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H Lehrach

Publications and source records attributed to H Lehrach.

At least 181 records · Page 10Linked to original sources
PubMed1995

Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo.

A total of 5 conventional, directionally cloned plasmid cDNA libraries have been constructed from the entire embryonic region of the mid-gastrulation mouse embryo and from its four principal tissue constituents (ectoderm, mesoderm, endoderm and primitive streak). These libraries have been validated with respect to the number of independent clones, insert-size and appropriate representation of diagnostic marker genes. Subtractive hybridisation has been used to remove clones common to the Endoderm and Mesoderm cDNA libraries resulting in an Endoderm minus Mesoderm subtracted library. Probe prepared from this subtracted library has been hybridised to a grid containing approximately 18,500 Embryonic Region library clones. Three novel clones have been recovered as well as expected genes already known to be highly expressed in the primitive endoderm lineage at this stage of development. In situ hybridisation to early postimplantation embryos has revealed the expression patterns of these novel genes. One is highly expressed exclusively in visceral endoderm, one is expressed in ectodermal and endodermal tissues, and the third proves to be an early marker of prospective and differentiated surface ectoderm as well as being expressed in endoderm and its derivatives.

Animals↗
PubMed1994

The construction of a beta-lactamase-encoding ApR gene cassette flanked by recognition sites for NotI, SacII, MluI, SplI, BssHII and NarI.

An ApR gene cassette was constructed using the gene present in the PBR322 derivative pTZ18. The cassette is maintained in the plasmid pcLINK-1 and may be excised with any of the six rare cutting enzymes NotI, SacII, MluI, SplI, BssHII or NarI. By using a double-digestion procedure, the ApR gene may be excised with two different protruding ends. In the process of constructing the pcLINK-1 plasmid the multiplecloning site of pTZ18 was extended with recognition sites for the enzymes NotI, SacII, MluI, SplI, BssHII and NarI.

Base Sequence↗
PubMed1994

A YAC contig spanning the ataxia-telangiectasia locus (groups A and C) at 11q22-q23.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease involving cerebellar degeneration, immunodeficiency, cancer predisposition, chromosomal instability and radiosensitivity. A-T is heterogeneous, and the majority of A-T cases are associated with two complementation groups, A and C. The ATA and ATC loci are closely linked at chromosome 11q22-q23. Recombination mapping and linkage disequilibrium analysis have confined both loci between the markers D11S1817 and D11S927, spaced approximately 3.5 Mb apart. Isolation in yeast artificial chromosomes of the genomic segment defined by these loci is essential to identify the gene or genes containing the ATA and ATC mutations. A YAC contig spanning 4.5 Mb, which includes the D11S1817-D11S927 interval, was constructed using two whole genome libraries (ICRF and St. Louis), and a chromosome 11-specific library. Construction of this contig was expedited by prior generation of a region-specific ICRF sublibrary using Alu-PCR products derived from a radiation hybrid. The contig was expanded further by screening the libraries with Alu-PCR products derived from YAC clones and with STSs from YAC ends. YAC clones were aligned by fingerprinting with moderately repetitive probes.

Ataxia Telangiectasia↗
PubMed1994

Genomic organization and expressed sequences of the mouse extended H-2K region.

The mouse major histocompatibility complex (MHC) has long been of great interest to many biologists because of not only its critical role in the immune system, but also its association with at least three embryonic lethal genes. Here, we present an analysis of the mouse extended H-2K region using YAC technology. Six new expressed sequences were identified, demonstrating that the high gene density previously described continues. Restriction mapping of a YAC clone extending proximal of the MHC region defined a CpG-rich region located up to 320 kb away from H-2K. The absence of any CpG-rich region for a distance spanning approximately 200 kb near the YAC's proximal end suggests that the high gene density probably diminishes at a distance of 360 kb away from H-2K. The description of genomic organization of both H-2K and the extended H-2K region provides insight into the characteristics of this whole region with respect to gene diversity and density.

Animals↗
PubMed1994

Physical map of mouse chromosome 17 in the region relevant for positional cloning of the Hybrid sterility 1 gene.

Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mapped and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene.

Animals↗
PubMed1994

An algorithm to detect chimeric clones and random noise in genomic mapping.

Experimental noise and noncontiguous clone inserts can pose serious problems in reconstructing genomic maps from hybridization data. We describe an algorithm that easily identifies false positive signals and clones containing chimeric inserts/internal deletions. The algorithm "dechimerizes" clones, splitting them into independent contiguous components and cleaning the initial library into a more consistent data set for further ordering. The effectiveness of the algorithm is demonstrated on both simulated data and the real YAC map of the whole genome of the fission yeast Schizosaccharomyces pombe.

Algorithms↗
PubMed1994

Relational genome analysis using reference libraries and hybridisation fingerprinting.

The genomes of eukaryotic organisms are studied by an integrated approach based on hybridisation techniques. For this purpose, a reference library system has been set up, with a wide range of clone libraries made accessible to probe hybridisation as high density filter grids. Many different library types made from a variety of organisms can thus be analysed in a highly parallel process; hence, the amount of work per individual clone is minimised. In addition, information produced on one analysis level instantly assists in the characterisation process on another level. Genetic, physical and transcriptional mapping information and partial sequencing data are obtained for the individual library clones and are cross-referenced toward a comprehensive molecular understanding of genome structure and organisation, of encoded functions and their regulation. The order of genomic clones is established by hybridisation fingerprinting procedures. On these physical maps, the location of transcripts is determined. Complementary, partial sequence information is produced from corresponding cDNAs by hybridising short oligonucleotides, which will lead to the identification of regions of sequence conservation and the constitution of a gene inventory. The hybridisation analysis of the cDNA clones, and the genomic clones as well, could potentially be expanded toward a determination of (nearly) the complete sequence. The accumulated data set will provide the means to direct large-scale sequencing of the DNA, or might even make the sequence analysis of large genomic regions a redundant undertaking due to the already collected information.

Animals↗
PubMed1994

Application of robotic technology to automated sequence fingerprint analysis by oligonucleotide hybridisation.

We describe our production line for the rapid analysis of large cDNA libraries applying robotic techniques to automatically pick, amplify, array, hybridise and analyse the clones. We also outline the current state of the hybridisation techniques and describe anticipated future developments of the system. Our approach faces the large-scale analysis of cDNA clones with partial sequence analysis by oligonucleotide fingerprinting in the following way: after picking of individual colonies and arraying them automatically in quadruple density (384-well) microtitre plates, the cDNA clones are amplified by an automated waterbath polymerase chain reaction (PCR), which allows us to run about 46,000 reactions in parallel. The PCR products are automatically transferred to nylon membranes in a high density pattern using a robotic device. We routinely produce twelve 22 cm x 22 cm membranes in 90 min. Each membrane contains 20,736 clones, although much higher densities might be feasible using both miniaturized glass matrices and fluorescence based hybridisation techniques. Theoretical analysis and preliminary computer simulations indicate that about 100-200 sequence specific hybridisations of octanucleotides to about 100,000 PCR products of 1000-1500 base-pairs length will generate sufficient information for classifying the clones into groups of identical or related genes and to identify a large number of previously uncharacterized cDNA clones.

Biotechnology↗
PubMed1994

A 1.2-Mb YAC contig spans the quaking region.

We describe here a 1.2-Mb yeast artificial chromosome (YAC) contig within the region of mouse chromosome 17 between Brachyury (T) and D17Rp17e, and spanning the quaking (qk) region. We describe six new probes distributed across 1.2 Mb: D17Leh502, D17Leh503, D17Leh504, D17Leh505, D17Leh506, and D17Leh507. Probes D17Leh502 and D17Leh507 are at the extreme ends of the YAC contig. With the exception of D17Leh507, all of these probes are within a deletion associated with the quaking(viable) (qkv) allele of quaking. We have positioned these probes on a detailed YAC physical map together with two previously published probes, D17Leh508 and D17Aus119. We show here that D17Leh508 is also within the qkv deletion. Genetic mapping of D17Leh504 and D17Leh507 on two high-resolution genetic crosses carrying qkv and quaking(lethal-1) (qkl-1) alleles shows that these probes do not recombine with quaking and are therefore within 0.04 cM of qkv and 0.05 cM of qkl-1 mutations. The deletion breakpoint contained within the YAC contig has been positioned to within 90 kb by restriction mapping of wildtype and mutant DNA. This contig will form the basis for identification and mapping of expressed sequences and for an investigation of genome organization.

Alleles↗
PubMed1994

A YAC contig spanning the hypophosphatemic rickets disease gene (HYP) candidate region.

Dominant X-linked hypophosphatemic rickets (HYP) is the most common form of familial rickets. Linkage studies have localized the gene for this disorder to Xp22.1 between the markers DXS365 and DXS274, a region estimated to be approximately 3.5 cM. We have constructed a 1.5-Mb YAC contig encompassing this region by hybridization screening of high-density YAC clone filters. Rapid chromosome walking was achieved by direct hybridization of a pool of Alu-PCR products derived from a YAC containing DXS365 to the filter grids. Overlaps between YACs in the contig were estimated by hybridization of end probes to YAC digest blots and by analysis of cosmid fingerprints obtained by hybridization of YAC inserts to a flow-sorted chromosome X cosmid library. All YACs in the contig have been verified by fluorescence in situ hybridization. Several YACs spanning the HYP gene candidate region were selected for further analysis by rare-cutter enzyme digestion and pulsed-field gel electrophoresis. We estimate that the markers flanking the disease region, DXS365 and DXS274, are less than 1 Mb apart. This clone contig map provides an essential resource for the isolation of the HYP gene.

Animals↗
PubMed1994

Assignment of 55 novel cosmids to seven subregions of chromosome 13 using fluorescence in situ hybridization.

Fifty-five individual cosmids isolated from a chromosome 13-specific library have been regionally assigned using fluorescence in situ hybridization. These cosmids represent novel DNA markers that can be added to the limited number of DNA probes already available for this chromosome. Individual cosmids mapped along the length of the chromosome with almost equal distribution except that there appears to be an excess of probes from the distal 13q33-q34 region. Only two cosmids did not map back to chromosome 13, indicating that at least 96% of cosmids in this library are from this chromosome. The library is generally available as gridded colonies on nylon membranes suitable for hybridization screening.

Chromosome Walking↗
PubMed1994

Physical mapping in a YAC contig of 11 markers on the human X chromosome in Xp11.23.

By using yeast artificial chromosome (YAC) clones, we have generated a physical map of the short arm of the human X chromosome at Xp11.23. The region analyzed spans the distal marker UBE1 and the ARAF1/TIMP/SYN1/PFC gene cluster and further extends proximally to include ELK1, ZNF21, ZNF81, and OATL1 in a single contig. We have uniquely placed UBE1 and the SYN1 gene cluster in a 330-kb YAC within the contig. In addition, the genes SYP and TFE3 are physically linked in a YAC of 400 kb from the more centromeric portion of Xp11.23. We have refined the map by using fragmentation vectors to generate YAC deletion derivatives. These derivatives show that ZNF81 and ZNF21 lie within an approximately 130-kb segment and that SYN1.2 and ELK1 are less than 50 kb apart. Together these data reveal the following order for the markers in this region: Xpter-UBE1-ARAF1-TIMP-SYN1.2-PFC-ELK1++ +-ZNF81-ZNF21-OATL1-(SYP,TFE3)-Xcen.

Base Sequence↗
PubMed1994

Identification of YAC and cosmid clones encompassing the ZFX-POLA region using irradiation hybrid cell lines.

The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions. To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used. Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH). Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chimerism by FISH. Cosmid content analysis of the YACs defined two contigs, one with 12 YACs covering about 1.5 Mb and one with 3 YACs. Five YACs from the 12-YAC cluster had been previously recognized by DNA polymerase alpha (POLA). ZFX identified a single YAC; hence, the physical linkage of ZFX and POLA was demonstrated within the contig. Four YACs had been isolated previously with ZFX and these extend the contig to 2 Mb. Restriction mapping of several YACs demonstrates that ZFX and POLA are about 700 kb apart, a distance similar to that reported in the mouse between Zfx and Pola. The order of these two loci and two additional loci identified by homologous mouse linking clones was found to be conserved between human and mouse: tel-ZFX-DXCrc57-DXCrc140-POLA-cen. We have shown that YAC contigs can be rapidly constructed from targeted regions without the need for time-consuming YAC end rescue and chromosomal walking.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A 6.5-Mb yeast artificial chromosome contig incorporating 33 DNA markers on the human X chromosome at Xq22.

The Xq22 region of the human X chromosome contains genes for a number of inherited disorders. Sixty-nine yeast artificial chromosome clones have been isolated and assembled into a 6.5-Mb contig that contains 33 DNA markers localized to this region. This contig extends distally from DXS366 to beyond DXS87 and includes the genes involved in X-linked agammaglobulinemia (btk), Fabry disease (GLA), and Pelizaeus-Merzbacher disease (PLP). The order of markers in this contig is consistent with the known genetic and physical mapping information of Xq22. This cloned material provides a source from which to isolate other genes located in this part of the X chromosome.

Adult↗
PubMed1994

Trinucleotide repeat expansions and human genetic disease.

Trinucleotide repeat expansions are now a well-established mutational mechanism in human genetic disease. An unstable CAG repeat is known to be responsible for three neurodegenerative disorders: Huntington's disease, spinal and bulbar muscular atrophy and spinocerebellar ataxia type 1. Similarities in the genetics of these diseases, the size of the repeat expansions and the position of the unstable repeat within the gene (when known) suggest a common basis to the observed phenotypes. The cloning of two regions at which chromosome breakage can be induced (FRAXA and FRAXE) has in each case uncovered an unstable CG-rich triplet repeat which becomes methylated when fully expanded. In addition to these two classes of mutation, the presence of an expanded CTG repeat in the 3' untranslated region of a protein kinase causes myotonic dystrophy. The size of the respective expansions, repeat stability, mutational origins and possible mechanisms of action are discussed.

Adult↗