Ionizing radiation is known to induce genomic instability that is transmitted across many generations of the progeny of surviving cells. However, the mechanism underlying the initiation, perpetuation and manifestation of radiation-induced genomic instability remains unclear. We expect that large radiation-induced deletions destabilize the structure of chromatin and that this destabilization is transmitted across many generations and plays a role in the perpetuation of genomic instability. Therefore, in this study, we examined the relationship between deletion size and the frequency of delayed chromosomal aberrations in SV40-immortalized normal human fibroblast (GM638) cells. GM638 cells were irradiated with 3 Gy of X rays, and chromosomal aberrations were analyzed in clones derived after irradiation. To determine the size of each deletion, we isolated mutants of the HPRT gene from the X-irradiated cell population and examined the genes around the HPRT locus, which is located in the q-arm of chromosome X. The results indicated that X chromosomes with large (>0.5 Mb) deletions have a higher probability of exhibiting delayed chromosomal aberrations and that these aberrations were induced more frequently in q-arms than in p-arms. Because no induction of X-chromosomal instability was observed in clones that lacked such large deletions, the present findings suggest that chromosomes with large radiation-induced deletions can be genomically unstable.

The GenBank((R))sequence database incorporates publicly available DNA sequences of >55 000 different organisms, primarily through direct submission of sequence data from individual laboratories and large-scale sequencing projects. Most submissions are made using the BankIt (Web) or Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage. GenBank data is accessible through NCBI's integrated retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping and protein structure information, plus the biomedical literature via PubMed. Sequence similarity searching is provided by the BLAST family of programs. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. NCBI also offers a wide range of WWW retrieval and analysis services based on GenBank data. The GenBank database and related resources are freely accessible via the NCBI home page at http://www.ncbi.nlm.nih.gov

The GenBank (Registered Trademark symbol) sequence database incorporates DNA sequences from all available public sources, primarily through the direct submission of sequence data from individual laboratories and from large-scale sequencing projects. Most submitters use the BankIt (Web) or Sequin programs to format and send sequence data. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage. GenBank data is accessible through NCBI's integrated retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome and protein structure information. MEDLINE (Registered Trademark symbol) s from published articles describing the sequences are included as an additional source of biological annotation through the PubMed search system. Sequence similarity searching is offered through the BLAST series of database search programs. In addition to FTP, Email, and server/client versions of Entrez and BLAST, NCBI offers a wide range of World Wide Web retrieval and analysis services based on GenBank data. The GenBank database and related resources are freely accessible via the URL: http://www.ncbi.nlm.nih.gov

The GenBank sequence database incorporates publicly available DNA sequences of more than 105 000 different organisms, primarily through direct submission of sequence data from individual laboratories and large-scale sequencing projects. Most submissions are made using the BankIt (web) or Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage. GenBank data is accessible through NCBI's integrated retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical literature via PubMed. Sequence similarity searching is provided by the BLAST family of programs. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. NCBI also offers a wide range of World Wide Web retrieval and analysis services based on GenBank data. The GenBank database and related resources are freely accessible via the NCBI home page at http://www.ncbi.nlm.nih.gov.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

MOTIVATION: To meet the demands of large-scale sequencing, thousands of clones must be fingerprinted and assembled into contigs. To determine the order of clones, a typical experiment is to digest the clones with one or more restriction enzymes and measure the resulting fragments. The probability of two clones overlapping is based on the similarity of their fragments. A contig contains two or more overlapping clones and a minimal tiling path of clones is selected to be sequenced. Interactive software with algorithmic support is necessary to assemble the clones into contigs quickly. RESULTS: FPC (fingerprinted contigs) is an interactive program for building contigs from restriction fingerprinted clones. FPC uses an algorithm to cluster clones into contigs based on their probability of coincidence score. For each contig, it builds a consensus band (CB) map which is similar to a restriction map; but it does not try to resolve all the errors. The CB map is used to assign coordinates to the clones based on their alignment to the map and to provide a detailed visualization of the clone overlap. FPC has editing facilities for the user to refine the coordinates and to remove poorly fingerprinted clones. Functions are available for updating an FPC database with new clones. Contigs can easily be merged, split or deleted. Markers can be added to clones and are displayed with the appropriate contig. Sequence-ready clones can be selected and their sequencing status displayed. As such, FPC is an integrated program for the assembly of sequence-ready clones for large-scale sequencing projects.

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.