Genetic, physical, and phenotypic characterization of the Del(13)Svea36H mouse.

The Del(13)Svea36H deletion was recovered from a radiation mutagenesis experiment and represents a valuable resource for investigating gene content and function at this region of mouse Chromosome (Chr) 13 and human Chr 6p21.3-23 and 6p25. In this paper we examine the physical extent of chromosome loss and construct an integrated genetic and radiation hybrid map of the deleted segment. We show that embryos which are homozygous for the deletion die at or before implantation and that heterozygotes are subviable, with a substantial proportion of carriers dying after mid-gestation but before weaning. The majority of viable carriers exhibit a variety of phenotypes including decreased size, eyes open at birth, corneal opacity, tail kinks, and craniofacial abnormalities. Both the heterozygous viability and the penetrance of the visible phenotypes vary with genetic background.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Large-scale production of mouse phenotypes: the search for animal models for inherited diseases in humans.

This paper is aimed principally at bioinformaticians and biologists as an introduction to recent advances in mouse mutagenesis, concentrating on genome-wide screens utilising the powerful mutagen N-ethyl-N-nitroso-urea (ENU). It contains a brief background to the underlying genetics as well as details of the practical aspects of organisation and data capture for such projects.

A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways.

Informatics for mutagenesis: the design of mutabase--a distributed data recording system for animal husbandry, mutagenesis, and phenotypic analysis.

The increasing use of high-throughput methods for the production of biologically important information and the increasing diversity of that information pose considerable bioinformatics challenges. These challenges will be met by implementing electronic data management systems not only to capture the data, but increasingly to provide a platform for data integration and mining as we enter the post-genomic era. We discuss the design and implementation of such a data capture system, 'Mutabase', as a model of how such electronic systems might be designed and implemented. Mutabase was created in support of a large-scale, phenotype-driven mouse mutagenesis program at MRC Mammalian Genetics Unit, Harwell, in collaboration with SmithKline Beecham Pharmaceuticals, Queen Mary and Westfield College, London, and Imperial College of Science, Technology and Medicine, London. The aim of this mutagenesis project is to make a significant contribution to the existing mouse mutant resource, closing the phenotype gap and providing many more models for fundamental research and disease modeling. Mutabase records experimental details at the 'point of generation' and provides a number of dissemination and analysis tools for the experimental data, as well as providing a means of assessing various aspects of progress of the program. Mutabase uses a hypertext-based interface to provide interaction between a number of intranet-based client workstations and a central industrial strength database. Mutabase utilizes a variety of techniques in order to implement the user interface system including Perl/CGI, Java Servlets, and an experimental CORBA server. We discuss the relative merits of these methods in the context of the need to provide sound informatics approaches for the support of systematic mutagenesis programs.

Comparative genome sequence analysis of the Bpa/Str region in mouse and Man.

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.

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.

The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

HOSEpipe--a WWW-hosted data management and analysis system for STS content mapping projects.

We have developed a data management system, 'HOSEpipe' (High Output STS Evaluation pipeline) to aid sample tracking and data analysis in STS content mapping projects. The system is based around a World Wide Web (WWW) server that provides a number of pages including forms for sample processing and data entry accessible via a standard WWW browser application. The system is split into two main modules: firstly, a sequence evaluation and annotation module that takes de novo sequence for a potential STS, screens it against existing STSs and DNA sequence databases, followed by appropriate primer sequence design; secondly, a module that handles YAC library STS screening and includes facilities for both sample tracking and experimental data analysis. We present the design and rationale of the HOSEpipe system and its development to support a whole chromosomal physical mapping project. This software and design approach is potentially applicable to physical mapping projects of varying sizes and resolution and to similar projects, such as sample sequencing and the construction of sequence-ready maps.

The generation of ordered sets of cosmid DNA clones from human chromosome region 11p.

We describe progress in a continuing project aimed at the generation of an overlapping cosmid DNA clone map of the short arm of human chromosome 11. The automated procedures used to prepare DNA samples and the computerized data collection and recording systems are described. We also demonstrate the use of the clones as reagents for the rapid isolation of genomic DNAs containing smaller probed regions. We have isolated approximately 4700 human cosmid DNA clones from mouse/human hybrid cell lines that contain predominantly human chromosomal region 11p. Of the DNA in the cell lines, 60% is derived from this chromosomal region, and the remaining 40% is derived from regions of chromosomes 3, 19, and 20. A total of 4159 clones have been fingerprinted to identify potential overlaps, and we have developed 535 sets ("contigs"). Using random modeling, it is estimated that 65% of 11p must be contained in the analyzed cosmids. The database of clones has been used to identify single or overlapping clones from noncosmid DNA probes. Examples are presented. It is proposed that cosmid reference filters be distributed to requesting laboratories.