Characterization of a murine gene expressed from the inactive X chromosome

In mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.     

The mapping of a cDNA from the human X-linked Duchenne muscular dystrophy gene to the mouse X chromosome

The recent discovery of sequences at the site of the Duchenne muscular dystrophy (DMD) gene in humans has opened up the possibility of a detailed molecular analysis of the genes in humans and in related mammalian species. Until relatively recently, there was no obvious mouse model of this genetic disease for the development of therapeutic strategies. The identification of a mouse X-linked mutant showing muscular dystrophy, mdx, has provided a candidate mouse genetic homologue to the DMD locus; the relatively mild pathological features of mdx suggest it may have more in common with mutations of the Becker muscular dystrophy type at the same human locus, however. But the close genetic linkage of mdx to G6PD and Hprt on the mouse X chromosome, coupled with its comparatively mild pathology, have suggested that the mdx mutation may instead correspond to Emery Dreifuss muscular dystrophy which itself is closely linked to DNA markers at Xq28-qter in the region of G6PD on the human X chromosome. Using an interspecific mouse domesticus/spretus cross, segregating for a variety of markers on the mouse X chromosome, we have positioned on the mouse X chromosome sequences homologous to a DMD cDNA clone. These sequences map provocatively close to the mdx mutation and unexpectedly distant from sparse fur, spf, the mouse homologue of OTC (ornithine transcarbamylase) which is closely linked to DMD on the human X chromosome.     

Localization of the region homologous to the Duchenne muscular dystrophy locus on the mouse X chromosome

Recent progress has resulted in part of the gene mutated in Duchenne and the milder Becker muscular dystrophies being cloned and has suggested that the gene itself extends over 1,000 to 2,000 kilobases (kb). To study how mutations in this gene affect muscle development and integrity, it would be of interest to have available a mouse model of the human disease. The mouse mdx mutation affects muscle and confers a mild dystrophic syndrome, but it is not clear whether this mutation is equivalent to Duchenne/Becker muscular dystrophy in man. Here we describe the use of two sequences from the human Duchenne muscular dystrophy (DMD) gene that cross-hybridize to mouse X-linked sequences to localize the gene homologous to DMD in the mouse. Both sequences map to the region of 10 centimorgan lying between the Tabby (Ta) and St14-1 (DxPas8) loci, close to the phosphorylase b kinase locus (Phk). By analogy with the human X-chromosome, we conclude that the region in the mouse around the G6pd and St14-1 loci may contain two genes corresponding to distinct human myopathies: Emery Dreifuss muscular dystrophy which is known to be closely linked to St14-1 in man and the DMD homologue described here.     

A gene expression map of human chromosome 21 orthologues in the mouse

The DNA sequence of human chromosome 21 (HSA21) has opened the route for a systematic molecular characterization of all of its genes. Trisomy 21 is associated with Down's syndrome, the most common genetic cause of mental retardation in humans. The phenotype includes various organ dysmorphies, stereotypic craniofacial anomalies and brain malformations. Molecular analysis of congenital aneuploidies poses a particular challenge because the aneuploid region contains many protein-coding genes whose function is unknown. One essential step towards understanding their function is to analyse mRNA expression patterns at key stages of organism development. Seminal works in flies, frogs and mice showed that genes whose expression is restricted spatially and/or temporally are often linked with specific ontogenic processes. Here we describe expression profiles of mouse orthologues to HSA21 genes by a combination of large-scale mRNA in situ hybridization at critical stages of embryonic and brain development and in silico (computed) mining of expressed sequence tags. This chromosome-scale expression annotation associates many of the genes tested with a potential biological role and suggests candidates for the pathogenesis of Down's syndrome.     

In situ nick-translation distinguishes between active and inactive X chromosomes

Template-active regions of chromatin are structurally distinct from nontranscribing segments of the genome. Recently, it was suggested that the conformation of active genes which renders them sensitive to DNase I may be maintained even in fixed mitotic chromosomes. We have developed a technique of mitotic cell fixation and DNase I-directed nick-translation which distinguishes between active and inactive X chromosomes. We report here that Gerbillus gerbillus (rodent) female cells contain easily identified composite X chromosomes each of which includes the original X chromosome flanked by two characteristic autosomal segments. After nick-translation the active X chromosome in each cell is labelled specifically in both the autosomal and X-chromosomal regions. The inactive X chromosome is labelled only in the autosomal regions and in a small early replicating band within the late replicating 'original X' chromosome. Our technique opens the possibility of following the kinetics of X-chromosome inactivation and reactivation during embryogenesis, studying active genes in the inactive X chromosome and mapping tissue-specific gene clusters.     

The DNA sequence of the human X chromosome

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.     

Homoeobox gene expression in mouse embryos varies with position by the primitive streak stage

Pattern formation in animal development requires that genes be expressed differentially according to position in the sheets of cells that make up the early embryo. The homoeobox-containing genes of Drosophila are control genes active both in the establishment of a segmentation pattern and in the specification of segment identity. In situ hybridization experiments confirm that these genes are expressed in a segmentally-restricted manner and that their expression presages morphological differentiation of segmental structures. Homoeobox genes have recently been isolated from the mouse and have been shown to be expressed during mouse development. Using in situ hybridization, we show here that expression of the mouse homoeobox gene Mo-10 (ref. 7) is spatially restricted in the developing embryo and that localization of expression is already evident within the germ layers before their morphological differentiation. These findings support the suggestion that the homoeobox genes of mammals, like those of Drosophila, may be important in pattern formation.     

Localization of the X inactivation centre on the human X chromosome in Xq13

X-chromosome inactivation results in the strictly cis-limited inactivation of many but not all genes on one of the two X chromosomes during early development in somatic cells of mammalian females. One feature of virtually all models of X inactivation is the existence of an X-inactivation centre (XIC) required in cis for inactivation to occur. This concept predicts that all structurally abnormal X chromosomes capable of being inactivated have in common a defineable region of the X chromosome. Here we report an analysis of several such rearranged human X chromosomes and define a minimal region of overlap. The results are consistent with models invoking a single XIC and provide a molecular foothold for cloning and analysing the XIC region. One of the markers that defines this region is the XIST gene, which is expressed specifically from inactive, but not active, X chromosomes. The localization of the XIST gene to the XIC region on the human X chromosome implicates XIST in some aspect of X inactivation.