S.D.M. Brown

Isolation, physical mapping, and Northern analysis of the X-linked human gene encoding methyl CpG-binding protein, MECP2

Methylation ofcytosine residues in mammalian genomes is associated with transcriptional repression and inactive chromatin. It has been shown that methylation of DNA is essential for normal mouse development (Li et al. 1992), possibly by preventing the inappropriate expression of genes. The mechanism of transcriptional repression is thought to involve nuclear factors that bind preferentially to methylated DNA. Two activities have been identified, MeCPI and MeCP2, which bind specifically to DNA templates containing methyl-CpG pairs (Meehan et al. 1989; Lewis et al. 1992). It has recently been reported that MeCP2, in common with DNA methyltransferase (Li et al. 1992), is dispensable in stem cells but essential for embryonic development in the mouse (Tate et al. 1996). Rat MeCP2 has been isolated and well characterized (Lewis et al. 1992; Meehan et al. 1992a; Nan et al. 1993). The Mecp2 gene encodes a 53-kDa chromatin-associated nuclear protein. Immunological studies have shown that it colocalizes with 5-methyl cytosine in mouse L cells (Lewis et al. 1992). MeCP2 contains an 85 amino acid methyl-binding domain (MBD) that, in vitro, binds exclusively to DNA containing one or more symmetrically methylated CpGs. MeCP2 also has a weak affinity for non-methylated DNA through nonspecific DNA binding domain (Meehan et al. 1992a; Nan et al. 1993). The protein is abundant in somatic tissues (100,000-500,000 molecules per nucleus) but is barely detectable in embryonic tissues (Meehan et al, 1992a). The mouse Mecp2 gene has been mapped to a 40-kb interval between Llcam and Rsvp in the central span of the mouse X Chromosome (Chr) (Quaderi et al. 1994). This region is known to be syntenically equivalent to human Xq28 (Brown et al. 1993), and, with the exception of F8a (Levinson et al. 1990, 1992), locus order is conserved between the two species (Brown et al. 1993; Davies et al. 1992; Dietrich et al. 1992). Therefore, we expected the human MECP2 gene to be located between L1CAM and RCP/ GCP. We have previously published an extensive physical map of the Xq28 region (Palmieri et al. 1994). A filter containing all YACs localized between L1CAM and RCP/GCP was hybridized with a rat Mecp2 probe, and three overlapping YACs (1183, 4291, and 411) were positive (Fig. la), placing the MECP2 gene about 70 kb centromeric to the RCP/GCP locus. A rat Mecp2 probe containing 1312 bp (80%) of coding sequence of the rat Mecp2 was used to screen a human skeletal muscle cDNA library in Xgtl0. From a total of 600,000 plaques analyzed, eight positive clones were identified ranging in size from 1.3 kb to 1.7 kb.

Genes and deafness

Many different genes appear to be involved in the development and function of the mammalian inner ear. Some of the genes involved during early inner ear morphogenesis have been identified using mutations or targetted transgenic interruption, while a handful of genes involved in pigmentation anomalies associated with hearing impairment have been cloned. Several genes involved in syndromic late-onset hearing loss have also been identified. However, the majority of cases of hereditary hearing impairment from childhood probably involve genes expressed in the sensory neuroepithelia of the inner ear, and none of the genes or mutations causing this type of deafness have yet been identified. Here, we review the progress that has been made in finding genes for deafness and in using mouse mutants to elucidate the biological basis of the hearing deficit.

Isolation of a sequence common to A- and B-chromosomes of rye (Secale cereale) by microcloning

The techniques of microdissection and microcloning have been applied to the isolation of B-chromosome DNA from rye. We have identified a DNA sequence on the rye B-chromosome which is homologous to an A-chromosome sequence, and which is dispersed and moderately repeated on the A- and B-chromosomes. This demonstrates that the rye B-chromosome is heterogeneous in the nature of its DNA sequence composition, containing sequences which are present on the A-chromosomes in addition to those not present on the A-chromosomes.

Genetics of deafness.

The genetics of deafness is a rapidly expanding area of research. A remarkable total of twenty-two genes involved in non-syndromic deafness in humans have been localized within the past two years, compared with only one known previously. Some of the genes involved in neuroepithelial deafness, the most common type of pathology, have been identified in the past year. Two of these genes encode unconventional myosin molecules. The roles of these and other molecules identified by genetic approaches as important in hearing are being explored.

Mouse chromosome 7

Center for Neuroscience, University of Tennessee, Memphis, 855 Monroe Avenue, Memphis, Tennessee 38163, USA Department of Carcinogenesis, University of Texas Science Park, Research Division, Smithville, Texas 78957, USA Department of Biochemistry and Cell Biology, SUNY at Stony Brook, Stony Brook, New York 11794-5215, USA Division of Genetics, Children’s Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA

The X-linked methylated DNA binding protein, Mecp2, is subject to X inactivation in the mouse

DNA methylation at the promoter region of X-linked genes is associated with the maintenance of X inactivation in mammals. One of the methylated DNA binding proteins, MECP2, that binds to methylated bases in DNA is encoded by a gene (Mecp2) located on the mouse X Chromosome (Chr). To determine whether this gene was expressed from the inactive X Chr, and X-autosome translocation (T(X;16)16H) system in which expression from the Mecp2 allele on the inactive X Chr could be assayed was used. Results from these experiments indicate that Mecp2 is subject to X inactivation in mouse.

Molecular and genetic mapping of the mouse mdx locus

mdx is an X-linked muscular dystrophy mutant of the mouse and a putative homolog of the human X-linked muscular dystrophy locus--Duchenne muscular dystrophy (DMD). Utilizing a C57BL/10/Mus Spretus interspecific cross in which the mdx mutation was segregating, we have constructed a detailed genetic map around the mdx locus on the mouse X chromosome. We were unable to detect recombinants between mdx and exonic probes derived from the human DMD gene. These genetic data support the contention from biochemical studies (E.P. Hoffman, R. H. Brown, and L. M. Kunkel, 1987, Cell 51: 919-928) that DMD and mdx are homologous genes.

High-density molecular map of the central span of the mouse X chromosome.

A total of 17 linking clones previously sublocalized to the central span of the mouse X chromosome have been ordered by detailed analysis through interspecific Mus spretus/Mus musculus domesticus backcross progeny. These probes have been positioned with respect to existing DNA markers utilizing a new interspecific backcross segregating for the Tabby (Ta) locus. The density of clones within this 11.5-cM interval is now, on average, one clone every 1000 kb. This high-density map provides probes in the vicinity of a number of important genetic loci in this region which include the X-inactivation center, the Ta locus, and the mottled (Mo) locus, and therefore provides a molecular framework for identification of the genes encoded at these loci.

Mapping of a mouse homolog of a Heterochromatin protein gene to the X Chromosome

Modifiers of position-effect-variegation inDrosophila are thought to encode proteins that are either structural components of heterochromatin or enzymes that modify these components. We have recently shown that a sequence motif found in oneDrosophila modifier gene, Heterochromatin protein 1 (HP1), is conserved in a wide variety of animal and plant species (Singh et al. 1991). Using this motif, termed chromo box, we have cloned a mouse candidate modifier gene,M31, that also shows considerable sequence homology toDrosophila HP1. Here we report evidence of at least four independently segregating loci in the mouse homologous to theM31 cDNA. One of these loci—Cbx-rsl—maps to the X Chromosome (Chr), 1 cM proximal toAmg and outside the X-inactivation center region.

Genetic mapping in the region of the mouse X-inactivation center

The mouse X-inactivation center lies just distal to the T16H breakpoint. Utilizing pedigree analysis of backcross progeny from a Mus domesticus/Mus spretus interspecific cross, we have mapped a number of genetic loci, gene probes, microclones, and EagI linking clones distal to the T16H breakpoint. The genetic analysis provides a detailed genetic map in the vicinity of the mouse X-inactivation center. Comparative mapping data from the human X chromosome indicate that the most probable location of the mouse X-inactivation center is distal to Ccg-1 and in the region of the Pgk-1 locus. We report the assignment of two new loci, EM13 and DXSmh44, to the Ccg-1/Pgk-1 interval.