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P Kioschis

Publications and source records attributed to P Kioschis.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular cloning of tissue-specific transcripts of a transketolase-related gene: implications for the evolution of new vertebrate genes.

As part of a systematic search for differentially expressed genes, we have isolated a novel transketolase-related gene (TKR) (HGMW-approved symbol TKT), located between the green color vision pigment gene (GCP) and the ABP-280 filamin gene (FLN1) in Xq28. Transcripts encoding tissue-specific protein isoforms could be isolated. Comparison with known transketolases (TK) demonstrated a TKR-specific deletion mutating one thiamine binding site. Genomic sequencing of the TKR gene revealed the presence of a pseudoexon as well as the acquisition of a tissue-specific spliced exon compared to TK. Since it has been postulated that the vertebrate genome arose by two cycles of tetraploidization from a cephalochordate genome, this could represent an example of the modulation of the function of a preexisting transketolase gene by gene duplication. Thiamine defiency is closely involved with two neurological disorders, Beriberi and Wernicke-Korsakoff syndromes, and in both of these conditions TK with altered activity are found. We discuss the possible involvement of TKR in explaining the observed variant transketolase forms.

Alternative Splicing↗
PubMed1996

X-linked myotubular myopathy: refinement of the gene to a 280-kb region with new and highly informative microsatellite markers.

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the off-spring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.

Alleles↗
PubMed1996

A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.

X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man.

Amino Acid Sequence↗
PubMed1996

Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region.

We have recently described a female patient with myotubular myopathy (MTM1) and an interstitial deletion at Xq28. Characterisation of the deletion allowed us to position the MTM1 gene to a 600 kb region between DXS304 and DXS497. In order to further restrict the region we screened for deletions in a set of 38 patients. We found two overlapping deletions in boys that in addition to MTM1 showed an unexpected abnormal genital development. As the latter phenotype is not found in the other non-deleted MTM1 patients, our observations are best explained by a contiguous gene syndrome. The deletions define a 430 kb region that contains the MTM1 gene and most likely a gene implicated in male sexual development. A high resolution physical map of this region is presented.

Base Sequence↗
PubMed1996

Transcriptional analysis of the candidate region for incontinentia pigmenti (IP2) in Xq28.

The hereditary form of incontinentia pigmenti (IP2) is a rare disorder characterized by abnormalities of the tissues and organs derived from the ectoderm and neuroectoderm and has been linked to Xq28 distal to the factor VIII gene (F8C). Four YAC clones covering the 1.1-Mb candidate region at the telomere of Xq28 were subjected to direct cDNA selection and Alu long-range PCR. The products of both methods were subsequently used to isolate 154 cosmid clones that were assembled into five cosmid contigs. This first-generation cosmid map covered the region almost entirely and was used as a basis for constructing a transcript map that was in turn integrated with the physical YAC and cosmid maps. To isolate specifically coding sequences, exon trapping and cDNA selection methods were combined. Exon trapping was carried out on YAC Alu-PCR products, YAC Alu long-range PCR products, and on pools of cosmids. The region-specific enriched cDNA library was then screened by using the exon trap products as complex probes. To ensure a more complete analysis, the products from cDNA selection experiments were also used to screen conventional oligo(dT) primed cDNA libraries. Twenty overlapping cDNA contigs were assembled and computer analyses were performed to identify EST hits, open reading frames, protein motifs, and protein sequence homologies. Five of the cDNA contigs corresponded to known sequences such as the factor VIII, c6.1A, and c6.1B. genes, and both distal copies of the factor VIII intron 22 repeat sequence. Expression patterns of the 15 new cDNA contigs were analyzed by Northern blot and RT-PCR studies and these data were integrated with expression data obtained from known EST sequences. Although a more detailed analysis of this 1.1-Mb region with respect to the structure and function of the genes will only ultimately be possible by a global sequencing approach, an analysis of all novel transcripts as candidate genes for incontinentia pigmenti is already in progress.

Adult↗
PubMed1996

Transcription mapping in a 700-kb region around the DXS52 locus in Xq28: isolation of six novel transcripts and a novel ATPase isoform (hPMCA5).

The chromosomal band Xq28 has been a focus of interest in human genetics because > 20 hereditary diseases have been mapped to this region. However, about two-thirds of the disease genes remain uncloned. The region around the polymorphic DXS52 locus (ST14) within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes. So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN) gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region. By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus. In addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAP103-XAP108), the caltractin gene, and a gene encoding a novel Ca(2+)-transporting ATPase isoform (hPMCA5). The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag (EST) from sequence data bases and if, in addition, they showed evidence of expressed RT-OCT and/or Northern blot analysis. Our data facilitated the integration of the transcription map with the physical map around the DXS52 locus. Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata (CDPX2) is in progress.

Amino Acid Sequence↗
PubMed1994

Genomic organization of the adrenoleukodystrophy gene.

Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, is a severe neurodegenerative disease associated with an impairment of very long chain fatty acids beta-oxidation. We have recently identified by positional cloning the gene responsible for ALD, located in Xq28. It encodes a new member of the "ABC" superfamily of membrane-associated transporters that shows, in particular, significant homology to the 70-kDa peroxisomal membrane protein (PMP70). We report here a detailed characterization of the ALD gene structure. It extends over 21 kb and consists of 10 exons. To facilitate the detection of mutations in ALD patients, we have determined the intronic sequences flanking the exons as well as the sequence of the 3' untranslated region and of the immediate 5' promoter region. Sequences present in distal exons cross-hybridize strongly to additional sequences in the human genome. The ALD gene has been positioned on a pulsed-field map between DXS15 and the L1CAM gene, about 650 kb upstream from the color pigment genes. The frequent occurrence of color vision anomalies observed in patients with adrenomyeloneuropathy (the adult onset form of ALD) thus does not represent a contiguous gene syndrome but a secondary manifestation of ALD.

Adrenoleukodystrophy↗
PubMed1994

Identification of tissue-specific expressed sequences in human band Xq28 with complex pig cDNA probes.

As a part of the functional analysis of the region from the position of the fragile X mutation to the telomere of the long arm of the human X Chromosome (Chr), we have developed a number of different approaches to identify genes located in this area. We describe here a procedure allowing the rapid identification of expressed sequences based on the hybridization of radioactively labeled complex cDNA probes derived from different pig and human tissues to cosmid clones gridded onto nylon filters and to restriction fragments of these clones. This technique has allowed the identification of a number of differentially expressed sequences in cosmid clones covering most of the Xq27.3 to Xqter region. Using these sequences as hybridization probes, cDNA clones for new genes expressed in a tissue-specific manner were isolated. Applied to genomic regions defined by overlapping cosmid clones, this method will serve as a major component in our strategy to establish integrated physical and transcription maps.

Animals↗
PubMed1994

Rapid identification of gene sequences for transcriptional map assembly by direct cDNA screening of genomic reference libraries.

We have used the direct cDNA screening protocol to identify sequences transcribed in cerebral cortex from a reference library of human Xq28. To derive coding sequences from these genomic clones, we first identified fragments containing transcribed sequences and subjected these to exon trapping or to partial sequencing and analysis by Grail. In a preliminary analysis of three clones, coding sequences from two novel genes expressed in brain were identified. This method allows the rapid identification of coding sequences of genes expressed in specific tissues without recourse to cDNA libraries. The approach is amenable to large scale applications and should be useful for isolating candidate disease genes and in particular for assembling integrated transcriptional maps from large genomic regions.

Amino Acid Sequence↗
PubMed1994

A YAC clone map spanning 7.5 megabases of human chromosome band Xq28.

Xq28 has been of special interest in human genetics because a large number of diseases map to this region. As a step in the molecular analysis of the as yet uncloned disease genes, and as a test for the detailed analysis of larger regions of the genome, we have constructed YAC clone contigs covering the 7.5 Mb region between IDS to the telomere on the long arm of the human X chromosome. Contigs were assembled and verified by an integrated hybridization-based strategy. Data was combined from the physical map, from YAC and cosmid mapping experiments, and from the localization of specific transcripts in the region. Two gaps in the YAC map of 250 and 100 kb were covered in part by the aid of cosmid clones, but small gaps of 50 kb each remain. The cloned region is expected to contain yet unidentified genes for at least ten genetic diseases. The construction of ordered YAC clone contigs of Xq28 represents an important step in the molecular identification of these genes, and the further analysis of one of the genetically most interesting regions of the human genome.

Chromosomes, Artificial, Yeast↗
PubMed1994

X linked myotubular myopathy (MTM1) maps between DXS304 and DXS305, closely linked to the DXS455 VNTR and a new, highly informative microsatellite marker (DXS1684).

The locus for X linked recessive myotubular myopathy (MTM1) has previously been mapped to Xq28 by linkage analysis. We report two new families that show recombination between MTM1 and either DXS304 or DXS52. These families and a third previously described recombinant family were analysed with two highly polymorphic markers in the DXS304-DXS52 interval, the DXS455 VNTR and a newly characterised microsatellite, DXS1684 (82% heterozygosity). These markers did not recombine with MTM1 in the three families. Together with the recent mapping of an interstitial X chromosome deletion in a female patient with moderate signs of myotubular myopathy, our data suggest the following order of loci in Xq28: cen-DXS304-(DXS455, MTM1)-DXS1684-DXS305-DXS52-tel. This considerably refined localisation of the MTM1 locus should facilitate positional cloning of the gene. The availability of highly polymorphic and very closely linked markers will markedly improve carrier and prenatal diagnosis of MTM1.

Base Sequence↗
PubMed1993

Direct selection of DNA sequences conserved between species.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.

Animals↗
PubMed1993

Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.

Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1993

Construction of a transcription map of a 300 kb region around the human G6PD locus by direct cDNA selection.

A transcription map covering a 300 kb region around the G6PD gene in the human Xq28 region was constructed by the direct cDNA selection method and the analysis of the resulting region-specific enriched cDNA sublibrary. Seven new genes and two loci of endogenous retrovirus HERV-K were identified. The distribution of the genes across the region is strongly non-uniform and follows the non-uniform distribution of GpG islands in the area. While one of the novel genes was found to be highly homologous to bovine smg p25A GDP-dissociation inhibitor, the remaining genes did not detect any homology to known genes. The analysis of region-specific cDNA sublibraries represents a simple, rapid and efficient tool for the generation of a regional transcription map.

Animals↗
PubMed1992

A strategy for the selection of transcribed sequences in the Xq28 region.

As an essential step towards an exhaustive analysis of the coding potential of large regions of the genome, we have developed a protocol allowing the rapid isolation of transcripts defined by overlapping clone libraries. The method is based on the hybridisation of cDNA inserts, which had been amplified by PCR from cDNA libraries, to biotinylated DNA from cosmids or cosmid pools. Nonspecific hybrids are then removed, the selected cDNAs are eluted and reamplified by PCR. Using a cosmid containing part of the FMR-1 gene as test, we were able to demonstrate an eighty thousand fold enrichment of cDNAs for this gene after two rounds of selection-amplification. The technique was applied to the analysis of transcripts from two cosmid contigs, together encompassing a region of 900 kb in Xq28. These experiments have thus far resulted in the identification of 81 cDNA clones, of which 54 clones were mapped back to the cosmid contigs. Of the 54 clones placed on the contig maps, 12 cDNA clones can be shown to belong to two genes which have been previously reported (L1CAM and QM).

Base Sequence↗
PubMed1992

Identification and characterization of a new gene in the human Xq28 region.

A human Xqter chromosome cosmid library was screened with a mixed probe derived from porcine kidney mRNA. A new expressed gene was identified in a cosmid clone known to be part of a G6PD cosmid contig. This gene is most likely a housekeeping gene because the cDNA clone recognizes a 1 kb mRNA transcript in all cell lines and tissues tested. Hybridizing genomic DNA of several species with a cDNA probe indicated that the gene is highly conserved during evolution and that it belongs to a gene family. The genomic sequence shows a 100% homology with the recently identified QM cDNA sequence.

Adrenoleukodystrophy↗
PubMed1992

An archipelago of CpG islands in Xq28: identification and fine mapping of 20 new CpG islands of the human X chromosome.

19 probes for CpG islands, mapping to Xq28, have been used as probes to construct a physical map of genes of this band of the human X chromosome. A total of 22 CpG islands have been precisely mapped in respect to known loci along the 9-10 Mb of Xq28. The fine mapping of such a large number of CpG islands has demonstrated that also in gene rich Giemsa light bands, like Xq28, gene distribution is non uniform: the CpG islands are clustered in the distal portion of the band in a 2 Mb region between the G6PD gene and the DXS15 locus. Moreover, 16 CpG islands were found between the G6PD and the RCP/GCP genes, a region of DNA of only about 300 kb. If this structural organization has a biological function it has yet to be determined. However, the isolation of large genomic regions enriched in gene sequences and the availability of cosmid or YAC contigs will provide the means to test the significance of such gene organization, as well as the material for large sequencing projects and gene search, for the identification of candidate genes for inherited disorders mapped to Xq28 and for comparative mapping.

Animals↗