Isolation of a candidate gene for Norrie disease by positional cloning.

The gene for Norrie disease, an X-linked disorder characterized by progressive atrophy of the eyes, mental disturbances and deafness, has been mapped to chromosome Xp11.4 close to DXS7 and the monoamine oxidase (MAO) genes. By subcloning a YAC with a 640 kilobases (kb) insert which spans the DXS7-MAOB interval we have generated a cosmid contig which extends 250 kb beyond the MAOB gene. With one of these cosmids, microdeletions were detected in several patients with Norrie disease. Screening of cDNA libraries has enabled us to isolate and sequence a likely candidate gene for Norrie disease which is expressed in retina, choroid and fetal brain. No homologous sequences were found in DNA and protein databases indicating that this cDNA is part of a gene encoding a 'pioneer' protein.     

Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.     

小盘藻属二新种和小环藻属一新变种

在厦门港微型硅藻分类研究中,发现小盘藻属二新种和小环灌属一新变种,用检索表对小盘藻的二新种与已报道的另4种作了比较。     

Ornithine decarboxylase activity is critical for cell transformation.

The enzyme ornithine decarboxylase is the key regulator of the synthesis of polyamines which are essential for cell proliferation. Expression of this enzyme is transiently increased upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes. To test whether ornithine decarboxylase could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 cells with expression vectors carrying the complementary DNA encoding human ornithine decarboxylase in sense and antisense orientations. The increased expression of the enzyme (50-100-times endogenous levels) induced not only cell transformation, but also anchorage-independent growth in soft agar and increased tyrosine phosphorylation of a protein of M(r) 130K. Expression of ornithine decarboxylase antisense RNA was associated with an epithelioid morphology and reduced cell proliferation. Moreover, blocking the endogenous enzyme using specific inhibitor or synthesizing antisense RNA prevented transformation of rat fibroblasts by temperature-sensitive v-src oncogene. Our results imply that the gene encoding ornithine decarboxylase is a proto-oncogene central for regulation of cell growth and transformation.     

Different beta-subunits determine G-protein interaction with transmembrane receptors.

Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.     

Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.     

Human aminopeptidase N is a receptor for human coronavirus 229E.

Human coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections. Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells, is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.