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.

MSG1 (melanocyte-specific gene 1) is a recently isolated gene predominantly expressed in cultured normal melanocytes and pigmented melanoma cells. MSG1 encodes a 27-kDa nuclear protein that has strong intrinsic transcriptional transactivating activity. In this report, the human MSG1 gene was mapped to chromosome Xq13.1 using X chromosome-specific somatic cell hybrids, and the mouse Msg1 gene was mapped 1.9 +/- 1.3 cM proximal to Xist using an interspecific backcross panel. Both the human and the mouse MSG1 genes consist of three exons and two introns within 5 kb of genomic DNA, and their genomic structures are highly conserved. Southern blot analysis suggests the existence of MSG1 homologues in chicken, zebrafish, and Drosophila. A 2.0-kb fragment of the 5'-flanking region of the mouse Msg1 gene contains a TATA box and potential binding sites for several transcription factors including USF, Brn-3, Brn-2, TFE3, Oct-1, AP-2, and Spl. This promoter fragment activates transcription of a reporter gene in pigmented melanoma cells, but not in amelanotic melanoma cells or nonmelanocytic cells, indicating that Msg1 expression is at least partially regulated at the transcriptional level.

Mutations in the emerin gene, also referred to as the STA- or EMD-gene, have been found to be the cause of X-linked Emery-Dreifuss muscular dystrophy (EMD). For the present study an optimized set of primers was designed to amplify and sequence each of the six emerin gene exons, including the intron/exon boundaries. All emerin gene exons of 30 unrelated EMD patients have been screened by heteroduplex analysis. Aberrant patterns of single exons were found in seven patients. Direct sequencing of the respective exons revealed six novel mutations distributed in the promotor region and exons 3-6 (delta nt -19 to -40; delta AG nt 620-621; ins A nt 895; delta AT nt 908-909; C-->A nt 1420; ins TA nt 1570). By this study, the first mutations in the promotor region and in exon 5 have been identified. Each of the 25 mutations that have been described so far, including those from the present study, abolishes the synthesis of functional emerin. The mutations were submitted to the EMD Mutation database (http://www.path.cam.ac.uk/emd).

Incontinentia Pigmenti 2 (IP2) is an X-linked dominant disorder with male lethality. Affected females display a characteristic skin eruption that evolves through four classic stages, frequently accompanied by dental and retinal abnormalities. Non-random (skewed) X-inactivation in peripheral blood leukocytes and in fibroblasts has been observed in females with IP2; however, sample sizes have been small and methods of analysis varied. We have examined X-inactivation in a large group of multigenerational IP2 families, in smaller families, and in isolated cases. Ninety-eight percent of affected females in multigenerational IP2 pedigrees show completely skewed patterns of X-inactivation, while only approximately 10% of a normal control population is skewed. Results both in small families and in new mutation cases with subsequent segregation consistent with Xq28 linkage are similar. Isolated cases show a lower percentage (85%) of skewed affected individuals; this difference may be due to inaccurate clinical ascertainment. The parent of origin of new mutations could be determined in 15 families; paternal new mutations were twice as common as maternal. Fibroblast subclones from a biopsy at the boundary of a skin lesion in a newborn IP2 patient were isolated, and clones with either one or the other X active were identified, demonstrating that cells with the active disease-bearing X chromosome are still present in stage I skin lesions.

A novel cDNA which maps to human Xq28 has been isolated and characterized. Sequence similarity to DNase I is high at the DNA and peptide sequence levels. The transcript is present at highest levels in skeletal and cardiac muscle, with lower expression in other tissues. Mutation analysis has been performed using DNA samples from two unrelated patients with Barth syndrome, and from 11 unrelated patients with Emery-Dreifuss muscular dystrophy, two genetic disorders linked to Xq28. No disease-associated mutations were detected in the coding region of the gene; however, a novel 190 base pair insertion/deletion polymorphism was found in the 3' untranslated region. Translation of the long open reading frame found in the cDNA yields a putative 302 amino acid protein with 37.6% identity to human DNase I. The protein is predicted to contain a signal sequence at the amino terminus, a transmembrane domain near the carboxyl terminus, and a helix-loop-helix domain.

Caltractin belongs to a family of calcium-binding proteins and is a structural component of the centrosome. A human caltractin cDNA (CALT) has recently been mapped by fluorescence in situ hybridization (FISH) to Xq28. We report here refined mapping of the human CALT gene and its murine homolog between the loci DXS1104 (DXHXS1104) and DXS52 (DXHXS52) by PCR and Southern analysis of YACs and somatic cell hybrids from the region in both species. These mapping studies place the gene within the critical region for the murine X-linked dominant, male lethal mutations bare patches and striated.

The Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome type II, TRPS II) is characterized by craniofacial dysmorphism and skeletal abnormalities, It combines the clinical features of TRPS I and multiple cartilaginous exostoses (EXT), We have used YAC cloning, Southern blotting, PCR analysis, and fluorescence in situ hybridization to study chromosome 8 deletions, translocations, an inversion, and an insertion in patients with TRPS I, TRPS II or EXT. Our results indicate that the TRPS gene maps more than 1,000 kb proximal to the EXT1 gene and that both genes are affected in TRPS II, We conclude that TRPS II is not due to pleiotropic effects of mutations in a single gene, but that it is a true contiguous gene syndrome,

Three folate-sensitive fragile sites, termed FRAXA, FRAXE and FRAXF, have been identified on the distal end of chromosome Xq. The first two contain expanded, hypermethylated and unstable CGG (or GCC) repeats within CpG islands. We now report the isolation of similar sequences responsible for the third fragile site, FRAXF. A 5-kilobase EcoRI fragment derived from a cosmid coincident with the cytogenetic anomaly detects expanded, methylated and unstable sequences in five individuals who exhibit fragile sites in distal Xq; these individuals have normal repeat lengths at both FRAXA and FRAXE. By sequence analysis, the expanded region contains a GCC repeat. PCR and sequence analysis of chromosomes from the general population indicates that the repeat is polymorphic (6 to 29 triplets), and is stable upon transmission.

Three folate-sensitive fragile sites have been identified in distal Xq, and have been termed FRAXA, FRAXE and FRAXF. The former two have been characterized at the molecular level and found to contain expanded stretches of CGG repeats within hypermethylated CpG islands. We have isolated a cosmid clone that likely represents FRAXF. Somatic cell hybrid analysis maps the cosmid to Xq28 between IDS and GABRA3, the region to which FRAXF has been localized. A 5kb EcoRI fragment of the cosmid detects a fragment increased by 900 base pairs in a mentally retarded male exhibiting 28% fragile site expression. By DNA analysis this patient demonstrates normal alleles at both FRAXA and FRAXE. The proband`s DNA at this locus is methylated at three sites (SacII, EagI, and BssHII) within the CpG island. Additional normal and retarded family members were tested both for fragile site expression and for expansion and methylation at this locus; the expansion and methylation are found only in individuals expressing the fragile site, suggesting that this clone does represent the fragile site. Inheritance of the fragile site does not show a direct correlation with the mental impairment in this pedigree. The site of variation was localized to within 300 basemore » pairs. By sequence analysis, the plasmid subclone contains (CGG){sub 8}. PCR primers were designed to amplify across the repeat. Expanded alleles amplify poorly or not at all, which is consistent with difficulties observed with FRAXA. Alleles in the normal population vary from 6 to 29 repeats. Fluorescence in situ hybridization studies are currently under way to demonstrate unequivocally that this clone spans the fragile site in the patient mentioned above. Efforts to define the gene content of the region are under way, in order to determine the potential involvement of this repeat in regulation of genes in its vicinity.« less

We describe a mapping panel for the 8q23-qter region composed of human-hamster hybrid cell lines carrying deletion and translocation derivatives of human chromosome 8. The panel divides this region of the chromosome into nine intervals and has been used to map 40 loci by Southern blot hybridization and PCR. Use of this mapping panel has allowed us to align the terminal portions of two different genetic maps of chromosome 8 with each other and with the physical map of the chromosome.

Subchromosomal localizations for 19 X-linked expressed sequence tags (ESTs) have been determined. Two ESTs are located in Xq28, adding two novel genes to this disease-rich region. The remaining ESTs are located primarily in the pericentromeric region, with most mapping to Xp11.1-p21.1. YAC and cosmid genomic clones have been isolated for several of these loci. Available cDNAs have been used to characterize the corresponding transcripts by Northern analysis in multiple human tissues.

Identification of transcribed sequences from within genomic regions has been a major rate-limiting step in the pursuit of genes involved in many human genetic diseases. Early efforts focused primarily on screening of cDNA libraries, identification of evolutionarily conserved sequences, and northern blot hybridization. In recent years, several innovative techniques for gene identification have been devised. These techniques expand the size of the genomic region capable of being scanned for genes, while also allowing detection of genes regardless of their expression patterns. This article reviews several new and older techniques and discusses the advantages and limitations of each.

We have obtained lymphoblastoid cell lines from three patients with Langer-Giedion syndrome who have overlapping deletions in 8q24.1. To isolate the deletion chromosomes from their normal homologs, patient cell lines were fused with hamster cells and hybrid cells were selected for retention of human chromosome 8. These hybrid cell lines were screened for the presence of chromosome 8 by fluorescence in situ hybridization and by Southern blot hybridization. We have hybridized 31 recombinant DNA clones derived from the 8q22-qter region to Southern blots of the hybrid cell lines; 8 were found to lie within the deletion of at least one patient. One clone identified sequences that were missing from one copy of chromosome 8 in all three patients. These clones help to further define the deletions in these patients and will serve as starting points for detailed characterization of the region.

Diet-induced magnesium deficiency in puppies resulted in an increased rate of glucose removal from the blood after intravenous glucose infusions. The levels of immunoassayable insulin in the plasma of these animals were comparable to those of the controls. The accelerated removal of glucose from the plasma was reversed with magnesium treatment. Incubation of intact diaphragms from magnesium-deficient rats demonstrated an increased sugar (2-DG) and amino acid (AIB) uptake from magnesiumfree buffer. Although the plasma magnesium concentration rapidly decreased in the deficient rats, a considerably longer period of time elapsed before the changes in sugar transport became apparent. Kinetic studies suggested that the enhanced permeability was due to carrier mediated transport rather than to diffusion. Tissue magnesium levels remained normal despite a significant decrease in the serum magnesium. These studies suggest that some general characteristic of membrane structure and function is affected by the extracellular concentration of magnesium.