assigned the skeletal myosin heavy chain gene cluster (Myh), the functional locus for the cellular tumor antigen p53 (Trp53-1), and the cellular homologue of the viral erb-B oncogene (Erbb) toMus musculus chromosome 11 (MMU11). Our results provide regional assignments ofMyh andTrp53-1 to chromosome bands B2C, and ofErbb to bands A1A4. Taken together with in situ mapping of three other loci on MMU 11 (Hox-2 homeobox-containing gene cluster, theSparc protein, and theColla-1 collagen gene), which have been reported elsewhere, these data allowed us to construct a physical map of MMU11 and to compare it with the linkage map of this chromosome. The map positions of the homologous genes on human chromosomes suggest evolutionary relationships of distinct regions of MMU11 with six different human chromosome arms: 1p, 5q, 7p, 16p, 17p, and 17q. The delineation of conserved chromosome regions has important implications for the understanding of karyotype evolution in mammalian species and for the development of animal models of human genetic diseases.     

A gene (Bmn) controllingβ-mannosidase activity in the mouse is located in the distal part of chromosome 3

A gene (Bmn) with a major effect on -mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substratep-nitrophenyl--d-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance,cdm, as well as to theAdh-3 locus.This work was supported by Swedish Natural Science Research Council Project B-BU 2992-108.     

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G. austeni Newstead and G. p. palpalis R-D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A 'refractory' line of G. m. morsitans Westwood took 170 h to kill 50% infections while a 'susceptible' line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo. Flies fed before an infective feed were able to remove trypanosomes from their midguts more quickly than flies infected as tenerals. Increasing the period of starvation before infection increased the susceptibility to trypanosome infection of non-teneral flies. Teneral flies showed little agglutinating activity in vitro, suggesting that lectin is produced in response to the bloodmeal. Feeding flies before infection also abolished the differences in rate of trypanosome killing found between teneral 'susceptible' and 'refractory' G. m. morsitans, suggesting that maternally inherited susceptibility to trypanosome infection is a phenomenon limited to teneral flies. Electron micrographs of midguts of G. m. morsitans suggest that procyclic trypanosomes are killed by cell lysis, presumably the result of membrane damage caused by lectin action.     

Osmoregulation in Dunaliella tertiolecta: effects of salt stress,and the external pH on the internal pH

The intracellular pH of the halotolerant green algae Dunaliella tertiolecta, was determined by the distribution of 5,5-dimethyl-2(¹⁴C)-oxalolidine-2,5-dione (DMO) between the cell and the surrounding medium. 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione was not metabolized by the algal cells. The intracellular pH of Dunaliella tertiolecta was 6.8 in the dark and 7.4 in the light. During a salt stress, after two hours, the intracellular pH was increased by 0.2 pH units in both light and dark. The salt stressed cells maintained a constant pH of about 7.5 over the pH range of 6.5 to 8.5. Because of the relatively low permeability coefficient of the plasma membrane for DMO, this technique does not permit rapid pH determinations during the induction period after a salt stress. The magnitude of the salt induced pH changes measured 2 h after the salt stress implies a minor importance of this alkalization in this time range, but does not exclude a larger importance of pH changes for osmoregulation during the induction period.Abbreviations Chl chlorophyll - DMO 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione - PCV packed cell volume - SDS sodium dodecyl sulfate     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

A model for gramicidin A′-phospholipid interactions in bilayers

A model is proposed for the effect of gramicidin A on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin A influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin A. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin A to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine.Abbreviations NMR nuclear magnetic resonance - DMPC dimyristoylphosphatidylcholine - S molecular order parameter - CSA chemical shift anisotropy - DPPC dipalmitoylphosphati-dylcholine - LPC lysophosphatidylcholine     

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage

Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at 180 copies per uninduced cell and was measured at 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.     

Immunocytochemical localization of conglutin γ and legumin-like globulin in developing and mature seeds ofLupinus albus L.

Summary The distribution of two seed proteins, namely conglutin and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin was present in all cell types.A precursor of the legumin-like protein accumulated transiently in the developing cotyledon, but was subsequently modified by proteolytic cleavage. The onset of such modification was concomitant with a transition in the predominant vacuolar forms within the storage parenchyma cells. No precursor molecules of conglutin have been detected in this study, thus indicating that this protein is deposited in the protein bodies in its mature form.Abbreviations LM light microscopy - EM electron microscopy - DAF days after flowering - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - GAR goat antirabbit antiserum     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.