Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation.

The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.

Scan of human genome reveals no new Loci under ancient balancing selection.

There has been much speculation as to what role balancing selection has played in evolution. In an attempt to identify regions, such as HLA, at which polymorphism has been maintained in the human population for millions of years, we scanned the human genome for regions of high SNP density. We found 16 regions that, outside of HLA and ABO, are the most highly polymorphic regions yet described; however, evidence for balancing selection at these sites is notably lacking--indeed, whole-genome simulations indicate that our findings are expected under neutrality. We propose that (i) because it is rarely stable, long-term balancing selection is an evolutionary oddity, and (ii) when a balanced polymorphism is ancient in origin, the requirements for detection by means of SNP data alone will rarely be met.

Inducible nitric oxide synthase activity is essential for inhibition of prostatic tumor growth by interferon-beta gene therapy.

We have previously reported that adenoviral vector-mediated interferon (IFN)-beta gene therapy inhibits orthotopic growth of human prostate cancer cells in nude mice. The purpose of this study was to determine efficacy and mechanisms of this therapy in immune-competent mice. TRAMP-C2Re3 mouse prostate cancer cells infected with 100 multiplicity of infection (MOI) of adenoviral vector encoding for mouse IFN-beta (AdmIFN-beta), but not AdE/1 (a control adenoviral vector), produced approximately 60 ng/10(5) cells/24 h of IFN-beta. The tumorigenicity of AdmIFN-beta-transduced cells was dramatically reduced in the prostates of C57BL/6 mice. A single intratumoral injection of 2 x 10(9) PFU (plaque-forming unit) of AdmIFN-beta inhibited tumor growth by 70% and prolonged survival of tumor-bearing mice. Intriguingly, this AdmIFN-beta therapy did not alter the growth of tumors in inducible nitric oxide synthase (iNOS)-null C57BL/6 mice. Immunohistochemical analysis revealed that treatment of tumors with AdmIFN-beta in wild-type C57BL/6 mice led to increased iNOS expression, decreased microvessel density, decreased cell proliferation, and increased apoptosis. Furthermore, quantitative reverse-transcriptional PCR analysis showed that AdmIFN-beta therapy, in C57BL/6 but not the iNOS-null counterparts, reduced levels of the mRNAs for angiopoietin, basic fibroblast growth factor, matrix metalloproteinase-9, transforming growth factor-beta1, vascular endothelial growth factor (VEGF)-A, and VEGF-B, as well as the antiapoptotic molecule endothelin-1. These data indicated that IFN-beta gene therapy could be effective alternative for the treatment of locally advanced prostate cancer and suggest an obligatory role of NO in IFN-beta antitumoral effects in vivo.

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.

Hypervariability, suppressed recombination and the genetics of individuality.

We define 'genetic individuality' as intraspecies variation that has substantial heritability and involves traits that are sufficiently common that they can be observed in any modest-sized sampling of individuals. We propose that genetic individuality is largely shaped by the combinatory shuffling of a modest number of genes, each of which exists as a family of functionally and structurally diverged alleles. Unequivocal examples of such allele families are found at the O-antigen-biosynthetic locus in Pseudomonas aeruginosa and the human leucocyte antigen locus in humans. We examine characteristic features of these allele families and explore the possibility that genetic loci with similar characteristics can be recognized in a whole-genome scan of human genetic variation.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

A genomic sequence analysis of the mouse and human microtubule-associated protein tau.

Microtubule associated protein tau (MAPT) encodes the microtubule associated protein tau, the primary component of neurofibrillary tangles found in Alzheimer's disease and other neurodegenerative disorders. Mutations in the coding and intronic sequences of MAPT cause autosomal dominant frontotemporal dementia (FTDP-17). MAPT is also a candidate gene for progressive supranuclear palsy and hereditary dysphagic dementia. A human PAC (201 kb) and a mouse BAC (161 kb) containing the entire MAPT and Mtapt genes, respectively, were identified and sequenced. Comparative DNA sequence analysis revealed over 100 conserved non-repeat potential cis-acting regulatory sequences in or close to MAPT. Those islands with greater than 67% nucleotide identity range in size from 20 to greater than 1700 nucleotides. Over 90 single nucleotide polymorphisms were identified in MAPT that are candidate susceptibility alleles for neurodegenerative disease. The 5' and 3' flanking genes for MAPT are the corticotrophin-releasing factor receptor (CRFR) gene and KIAA1267, a gene of unknown function expressed in brain.

Identification of a genomic island present in the majority of pathogenic isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for "P. aeruginosa genomic island 1"). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

Error checking and graphical representation of multiple-complete-digest (MCD) restriction-fragment maps.

Genetic and physical maps display the relative positions of objects or markers occurring within a target DNA molecule. In constructing maps, the primary objective is to determine the ordering of these objects. A further objective is to assign a coordinate to each object, indicating its distance from a reference end of the target molecule. This paper describes a computational method and a body of software for assigning coordinates to map objects, given a solution or partial solution to the ordering problem. We describe our method in the context of multiple-complete-digest (MCD) mapping, but it should be applicable to a variety of other mapping problems. Because of errors in the data or insufficient clone coverage to uniquely identify the true ordering of the map objects, a partial ordering is typically the best one can hope for. Once a partial ordering has been established, one often seeks to overlay a metric along the map to assess the distances between the map objects. This problem often proves intractable because of data errors such as erroneous local length measurements (e.g., large clone lengths on low-resolution physical maps). We present a solution to the coordinate assignment problem for MCD restriction-fragment mapping, in which a coordinated set of single-enzyme restriction maps are simultaneously constructed. We show that the coordinate assignment problem can be expressed as the solution of a system of linear constraints. If the linear system is free of inconsistencies, it can be solved using the standard Bellman-Ford algorithm. In the more typical case where the system is inconsistent, our program perturbs it to find a new consistent system of linear constraints, close to those of the given inconsistent system, using a modified Bellman-Ford algorithm. Examples are provided of simple map inconsistencies and the methods by which our program detects candidate data errors and directs the user to potential suspect regions of the map.

Multiple-complete-digest restriction fragment mapping: generating sequence-ready maps for large-scale DNA sequencing.

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of x15-30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a "shotgun" method.

Assembly of high-resolution restriction maps based on multiple complete digests of a redundant set of overlapping clones.

An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchronization between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that underlie multiple-complete-digest mapping--including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility--are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.

Physical calibration of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease (RARE) cleavage.

Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of yeast artificial chromosome clones (YACs). While STS-content mapping has proven to be an effective means of ordering clone ends and STSs along chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA-assisted restriction endonuclease (RARE) cleavage, a method that exploits the binding specificity on duplex DNA of a RecA-protein-oligodeoxynucleotide complex to enhance the cleavage specificity of a restriction endonuclease. This technique allows selective cleavage at individual members of a large set of restriction sites. RARE-cleavage mapping was applied to a contig comprising 5 overlapping YACs spanning 580 kb on human chromosome 14. An STS-content map comprising 10 YAC-end specific STSs and one internal STS was constructed. RARE cleavage was performed on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site within the STS that was initially used to screen the YAC library for the clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the STS-content map into a true physical map that indicates precise positions of clone ends and STSs.

The human genome project.

The Human Genome Project in the United States is now well underway. Its programmatic direction was largely set by a National Research Council report issued in 1988. The broad framework supplied by this report has survived almost unchanged despite an upheaval in the technology of genome analysis. This upheaval has primarily affected physical and genetic mapping, the two dominant activities in the present phase of the project. Advances in mapping techniques have allowed good progress toward the specific goals of the project and are also providing strong corollary benefits throughout biomedical research. Actual DNA sequencing of the genomes of the human and model organisms is still at an early stage. There has been little progress in the intrinsic efficiency of DNA-sequence determination. However, refinements in experimental protocols, instrumentation, and project management have made it practical to acquire sequence data on an enlarged scale. It is also increasingly apparent that DNA-sequence data provide a potent means of relating knowledge gained from the study of model organisms to human biology. There is as yet little indication that the infusion of technology from outside biology into the Human Genome Project has been effectively stimulated. Opportunities in this area remain large, posing substantial technical and policy challenges.

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs.

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae are presented. In order of increasing size, they are chromosomes I, VI, III, IX, V and VIII, comprising 2.49 megabase pairs of DNA. The maps are based on the analysis of an overlapping set of lambda and cosmid clones. Overlaps between adjacent clones were recognized by shared restriction fragments produced by the combined action of EcoRI and HindIII. The average spacing between mapped cleavage sites is 2.6 kb. Five of the six chromosomes were mapped from end to end without discontinuities; a single internal gap remains in the map of chromosome IX. The reported maps span an estimated 97% of the DNA on the six chromosomes; nearly all the missing segments are telomeric. The maps are fully cross-correlated with the previously published SfiI/NotI map of the yeast genome by A. J. Link and M. V. Olson. They have also been cross-correlated with the yeast genetic map at 51 loci.

Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells.

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.