Search PubMed⌕ Search

Biomedical subjects

H Lehrach

Publications and source records attributed to H Lehrach.

At least 163 records · Page 9Linked to original sources
PubMed1996

Survey of CAG/CTG repeats in human cDNAs representing new genes: candidates for inherited neurological disorders.

Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3'-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.

Brain↗
PubMed1996

An integrated YAC map of the human X chromosome.

The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males. With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map. We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones. Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources. We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones. These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques. This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps.

Chromosome Mapping↗
PubMed1996

Toward the construction of integrated physical and genetic maps of the mouse genome using interspersed repetitive sequence PCR (IRS-PCR) genomics.

Using two recently developed techniques, IRS-PCR YAC walking and IRS-PCR genotyping, a framework-integrated physical and genetic map of the mouse genome was constructed. The map consists of 821 contigs, containing 7746 YAC clones originating from three different YAC libraries. Three hundred eighty of the contigs have been anchored to the genetic map. Approximately 16% of the physical length of the mouse genome is estimated to be represented.

Animals↗
PubMed1996

An improved method for the detection of Down's syndrome aneuploidy in uncultured amniocytes.

We report a modified method for the rapid detection of aneuploidies directly on human uncultured amniocytes that simplifies and shortens the entire experimental procedure, yielding signals which allow correct diagnosis of trisomy 21 in 97% of cases. The improvement is based on two points: 1) use of cosmid pockets specific for the Down's syndrome minimal region as FISH probes, and 2) a modified protocol for the fixation and preparation of amniocytes.

Amniotic Fluid↗
PubMed1995

Construction of genetic maps using distance geometry.

The techniques of distance geometry, which generate coordinates from observed interpoint distances, have been applied to the problem of determining the relative positions of linked genetic loci from observed interlocus distances. Only the most precise data needed to join the loci are used, with missing distances substituted by sums of precise intermediate distances. Good initial positions (and therefore the order) of loci on a linear map are obtained in an operation of complexity O(N3). The method can therefore be used to generate good initial framework maps for the large numbers of markers encountered in current mapping projects. The locus positions can be subsequently refined to maximize the agreement with the originally observed distances, taking account of the weights of individual interlocus distances. By choosing only small distances from which to construct the map, the method reduces any error due to an incorrect choice of mapping function. It also prevents undue expansion of the map due to error-prone markers, since such markers are accommodated in higher dimensions. The method estimates the error in the positions of individual markers on the final map and identifies well- and ill-defined regions of the map.

Algorithms↗
PubMed1995

Expression of a Drosophila GATA transcription factor in multiple tissues in the developing embryos. Identification of homozygous lethal mutants with P-element insertion at the promoter region.

GATA transcription factors are DNA-binding proteins that recognize the core consensus sequence, WGATAR. Previous studies indicated that GATA factors play ann important role in the development of tissue-specific functions in vertebrates. Here we report the identification of a new Drosophila melanogaster GATA factor, dGATAc, which displays a distinct expression pattern in embryos. The local concentration of dGATAc transcripts varies at different stages, being most prominent in the procephalic region at stages 6-10 and in the posterior spiracles, the gut, and the central nervous system at stages 11-13. On the basis of its predicted sequence, DNA-binding assays were performed to confirm that the dGATAc gene encodes a zinc finger protein that can bind the GATA consensus motif with predicted specificity. Two independent mutants carrying a P-element insertion at the dGATAc gene promoter region were identified that are homozygous lethal at the embryonic stage. Using a genetic scheme, it was demonstrated that the lack of dGATAc function can block normal embryonic development. Our results suggest that the dGATAc protein is a tissue-specific transcription factor that is vital to the development of multiple organ systems in D. melanogaster.

Amino Acid Sequence↗
PubMed1995

Fine-mapping of shotgun template-libraries; an efficient strategy for the systematic sequencing of genomic DNA.

To test the effectiveness of ordering shotgun DNA-templates prior to sequence analysis, the 450 kb left arm of yeast chromosome XII was randomly subcloned into a phagemid vector. Clones were ordered by hybridisation to an average map density of one new insert every 125 bp and are currently used for sequencing the chromosomal fragment. An 11.5 kb overlap between the template map and a DNA fragment that had been sequenced earlier allowed an independent evaluation of the strategy's effectiveness. To this end, clones were selected from the map and tag-sequenced from either end, thus comparing the map position with the actual location within the 11.5 kb. Of 65 selected clones, taken mostly at random from a total of 423, 58 mapped on average about a quarter of a clone length around their predicted position, with the other seven being between 0.6 and 1.5 clone length off. 75-86 sequencing reactions on clones selected from the map would have been sufficient for completely sequencing both strands of the 11.5 kb fragment. The results demonstrate the efficacy of such template sorting, considerably assisting sequencing at relatively little cost on the mapping level.

Base Sequence↗
PubMed1995

Characterization of a YAC contig spanning the pseudoautosomal region.

Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest. In addition, an extremely high level of YAC instability has been observed in this region. We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region. Of these a subset of 22 YACs was analyzed in detail. YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered. All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig. Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages. This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.

Chimera↗
PubMed1995

Cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from Drosophila melanogaster.

Mammalian somatic angiotensin converting enzyme (EC 3.4.15.1, ACE) consists of two highly homologous (N- and C-) domains encoded by a duplicated gene. We have identified an apparent single-domain (67 kDa) insect angiotensin converting enzyme (AnCE) in embryos of Drosophila melanogaster which converts angiotensin I to angiotensin II (Km, 365 microM), removes Phe-Arg from the C terminus of bradykinin (Km, 22 microM), and is inhibited by ACE inhibitors, captopril (IC50 = 1.1 x 10(-9) M) and trandolaprilat (IC50 = 1.6 x 10(-8) M). We also report the cloning and expression of a Drosophila AnCE cDNA which codes for a single-domain 615-amino acid protein with a predicted 17-amino acid signal peptide and regions with high levels of homology to both the N- and C-domains of mammalian somatic ACE, especially around the active site consensus sequence. Northern analysis identified a single 2.1-kilobase mRNA in Drosophila embryos, and Southern analysis of Drosophila genomic DNA indicates that the insect gene is not duplicated. When expressed in COS-7 cells, the AnCE protein is a secreted enzyme, which converts angiotensin I to angiotensin II and is inhibited by captopril (IC50 = 5.6 x 10(-9) M) and trandolaprilat (IC50 = 2 x 10(-8) M). The evolutionary significance of these results is discussed.

Amino Acid Sequence↗
PubMed1995

Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1.

BRCA1 is a breast/ovarian cancer susceptibility gene on human chromosome 17q21. We describe a complete and detailed physical map of a 500-kb region of genomic DNA containing the BRCA1 gene and the partial cloning in phage P1 artificial chromosomes. Approximately 70 exons were isolated from this region, 11 of which were components of the BRCA1 gene. Analysis of the other exons revealed a rho-related G protein and the interferon-induced leucine-zipper protein IFP-35.

Amino Acid Sequence↗
PubMed1995

MHC class I gene organization in > 1.5-Mb YAC contigs from the H2-M region.

Sixteen yeast artificial chromosome (YAC) clones have been mapped to the H2-M region at the distal end of the mouse major histocompatibility complex (MHC) on chromosome 17. Analysis of the YACs with single- and multicopy probes yielded a proximal contig spanning a minimum of 800 kb and a distal contig of 700 kb. A probe for the conserved fourth exon of MHC class I genes detected 19 restriction fragments, including 6 of the 8 previously characterized H2-M class I genes, in the proximal contig. This contig spans the gap from the M to the T region and includes the T1 gene. By contrast, only two class I genes, M2 and M3, were found in the distal contig. These two genes, which are both expressed, may mark the end of the MHC. The order among nine class I genes and seven other markers was determined in the cloned DNA from the centromere as T1, Tu32A, (M1-M7-M8), Tu32B, B30, M6, M4, M5, Mog, Tu42A parallel M2, Leh525, M3, Tu42B, where the orientation with respect to the centromere is unknown for M1-M7-M8.

Animals↗
PubMed1995

Thioredoxin, a mediator of growth inhibition, maps to 9q31.

The human thioredoxin gene has been provisionally mapped to 3p11-p12. Recently thioredoxin cDNA has been isolated in a procedure that detects transcripts coding for growth-suppressing proteins, and thus the chromosomal location of the gene is of particular interest. Chromosome 3 is believed to harbor several tumor suppressor genes important in the development of lung and other common epithelial tumors. To establish more firmly the chromosomal location of the human thioredoxin gene, a somatic hybrid panel was used; it identified chromosome 9 as the location of the transcribed thioredoxin gene. Fluorescence in situ hybridization of a YAC encoding the transcribed thioredoxin gene refined the localization to 9q31.

Animals↗
PubMed1995

A 6-Mb YAC contig in Xp22.1-p22.2 spanning the DXS69E, XE59, GLRA2, PIGA, GRPR, CALB3, and PHKA2 genes.

We report the generation of an approximately 6-Mb contig of 70 overlapping yeast artificial chromosomes (YAC) covering the interval between DXS16 and DXS1229 in Xp22.1-p22.2. Within this region lie the genes for calbindin (CALB3), gastrin-releasing peptide receptor (GRPR), phosphatidyl-inositol glycan-class A protein (PIGA), glycine receptor alpha-2 (GLRA2), phosphorylase kinase alpha (PHKA2), XE59 (a gene escaping X chromosome inactivation), and DXS69E (71-7A). YACs were isolated initially from four libraries either by hybridization or using sequence tagged sites (STSs) for DXS16, DXS9, GLRA2, DXS207, DXS43, DXS1416, DXS1317, DXS1195, and DXS418. Additional STSs were obtained from the end fragments of the original YACs studied, thus allowing us to cover the contig with a series of 73 STSs, approximately 1 per 100 kb. YAC contig construction allowed the following locus order to be established: Xpter-DXS16-DXS69E-DXS414-XE59 - DXS9 - (GLRA2, DXS987) - (PIGA, DXS207) - DXS1053-DXS197-(GRPR,DXS43)-CALB3-DXS14 16- DXS1317 - DXS1195 - DXS418 - DXS257 - (PHKA2, DXS999)-DXS443-DXS1229-Xcen. Restriction mapping of the DXS16-DXS43 interval predicted the existence of several CpG islands, suggesting the presence of other genes in the region. This work provides a starting point for further mapping and positional cloning of several X-linked disease genes.

Base Sequence↗
PubMed1995

Trinucleotide repeat expansion and human disease.

Trinucleotide repeat expansions have been identified as the underlying mutation in an increasing number of human genetic diseases, such as fragile site syndromes, myotonic dystrophy and several neurodegenerative disorders including Huntington's disease. By an unknown mechanism, polymorphic GC-rich triplet repeats expand in all these diseases. The expansions of a CCG repeat in fragile-site-associated disorders and the CTG repeat (in the 3'-untranslated region of the myotonin kinase gene) causing myotonic dystrophy are very large, whereas small expansions of CAG repeats have been identified in the open reading frame of genes in a number of neurological genetic disorders.

Age of Onset↗
PubMed1995

Mapping around the Fused locus on mouse chromosome 17.

We have established a high-resolution genetic map of the region surrounding the Fused locus as a first step towards the molecular identification and analysis of this gene. The candidate region has been covered to a large extent by YAC and P1 contigs, and has been partly characterized by pulsed-field gel analysis.

Animals↗
PubMed1995

Techniques in mammalian genome mapping.

Increasing emphasis is being given to genomic cloning using Escherichia coli vectors of intermediate insert capacity, such as bacteriophage P1, P1-derived artificial chromosomes and the F factor based bacterial artificial chromosomes. These vectors are being used in addition to yeast artifical chromosomes (YACs) in recognition of the difficulties encountered with YAC stability and with handling of YAC DNAs (problems that will not easily be overcome). Nonetheless, YACs remain the most practical cloning system for global contig building. Efforts are currently under way to produce YAC contigs that represent the human and mouse genomes, and these will increasingly exploit extensive anchoring to detailed genetic maps. Intermediate capacity clone collections based on YAC contigs will follow, enabling the compilation of mapped gene catalogues. In this way, the era of big gene hunts will draw to a close.

Animals↗
PubMed1995

Model for a transcript map of human chromosome 21: isolation of new coding sequences from exon and enriched cDNA libraries.

The construction of a transcriptional map for human chromosome 21 requires the generation of a specific catalogue of genes, together with corresponding mapping information. Towards this goal, we conducted a pilot study on a pool of random chromosome 21 cosmids representing 2 Mb of non-contiguous DNA. Exon-amplification and cDNA selection methods were used in combination to extract the coding content from these cosmids, and to derive expressed sequences libraries. These libraries and the source cosmid library were arrayed at high density for hybridisation screening. A strategy was used which related data obtained by multiple hybridisations of clones originating from one library, screened against the other libraries. In this way, it was possible to integrate the information with the physical map and to compare the gene recovery rate of each technique. cDNAs and exons were grouped into bins delineated by EcoRI cosmid fragments, and a subset of 91 cDNAs and 29 exons have been sequenced. These sequences defined 79 non-overlapping potential coding segments distributed in 24 transcriptional units, which were mapped along 21q. Northern blot analysis performed for a subset of cDNAs indicated the existence of a cognate transcript. Comparison to databases indicated three segments matching to known chromosome 21 genes: PFKL, COL6A1 and S100B and six segments matching to unmapped anonymous expressed sequence tags (ESTs). At the translated nucleotide level, strong homologies to known proteins were found with ATP-binding transporters of the ABC family and the dihydroorotase domain of pyrimidine synthetases. These data strongly suggest that bona fide partial genes have been isolated. Several of the newly isolated transcriptional units map to clinically important regions, in particular those involved in Down's syndrome, progressive myoclonus epilepsia and auto-immune polyglandular disease. The study presented here illustrates the complementarity of exon-amplification and cDNA selection techniques for generating a large resource of new expressed landmarks, which contribute to the construction of a chromosome 21 transcript map.

Chromosome Mapping↗