Structure of Arc repressor in solution: evidence for a family of beta-sheet DNA-binding proteins

The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.     

Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum

Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis and enter a prelysosomal endosomal pathway for further processing. Fluorescence microscopy and video recording of living cells to trace the passage of ligand-receptor complexes has identified the endosomal compartment as an extensive network of tubular cisternae. Endocytosed material entering this reticulum enters discrete swellings, identified as multivesicular bodies by electron microscopy, which move along the reticulum towards the pericentriolar area.     

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.     

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.     

A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n

The instability of chromosomes with breaks induced by X-irradiation led to the proposal that the natural ends of chromosomes are capped by a specialized structure, the telomere. Telomeres prevent end-to-end fusions and exonucleolytic degradation, enable the end of the linear DNA molecule to replicate, and function in cell division. Human telomeric DNA comprises approximately 2-20 kilobases (kb) of the tandemly repeated sequence (TTAGGG)n oriented 5'----3' in towards the end of the chromosome, interspersed with variant repeats in the proximal region. Immediately subtelomeric lie families of unrelated repeat motifs (telomere-associated sequences) whose function, if any, is unknown. In lower eukaryotes the formation and maintenance of telomeres may be mediated enzymatically (by telomerase) or by recombination; in man the mechanisms are poorly understood, although telomerase has been identified in HeLa cells. Here we describe an alpha thalassaemia mutation associated with terminal truncation of the short arm of chromosome 16 (within band 16p13-3) to a site 50 kb distal to the alpha globin genes, and show that (TTAGGG)n has been added directly to the site of the break. The mutation is stably inherited, proving that telomeric DNA alone is sufficient to stabilize the broken chromosome end. This mechanism may occur in any genetic disease associated with chromosome truncation.     

Localization of the human GM-CSF receptor gene to the X-Y pseudoautosomal region

Mammalian sex chromosomes share a small terminal region of homologous DNA sequences, which pair and recombine during male meiosis. Alleles in this region can be exchanged between X and Y chromosomes and are therefore inherited as if autosomal. Genes from this so-called pseudoautosomal region (PAR) are present in two doses in both males and females, and escape inactivation of the X chromosome in females. Indirect evidence suggests that there must be several pseudoautosomal genes, and several candidates have been proposed. Until now, the only gene that has been unequivocally located in the PAR is MIC2, which encodes a cell-surface antigen of unknown function. We now report the localization of a gene of known function to this region--the gene for the receptor of the haemopoietic regulator, granulocyte-macrophage colony stimulating factor. The chromosomal localization of this gene may be important in understanding the generation of M2 acute myeloid leukaemia.     

Expression of a candidate sex-determining gene during mouse testis differentiation

The development of a eutherian mammal as a male is a consequence of testis formation in the embryo, which is thought to be initiated by a gene on the Y chromosome. In the absence of this gene, ovaries are formed and female characteristics develop. Sex determination therefore hinges on the action of this testis-determining gene, known as Tdy in mice and TDF in humans. In the past, several genes proposed as candidates for Tdy/TDF have subsequently been dismissed on the grounds of inappropriate location or expression. We have recently described a candidate for Tdy, which maps to the minimum sex-determining region of the mouse Y chromosome. To examine further the involvement of this gene, Sry, in testis development, we have studied its expression in detail. Fetal expression of Sry is limited to the period in which testes begin to form. This expression is confined to gonadal tissue and does not require the presence of germ cells. Our observations strongly support a primary role for Sry in mouse sex determination.     

Host-parasitoid associations in patchy environments

Studies of insect host-parasitoid interactions have contributed much to the consensus that spatial patchiness is important in the regulation of natural populations. A variety of theoretical models predict that host and parasitoid populations, although unstable in the absence of environmental heterogeneity, may persist at roughly steady overall densities in a patchy environment owing to variation in levels of parasitism from patch to patch. Observed patterns of parasitism, however, have a variety of forms (with variation in attack rates among patches depending directly or indirectly on host density, or showing variation uncorrelated with host density). There is some confusion about the dynamical consequences of these different forms. Here we first show how the dynamical effects of all these forms of environmental heterogeneity can be assessed by a common criterion. This 'CV2 greater than 1 rule' states that the overall population densities will remain roughly steady from generation to generation if the coefficient of variation squared (CV2) of the density of searching parasitoids in the vicinity of each host exceeds approximately unity. By partitioning CV2 into components, we show that both direct and inverse patterns of dependence on host density, and density-independent patterns, all contribute to population regulation in the same way. Second, we show how a maximum-likelihood method can be applied to the kind of field data that are usually available (that is, percentage parasitism versus local host density) to estimate the components of CV2. This analysis indicates that heterogeneity is large enough to stabilize dynamics in 9 of 34 published studies, and that density-independent heterogeneity is the main factor in most cases.     

Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins

A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.