A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

A gene map of the human genome.

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/.

Ecto-5'-nucleotidase. I. Expression in human normal and neoplastic lymphoid cell lines representing sequential stages of B-cell differentiation.

The possible association of ecto-5'-nucleotidase (5'-NT) with differentiation of B-cells was explored with the use of normal and neoplastic lymphoblastoid cell lines representing sequential stages of B-cell maturation. There was no relationship between patterns of enzyme expression in the cell lines and immunoglobulin (Ig) secretion, chromosome constitution, proliferative rate, cell volume, or the presence of B1 and B2 antigens. Pre-B-cell lines, which were negative for surface Ig or Ig secretion but positive for cytoplasmic mu-chains, showed the presence of 5'-NT, whereas 9 of 11 lymphoma cell lines, Burkitt's or non-Burkitt's type, both secreting and nonsecreting, did not exhibit enzyme activity. Four myeloma cell lines and 13 of 15 normal B-cell lines were positive for 5'-NT. These results suggested that 5'-NT was present in pre-B-cells and in some very early B-cells. 5'-NT usually disappeared from early and some intermediate B-cells and reappeared in mature B-cells and plasmacytoid cells.

JADE: an approach for interconnecting bioinformatics databases.

To achieve the integration of biological data available on the World Wide Web and maintained in diverse sources such as GDB, Genbank or Acedb, we have developed a software called Jade. Jade allows programmers to create analytic tools and graphical user interfaces for one or more existing bioinformatics data sources. These tools can then be interchanged, compared and reused without making modifications in the data sources themselves. The system is implemented in the Java programming language and will run equally well on Macintosh, Windows or Unix workstations. Jade is free and can be used immediately by all interested parties.

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells.

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (Pi) level. In contrast, normal cells excreted nucleosides only at low Pi level while at high Pi levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.

Benefit of intravenous IgG replacement in hypogammaglobulinemic patients with chronic sinopulmonary disease.

Seven patients with hypogammaglobulinemia and chronic sinopulmonary infections were treated with a preparation of intravenous gammaglobulin. In order to maintain levels of serum IgG at greater than 500 to 750 mg/dl four weeks after infusion, 0.6 g/kg was administered every month. Stable serum levels were achieved after three to eight months. After six to 12 months of this regimen, there was significant reduction in acute infections requiring hospitalization, amelioration of clinical and radiographic evidence of chronic maxillary sinusitis, and improvement in pulmonary symptoms and pulmonary function test results. The administration of increased amounts of IgG intravenously is of benefit in patients with chronic sinopulmonary infections.

Cloning of a developmentally regulated tegument antigen of Schistosoma mansoni.

We have cloned a gene encoding a 22.6 kDa antigen from a Schistosoma mansoni cDNA library. Northern blots indicate that transcription of this antigen occurs in adults and sporocysts but not in cercariae, eggs or in newly-transformed schistosomula. Immunoprecipitation and Western blotting with specific antisera indicate that the antigen is not detectable in the newly transformed schistosomulum but appears within 24 h of schistosomulum transformation. Indirect immunofluorescence of adult worms shows this protein to be located in the tegument.

Expression of Schistosoma mansoni genes involved in anaerobic and oxidative glucose metabolism during the cercaria to adult transformation.

Schistosomes switch rapidly from the use of stored glycogen to a reliance on host glucose during the transformation from free-living cercariae to parasitic schistosomula. We have cloned a set of cDNAs encoding proteins involved in glucose metabolism to allow us to examine the expression of these genes during this transformation. We first obtained and characterized Schistosoma mansoni cDNA clones encoding the tricarboxylic acid cycle enzyme, mitochondrial malate dehydrogenase (SMDH) and the mitochondrial encoded electron transport protein, cytochrome oxidase subunit 1 (SCOX1). Northern blots were then prepared using mRNA isolated from whole cercariae, cercarial tails, schistosomula, adult males and adult females. The Northern blots were successively hybridized with a variety of probes including those for SMDH, SCOX, the glycolytic enzymes, hexokinase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase and several control probes. Probes were additionally hybridized to mRNA dot blots and the signals were quantified using storage phosphor technology. These studies reveal that transcripts encoding these metabolic enzymes are localized at much higher levels in cercarial tails than in whole cercariae or transformed schistosomula, and support the notion of a dominant aerobic metabolism in tails. Male and female adult worms express each of the mRNAs at roughly equal levels. Adults express the metabolic mRNAs, including those involved in oxidative glucose metabolism, at relatively high levels suggesting that adult schistosomes retain a significant capacity to produce energy through aerobic metabolism.

Alternative splicing of the Schistosoma mansoni gene encoding a homologue of epidermal growth factor receptor.

The complete coding DNA for a Schistosoma mansoni homologue of the epidermal growth factor receptor (SER) was characterized from cDNA clones obtained by homology to the tyrosine kinase domain of erbB. The DNA sequence predicts a 200-kDa translation product that contains a secretory leader, a cysteine-rich extracellular domain, a hydrophobic transmembrane sequence, and an intracellular tyrosine kinase domain. The SER transcript is present in cercariae and adult schistosomes. In addition to SER transcripts, schistosomes produce at least 3 variant transcripts encoding truncated SER products that include the secretory leader and a small portion of the extracellular domain followed by short sequences of unrelated, C-terminal amino acids. Based on these sequences, 2 of the variant mRNAs (class 2 and 5) appear to encode soluble, secreted proteins while one (class 4) encodes an SER variant protein with a hydrophobic C-terminus that may serve as a membrane anchor. Class 2 SER variant transcripts are present at levels comparable to SER transcripts in adult worms but are not detected in cercariae. Class 4 and 5 SER variant transcripts are also found within adult worms but at lower levels. Genomic cloning and characterization demonstrate that the variant SER transcripts arise through alternative splicing of the SER gene.

Training pediatricians for the evolving generalist-specialist interface in the managed care era.

Managed care involves the linkage of service delivery and financing. One of the outgrowths of the rapid expansion of managed care over the past decade has been an increasing consensus that the generalist of the future will need to manage more of the patients traditionally cared for by subspecialists. Subspecialty education for pediatric residents becomes increasingly important as the role of the pediatric generalist enlarges to include independent outpatient management of some less complex but traditional subspecialty patients as well as collaborative management of more complex patients. To prepare for this role, a balanced exposure to subspecialty problems in outpatient as well as inpatient settings is required. At the same time, however, the growth of managed care has led to certain barriers for providing this enhanced training. This article describes the effects of managed care on the role and scope of the pediatric subspecialist as well as on educational strategies for coping with these changes while reshaping the roles of both generalists and subspecialists for maximal effectiveness in meeting the health care needs of children.

Gingival telangiectases: an underappreciated physical sign of juvenile dermatomyositis.

BACKGROUND: MEDLINE searches (1966-June 1969) failed to identify references that give detailed descriptions of the oral manifestations of dermatomyositis (DM). However, several reports predating MEDLINE provided more complete descriptions of oral lesions associated with DM. OBSERVATIONS: We describe 5 cases of juvenile DM with oral manifestations, primarily in the form of gingival telangiectases. These findings are compared with those descriptions found in earlier reports. CONCLUSIONS: Oral lesions in juvenile DM have rarely been reported. Mucous membrane involvement associated with DM may include telangiectases, edema, erosions, ulcers, and leukoplakia-like areas. In cases of DM, gingival telangiectases likely represent an underappreciated diagnostic finding analogous to nail-fold telangiectases.

Genetic analyses on DNA microarrays.

A great stir has coursed through the genetics community with the advent in 1996 of "chip technologies," a series of related techniques that allow DNA hybridization-based studies to be performed with unprecedented speed and parallelism. All chip technologies have in common a microarray, a small glass chip approximately one square centimeter in area, to which nucleotide sequences are bound. Fluorescently labeled nucleic acids are hybridized to the microarray and imaged with a laser scanner or fluorescence microscope. This unit offers an overview of the two dominant technologies, cDNA microarrays and oligonucleotide chips. It concludes with a discussion regarding how well microarrays perform real-world expression analysis.

Navigating public physical mapping databases.

This unit provides concise overviews of the many physical mapping resources available and relates them to the genetic and transcript maps. Useful information on resolution of the maps, how to access them, and how to interpret them is compiled and presented in a clear fashion. Especially useful is a set of detailed protocols describing how to construct an STS marker and how to map it by means of available yeast artificial chromosomes (YACs). An additional protocol describes accessing EST marker maps.

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production.

The major goal of this work is to establish a culture system for the growth of human B lymphocytes at the single-cell level so that the immunoglobulin secreted by the clonal progeny of that cell can be analyzed. A method which involves culturing small numbers (1-1000) of lymphocytes, which have been infected with Epstein-Barr virus (EBV) prior to plating, in round-bottom microtiter plates is described. A feeder layer of irradiated (2500 R) umbilical cord blood lymphocytes to which phytohemagglutinin has been added was found to be optimal. Culture supernatants collected from 3 to 6 weeks postinfection are assayed for the production of IgG and IgM by radioimmunoassay in order to determine the overall cloning efficiency of the system. We have shown that up to 33% of surface Ig-positive cells produce detectable clones in this system. Umbilical cord blood cells are superior to T-cell and macrophage cell lines as feeder layers. Furthermore, culture supernatants from phytohemagglutinin-stimulated umbilical cord lymphocytes do not adequately replace these cells. We also observed that while most IgM-secreting clones continued to produce immunoglobulin during the 7-week time period analyzed, the majority of IgG-secreting clones had a relatively short half-life in vitro. This culture system allows us to examine a significant proportion of the human B-cell population and carry out studies on the frequency of specific antibody- and isotype-producing clones.