High-resolution BAC-based map of the central portion of mouse chromosome 5.

The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec-Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50-200-kb stretches will be remarkably valuable.

A relational schema for both array-based and SAGE gene expression experiments.

MOTIVATION AND RESULTS: A relational schema is described for capturing highly parallel gene expression experiments using different technologies. This schema grew out of efforts to build a database for collaborators working on different biological systems and using different types of platforms in their gene expression experiments as well as different types of image quantification software. The tables are conceptually organized into three categories of information: Platform, Experiment (which includes image scanning and quantification), and Data. The strengths of the schema are: (i) integrating information on array elements using a gene index; (ii) describing samples using ontologies; (iii) reducing an experiment to a single RNA source for precise descriptions yet not losing the relationships between experiments done at the same time or for the same project; and (iv) maintaining both raw and processed (e.g. cleansed and normalized) data and recording how the data is processed. The result is a novel schema, which can hold both array and non-array data, is extensible for detailed experimental descriptions that are precise and consistent, and allows for meaningful comparisons of genes between experiments.

The genetic program of hematopoietic stem cells.

Blood cell production originates from a rare population of multipotent, self-renewing stem cells. A genome-wide gene expression analysis was performed in order to define regulatory pathways in stem cells as well as their global genetic program. Subtracted complementary DNA libraries from highly purified murine fetal liver stem cells were analyzed with bioinformatic and array hybridization strategies. A large percentage of the several thousand gene products that have been characterized correspond to previously undescribed molecules with properties suggestive of regulatory functions. The complete data, available in a biological process-oriented database, represent the molecular phenotype of the hematopoietic stem cell.

Sonic hedgehog controls epaxial muscle determination through Myf5 activation.

Sonic hedgehog (Shh), produced by the notochord and floor plate, is proposed to function as an inductive and trophic signal that controls somite and neural tube patterning and differentiation. To investigate Shh functions during somite myogenesis in the mouse embryo, we have analyzed the expression of the myogenic determination genes, Myf5 and MyoD, and other regulatory genes in somites of Shh null embryos and in explants of presomitic mesoderm from wild-type and Myf5 null embryos. Our findings establish that Shh has an essential inductive function in the early activation of the myogenic determination genes, Myf5 and MyoD, in the epaxial somite cells that give rise to the progenitors of the deep back muscles. Shh is not required for the activation of Myf5 and MyoD at any of the other sites of myogenesis in the mouse embryo, including the hypaxial dermomyotomal cells that give rise to the abdominal and body wall muscles, or the myogenic progenitor cells that form the limb and head muscles. Shh also functions in somites to establish and maintain the medio-lateral boundaries of epaxial and hypaxial gene expression. Myf5, and not MyoD, is the target of Shh signaling in the epaxial dermomyotome, as MyoD activation by recombinant Shh protein in presomitic mesoderm explants is defective in Myf5 null embryos. In further support of the inductive function of Shh in epaxial myogenesis, we show that Shh is not essential for the survival or the proliferation of epaxial myogenic progenitors. However, Shh is required specifically for the survival of sclerotomal cells in the ventral somite as well as for the survival of ventral and dorsal neural tube cells. We conclude, therefore, that Shh has multiple functions in the somite, including inductive functions in the activation of Myf5, leading to the determination of epaxial dermomyotomal cells to myogenesis, as well as trophic functions in the maintenance of cell survival in the sclerotome and adjacent neural tube.

EpoDB: a prototype database for the analysis of genes expressed during vertebrate erythropoiesis.

EpoDB is a database of genes expressed in vertebrate red blood cells. It is also a prototype for the creation of cell and tissue-specific databases from multiple external sources. The information in EpoDB obtained from GenBank, SWISS-PROT, Transfac, TRRD and GERD is curated to provide high quality data for sequence analysis aimed at understanding gene regulation during erythropoiesis. New protocols have been developed for data integration and updating entries. Using a BLAST-based algorithm, we have grouped GenBank entries representing the same gene together. This sequence similarity protocol was also used to identify new entries to be included in EpoDB. We have recently implemented our database in Sybase (relational tables) in addition to SICStus Prolog to provide us with greater flexibility in asking complex queries that utilize information from multiple sources. New additions to the public web site (http://www.cbil.upenn.edu/epodb) for accessing EpoDB are the ability to retrieve groups of entries representing different variants of the same gene and to retrieve gene expression data. The BLAST query has been enhanced by incorporating BLASTView, an interactive and graphical display of BLAST results. We have also enhanced the queries for retrieving sequence from specified genes by the addition of MEME, a motif discovery tool, to the integrated analysis tools which include CLUSTALW and TESS.

bioWidgets: data interaction components for genomics.

MOTIVATION: The presentation of genomics data in a perspicuous visual format is critical for its rapid interpretation and validation. Relatively few public database developers have the resources to implement sophisticated front-end user interfaces themselves. Accordingly, these developers would benefit from a reusable toolkit of user interface and data visualization components. RESULTS: We have designed the bioWidget toolkit as a set of JavaBean components. It includes a wide array of user interface components and defines an architecture for assembling applications. The toolkit is founded on established software engineering design patterns and principles, including componentry, Model-View-Controller, factored models and schema neutrality. As a proof of concept, we have used the bioWidget toolkit to create three extendible applications: AnnotView, BlastView and AlignView.

Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa.

To accelerate gene discovery and facilitate genetic mapping in the protozoan parasite Toxoplasma gondii, we have generated >7000 new ESTs from the 5' ends of randomly selected tachyzoite cDNAs. Comparison of the ESTs with the existing gene databases identified possible functions for more than 500 new T. gondii genes by virtue of sequence motifs shared with conserved protein families, including factors involved in transcription, translation, protein secretion, signal transduction, cytoskeleton organization, and metabolism. Despite this success in identifying new genes, more than 50% of the ESTs correspond to genes of unknown function, reflecting the divergent evolutionary status of this parasite. A newly recognized class of genes was identified based on its similarity to sequences known only from other members of the same phylum, therefore identifying sequences that are apparently restricted to the Apicomplexa. Such genes may underlie pathways common to this group of medically important parasites, therefore identifying potential targets for intervention.

EpoDB: a database of genes expressed during vertebrate erythropoiesis.

EpoDB is a database designed for the study of gene regulation during differentiation and development of vertebrate red blood cells. In building EpoDB, we have taken the in advance approach to the data integration problem: we have extracted data relevant to red blood cells from GenBank, SWISS-PROT, TRRD (transcriptional regulation data) and GERD (expression levels data) to create a single integrated, highly curated view. Tools have been developed to automate data extraction from online resources, cleanse data of errors, enter information manually from the primary literature, generate a uniform, canonical representation of information and maintain data currency. The database is organized around biological features, e.g., genes, rather than sequences, which are supported by a controlled and consistent vocabulary for gene names and gene family names. Beyond the standard database queries, the functionality of EpoDB includes the ability to extract features and subsequences, display sequences and features graphically using bioWidget viewers and integrated analysis tools. EpoDB may be accessed at: http://cbil.humgen.upenn.edu/epodb/

Regulated demethylation of the myoD distal enhancer during skeletal myogenesis.

myoD is one of a family of four related basic helix-loop-helix transcription factors involved in the specification and differentiation of skeletal muscle. We previously identified a 258-bp distal enhancer that is sufficient for embryonic activation of myoD and is highly conserved between humans and mice. In this paper, we show using a modified bisulfite deamination/PCR amplification method that the distal myoD enhancer is completely unmethylated at all the CpG sites tested in myogenic cells and a subpopulation of somite cells. Conversely, the distal enhancer in nonmuscle cells and tissues is methylated to an average level of > 50% and we find no chromosomes in these tissues with a completely unmethylated enhancer. We present evidence that demethylation of the distal enhancer in somites of mouse embryos precedes myoD transcription, suggesting that demethylation of the distal enhancer is an active, regulated process that is essential for myoD activation. We also show by analysis of transgenic mice carrying a human distal enhancer/reporter construct in which the three enhancer CpG sites have been mutated that methylation of the distal enhancer is not required to prevent precocious or ectopic embryonic myoD expression. We propose that a subset of somite cells demethylate the distal enhancer in response to specific developmental signals, thus making the enhancer accessible and able to respond to subsequent signals to activate the myoD gene.

Embryonic activation of the myoD gene is regulated by a highly conserved distal control element.

MyoD belongs to a small family of basic helix-loop-helix transcription factors implicated in skeletal muscle lineage determination and differentiation. Previously, we identified a transcriptional enhancer that regulates the embryonic expression of the human myoD gene. This enhancer had been localized to a 4 kb fragment located 18 to 22 kb upstream of the myoD transcriptional start site. We now present a molecular characterization of this enhancer. Transgenic and transfection analyses localize the myoD enhancer to a core sequence of 258 bp. In transgenic mice, this enhancer directs expression of a lacZ reporter gene to skeletal muscle compartments in a spatiotemporal pattern indistinguishable from the normal myoD expression domain, and distinct from expression patterns reported for the other myogenic factors. In contrast to the myoD promoter, the myoD enhancer shows striking conservation between humans and mice both in its sequence and its distal position. Furthermore, a myoD enhancer/heterologous promoter construct exhibits muscle-specific expression in transgenic mice, demonstrating that the myoD promoter is dispensable for myoD activation. With the exception of E-boxes, the myoD enhancer has no apparent sequence similarity with regulatory regions of other characterized muscle-specific structural or regulatory genes. Mutation of these E-boxes, however, does not affect the pattern of lacZ transgene expression, suggesting that myoD activation in the embryo is E-box-independent. DNase I protection assays reveal multiple nuclear protein binding sites in the core enhancer, although none are strictly muscle-specific. Interestingly, extracts from myoblasts and 10T1/2 fibroblasts yield identical protection profiles, indicating a similar complement of enhancer-binding factors in muscle and this non-muscle cell type. However, a clear difference exists between myoblasts and 10T1/2 cells (and other non-muscle cell types) in the chromatin structure of the chromosomal myoD core enhancer, suggesting that the myoD enhancer is repressed by epigenetic mechanisms in 10T1/2 cells. These data indicate that myoD activation is regulated at multiple levels by mechanisms that are distinct from those controlling other characterized muscle-specific genes.

Drosophila genes Posterior Sex Combs and Suppressor two of zeste encode proteins with homology to the murine bmi-1 oncogene.

The Polycomb group (Pc-G) genes are needed to maintain expression patterns of the homeotic selector genes of the Antennapedia (Antp-C) and bithorax (bx-C) complexes, and hence for the maintenance of segmental determination. We report the predicted protein sequence of the Pc-G gene Posterior Sex Combs (Psc), and of the neighbouring and related gene Suppressor two of zeste (Su(z)2). Both genes encode large proteins that contain a 200 amino-acid domain identical over 37.4% that is also conserved in the murine oncogene bmi-1. At the amino terminus of this domain is a cysteine-rich sequence that has been proposed as a novel type of zinc finger.

Molecular genetics of the Posterior sex combs/Suppressor 2 of zeste region of Drosophila: aberrant expression of the Suppressor 2 of zeste gene results in abnormal bristle development.

We report the molecular characterization of the Posterior sex combs-Suppressor 2 of zeste region of Drosophila melanogaster. The distal breakpoint of the Aristapedioid inversion divides the region into two parts. We have molecularly mapped the lesions associated with several loss of function mutations in the Polycomb group gene Posterior sex combs (Psc) proximal to this breakpoint. In addition, we have found that lesions associated with several loss of function mutations in the Suppressor 2 of zeste [Su(z)2] gene lie distal to this breakpoint. Since the breakpoint does not cause a loss of function in either gene, no essential sequences are shared by these two neighboring genes. There are three dominant gain of function mutations in the region that result in abnormal bristle development. We find that all three juxtapose foreign DNA sequences upstream of the Su(z)2 gene, and that at least two of these mutations (Arp1 and vgD) behave genetically as gain of function mutations in Su(z)2. Northern and in situ hybridization analyses show that the mutations result in increased accumulation of the Su(z)2 mRNA, which we argue is responsible for the bristle loss phenotype.

Keeping children on top of the states' policy agendas.

There is no longer any doubt that national consciousness of the truth that the future always depends on today's children has reached new levels of legislative action. This article cites Alaska's progress, and what has been and has not been achieved, and movements in other states, as a call to vigorous action.

Aristapedioid: a gain of function, homeotic mutation in Drosophila melanogaster.

The isolation of gain of function mutations has allowed the identification of a number of genes which are important in the normal development of the organism. We report here the isolation and characterization of Aristapedioid, a gain of function mutation which causes a partial transformation of arista towards tarsus and the loss or decrease in size of the dorso-central and scutellar bristles. Aristapedioid is the result of a P element mediated inversion which juxtaposes unrelated DNA adjacent to Suppressor 2 of zeste, causing a gain of function mutation in that gene.

Genetic interactions of the suppressor 2 of zeste region genes.

A wide variety of gain of function mutations have been induced in the Posterior Sex Comb (Psc)--Aristapedioid (Arp)--Suppressor 2 of zeste (Su(z)2) region of the second chromosome of Drosophila. This region contains at least three apparently related genes, two of which we have been studying. Psc1 has previously been used to identify Psc as a Pc group gene; however, it is a complex mutation with both gain and loss of function character. We report here that the Pc group character of Psc is not due to a gain of function and presumably reflects the function of the wild-type gene. We also provide evidence for a maternal function for Psc, as well as the neighboring Su(z)2 gene. Su(z)2 does not appear to be a Pc group gene as it does not act in a synergistic fashion with other Pc group genes in promoting posteriorly directed transformations. However, we have found that mutations in Su(z)2 do interact in a variety of interesting ways with mutations in Pc group genes.