Characterization of a yeast artificial chromosome contig spanning the Huntington's disease gene candidate region.

The Huntington's disease (HD) gene has been localized by recombination events to a region covering 2.2 megabases (Mb) DNA within chromosome 4p16.3. We have screened three yeast artificial chromosome (YAC) libraries in order to isolate and characterize 44 YAC clones mapping to this region. Approximately 50% of the YACs were chimaeric. Unstable YACs were identified across the whole region, but were particularly prevalent around the D4S183 and D4S43 loci. The YACs have been assembled into a contig extending from D4S126 to D4S98 covering roughly 2 Mb DNA, except for a gap of about 250 kilobases (kb). The establishment of a YAC contig which spans the region most likely to contain the HD mutation is an essential step in the isolation of the HD gene.     

Cloning of the alpha-adducin gene from the Huntington's disease candidate region of chromosome 4 by exon amplification.

We have applied the technique of exon amplification to the isolation of genes from the chromosome 4p16.3 Huntington's disease (HD) candidate region. Exons recovered from cosmid Y24 identified cDNA clones corresponding to the alpha-subunit of adducin, a calmodulin-binding protein that is thought to promote assembly of spectrin-actin complexes in the formation of the membrane cytoskeleton, alpha-adducin is widely expressed and, at least in brain, is encoded by alternatively spliced mRNAs. The alpha-adducin gene maps immediately telomeric to D4S95, in a region likely to contain the HD defect, and must be scrutinized to establish whether it is the site of the HD mutation.     

Isolation and characterization of a candidate gene for Norrie disease.

Previous analysis has refined the location of the gene for Norrie disease, a severe, X-linked, recessive neurodevelopmental disorder, to a yeast artificial chromosome subfragment of 160 kilobases (kb). This fragment was used to screen cDNA libraries from human fetal and adult retina. As a result, we have identified an evolutionarily conserved cDNA, which is expressed in fetal and adult brain and encodes a predicted protein of 133 amino acids. The cDNA detects genomic sequences which span a maximum of 50 kb, and which are partly deleted in several typical Norrie disease patients. An EcoRI polymorphism with a calculated heterozygosity value of 43% was observed. The locus identified is a strong candidate for the Norrie disease gene.     

Isolation of a candidate gene for Norrie disease by positional cloning.

The gene for Norrie disease, an X-linked disorder characterized by progressive atrophy of the eyes, mental disturbances and deafness, has been mapped to chromosome Xp11.4 close to DXS7 and the monoamine oxidase (MAO) genes. By subcloning a YAC with a 640 kilobases (kb) insert which spans the DXS7-MAOB interval we have generated a cosmid contig which extends 250 kb beyond the MAOB gene. With one of these cosmids, microdeletions were detected in several patients with Norrie disease. Screening of cDNA libraries has enabled us to isolate and sequence a likely candidate gene for Norrie disease which is expressed in retina, choroid and fetal brain. No homologous sequences were found in DNA and protein databases indicating that this cDNA is part of a gene encoding a 'pioneer' protein.     

The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a large DNA duplication on the short arm of human chromosome 17. The trembler (Tr) mouse serves as a model for CMT1A because of phenotypic similarities and because the Tr locus maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17. Recently, the peripheral myelin gene Pmp-22 was found to carry a point mutation in Tr mice. We have isolated cDNA and genomic clones for human PMP-22. The gene maps to human chromosome 17p11.2-17p12, is expressed at high levels in peripheral nervous tissue and is duplicated, but not disrupted, in CMT1A patients. Thus, we suggest that a gene dosage effect involving PMP-22 is at least partially responsible for the demyelinating neuropathy seen in CMT1A.     

Targeted breakage of a human chromosome mediated by cloned human telomeric DNA.

Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.     

Kallmann syndrome gene on the X and Y chromosomes: implications for evolutionary divergence of human sex chromosomes.

The recently identified gene for X-linked Kallmann syndrome (hypogonadotropic hypogonadism and anosmia) has a closely related homologue on the Y chromosome. The X and Y copies of this gene are located in a large region of X/Y homology, on Xp22.3 and Yq11.2, respectively. Comparison of the structure of the X-linked Kallmann syndrome gene and its Y homologue shed light on the evolutionary history of this region of the human sex chromosomes. Our data show that the Y homologue is not functional. Comparative analysis of X/Y sequence identity at several loci on Xp22.3 and Yq11.2 suggests that the homology between these two regions is the result of a complex series of events which occurred in the recent evolution of sex chromosomes.     

The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast

The Rhesus blood-group antigens are defined by a complex association of membrane polypeptides that includes the non-glycosylated Rh proteins (RhD and RhCE) and the RHag glycoprotein, which is strictly required for cell surface expression of these antigens. RhAG and the Rh polypeptides are erythroid-specific transmembrane proteins belonging to the same family (36% identity). Despite their importance in transfusion medicine, the function of RhAG and Rh proteins remains unknown, except that their absence in Rh(null) individuals leads to morphological and functional abnormalities of erythrocytes, known as the Rh-deficiency syndrome. We recently found significant sequence similarity between the Rh family proteins, especially RhAG, and Mep/Amt ammonium transporters. We show here that RhAG and also RhGK, a new human homologue expressed in kidney cells only, function as ammonium transport proteins when expressed in yeast. Both specifically complement the growth defect of a yeast mutant deficient in ammonium uptake. Moreover, ammonium efflux assays and growth tests in the presence of toxic concentrations of the analogue methylammonium indicate that RhAG and RhGK also promote ammonium export. Our results provide the first experimental evidence for a direct role of RhAG and RhGK in ammonium transport. These findings are of high interest, because no specific ammonium transport system has been characterized so far in human.     

Mice lacking the homologue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorge syndrome

Heterozygous deletions within human chromosome 22q11 are the genetic basis of DiGeorge/velocardiofacial syndrome (DGS/VCFS), the most common deletion syndrome (1 in 4,000 live births) in humans. CRKL maps within the common deletion region for DGS/VCFS (ref. 2) and encodes an SH2-SH3-SH3 adapter protein closely related to the Crk gene products. Here we report that mice homozygous for a targeted null mutation at the CrkL locus (gene symbol Crkol for mice) exhibit defects in multiple cranial and cardiac neural crest derivatives including the cranial ganglia, aortic arch arteries, cardiac outflow tract, thymus, parathyroid glands and craniofacial structures. We show that the migration and early expansion of neural crest cells is unaffected in Crkol-/- embryos. These results therefore indicate an essential stage- and tissue-specific role for Crkol in the function, differentiation, and/or survival of neural crest cells during development. The similarity between the Crkol-/- phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome.     

Identification of Stk6/STK15 as a candidate low-penetrance tumor-susceptibility gene in mouse and human

Linkage analysis and haplotype mapping in interspecific mouse crosses (Mus musculus x Mus spretus) identified the gene encoding Aurora2 (Stk6 in mouse and STK15 in human) as a candidate skin tumor susceptibility gene. The Stk6 allele inherited from the susceptible M. musculus parent was overexpressed in normal cells and preferentially amplified in tumor cells from F(1) hybrid mice. We identified a common genetic variant in STK15 (resulting in the amino acid substitution F31I) that is preferentially amplified and associated with the degree of aneuploidy in human colon tumors. The Ile31 variant transforms rat1 cells more potently than the more common Phe31 variant. The E2 ubiquitin-conjugating enzyme UBE2N was a preferential binding partner of the 'weak' STK15 Phe31 variant form in yeast two-hybrid screens and in human cells. This interaction results in colocalization of UBE2N with STK15 at the centrosomes during mitosis. These results are consistent with an important role for the Ile31 variant of STK15 in human cancer susceptibility.     

The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.     

Structure of the X-linked Kallmann syndrome gene and its homologous pseudogene on the Y chromosome.

The gene for the X-linked Kallmann syndrome (KAL), a developmental disorder characterized by hypogonadotropic hypogonadism and anosmia, maps to Xp22.3 and has a homologous locus, KALP, on Yq11. We show here that KAL consists of 14 exons spanning 120-200 kilobases that correlate with the distribution of domains in the predicted protein including four fibronectin type III repeats. The KALP locus reveals several large deletions and a number of small insertions, deletions and base substitutions which indicate it is a non-processed pseudogene. The sequence divergence between KAL and KALP in humans, and the chromosomal location of KAL homologous sequences in other primates, suggest that KALP and the steroid sulphatase pseudogene on Yq11 were involved in the same rearrangement event on the Y chromosome during primate evolution.     

Mutational and functional analyses reveal that ST7 is a highly conserved tumor-suppressor gene on human chromosome 7q31

Loss of heterozygosity (LOH) of markers on human chromosome 7q31 is frequently encountered in a variety of human neoplasias, indicating the presence of a tumor-suppressor gene (TSG). By a combination of microcell-fusion and deletion-mapping studies, we previously established that this TSG resides within a critical region flanked by the genetic markers D7S522 and D7S677. Using a positional cloning strategy and aided by the availability of near-complete sequence of this genomic interval, we have identified a TSG within 7q31, named ST7 (for suppression of tumorigenicity 7; this same gene was recently reported in another context and called RAY1). ST7 is ubiquitously expressed in human tissues. Analysis of a series of cell lines derived from breast tumors and primary colon carcinomas revealed the presence of mutations in ST7. Introduction of the ST7 cDNA into the prostate-cancer-derived cell line PC3 had no effect on the in vitro proliferation of the cells, but abrogated their in vivo tumorigenicity. Our data indicate that ST7 is a TSG within chromosome 7q31 and may have an important role in the development of some types of human cancer.