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H Lehrach

Publications and source records attributed to H Lehrach.

At least 73 records · Page 4Linked to original sources
PubMed2000

The centromeric part of the human NK gene complex: linkage of LOX-1 and LY49L with the CD94/NKG2 region.

The natural killer (NK) gene complex is a genomic region containing lectin-type receptor genes. We have established a contig of PAC and BAC clones comprising about 1 Mb of the centromeric part of the NK gene complex. This region extends from the LOX-1 gene, which encodes a receptor for oxidized LDL and was found within 100 kb telomeric of the STS marker D12S77, contains the CD94 and NKG2 NK receptor genes and reaches beyond D12S852 on the proximal side. In this part we have mapped the human LY49L gene, a homologue of the rodent Ly49 genes, which encode important MHC class I receptors for the regulation of NK cell activity in rodents. The LY49L gene is localized 100 to 200 kb centromeric of the NKG2 gene cluster and 300 to 400 kb telomeric of the STS marker D12S841. Genomic sequencing of the complete gene including promoter and intron sequences confirmed that the structure is similar to the mouse Ly49 genes. Screening of several cDNA libraries did not detect any transcripts of putative additional human LY49 genes. In addition, in the course of these studies several EST sequences were localized in the region, one immediately upstream of the LY49L gene.

Antigens, CD↗
PubMed2000

The NKG2 natural killer cell receptor family: comparative analysis of promoter sequences.

The NKG2 receptor family is crucially involved in target cell recognition by natural killer cells and comprises several activating as well as inhibitory family members. We have established approximately 3 kilobases of upstream promoter sequences of the human NKG2-C, -E and -F genes and have carried out a comparative analysis with available NKG2-A sequences. We found extended regions of homology which contain numerous putative transcription factor binding sites conserved in the NKG2 genes. However, variation in Alu insertion among family members has led to promoter structures unique to the respective family members, which could contribute to differences in transcriptional initiation as well as gene-specific regulation.

Base Sequence↗
PubMed2000

Construction and characterization of a gridded cattle BAC library.

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).

Animals↗
PubMed2000

Characterisation and tissue-specific expression of the two keratin subfamilies of intermediate filament proteins in the cephalochordate Branchiostoma.

The cloning of three intermediate filament proteins expressed at the gastrula stage (kl, Y1, X1) extends the size of the IF multigene family of Branchiostoma to at least 13 members. This is one of the largest protein families established for the lancelet. Sequence comparisons indicate five keratin orthologs, three of type I (E1, k1, Y1) and two of type II (E2, D1). This assignment is confirmed by the obligatory heteropolymeric polymerisation behaviour of the recombinant proteins. In line with the hetero-coiled-coil principle IF are formed by any stoichiometric mixture of type I and II keratin orthologs. In spite of the strong sequence drift chimeric IF are formed between K8, a human keratin II, and two of the lancelet type I keratins. We discuss whether the remaining 8 IF proteins reflect three additional and potentially cephalochordate-specific subfamilies. The tissue-specific expression patterns of the 5 keratins and some other IF proteins were analysed by immunofluorescence in the adult. Keratins are primarily present in ectodermally derived tissues. Developmental control of the expression of some IF proteins is observed, but three keratins (k1, Y1, D1) and an additional IF protein (X1) detected at the gastrula stage are expressed throughout the life cycle.

Amino Acid Sequence↗
PubMed2000

Information theoretical probe selection for hybridisation experiments.

MOTIVATION: The choice of probes is an important feature of hybridisation experiments. In this paper we present an algorithm that optimises probes with respect to a training set of sequences based on Shannon entropy as a quality criterion. The practical motivation for our algorithm is oligonucleotide fingerprinting, a method for the simultaneous identification of sequences (cDNA or genomic DNA) by their hybridisation tags according to a set of short probes such as octamers, although the algorithm is of course not restricted to that application. RESULTS: We can show that our method is superior to the selection of probes according to their frequencies, which is a widely used strategy, and to randomly chosen probe sets. The quality of probe sets is assessed by a simulation pipeline that entails the set of probes as a simulation parameter. The performance of probe sets trained on sequences from different organisms shows additionally that probes should be chosen with regard to the organism under analysis. Case studies are presented on how constraints (G+C-content, complexity of the individual probes) influence the selection process. AVAILABILITY: A description of the oligonucleotide fingerprinting pipeline is published on our web-page http://www.molgen.mpg.de/ approximately ag_onf/met.htm. An executable of the algorithm and probe lists designed for human and rodents can be downloaded from the ftp-site ftp://ftp.molgen.mpg.de/pub/mpimg/probe_design/.

Algorithms↗
PubMed2000

Tissue gene expression analysis using arrayed normalized cDNA libraries.

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.

Algorithms↗
PubMed1999

Toward the gene catalogue of sea urchin development: the construction and analysis of an unfertilized egg cDNA library highly normalized by oligonucleotide fingerprinting.

We describe the use of oligonucleotide fingerprinting for the generation of a normalized cDNA library from unfertilized sea urchin eggs and report the preliminary analysis of this library, which resulted in the establishment of a partial gene catalogue of the sea urchin egg. In an analysis of 21,925 cDNA clones by hybridization with 217 oligonucleotide probes, we were able to identify 6291 clusters corresponding to different transcripts, ranging in size from 1 to 265 clones. This corresponds to an average 3.5-fold normalization of the starting library. The normalized library represents about one-third of all genes expressed in the sea urchin egg. To generate sequence information for the transcripts represented by the clusters, representative clones selected from 711 clusters were sequenced. The construction and preliminary analysis of the normalized library are the first steps in the assembly of an increasingly complete collection of maternal genes expressed in the sea urchin egg, which will provide a number of insights into the early development of this well-characterized model organism.

Animals↗
PubMed1999

Aggregation of truncated GST-HD exon 1 fusion proteins containing normal range and expanded glutamine repeats.

We have shown previously by electron microscopy that the purified glutathione S-transferase (GST)-Huntington's disease (HD) exon 1 fusion protein with 51 glutamine residues (GST-HD51) is an oligomer, and that site-specific proteolytic cleavage of this fusion protein results in the formation of insoluble more highly ordered protein aggregates with a fibrillar or ribbon-like morphology (E. Scherzinger et al. (1997) Cell 90, 549-558). Here we report that a truncated GST HD exon 1 fusion protein with 51 glutamine residues, which lacks the proline-rich region C-terminal to the polyglutamine (polyQ) tract (GST-HD51 delta P) self-aggregates into high-molecular-mass protein aggregates without prior proteolytic cleavage. Electron micrographs of these protein aggregates revealed thread-like fibrils with a uniform diameter of ca. 25 nm. In contrast, proteolytic cleavage of GST-HD51 delta P resulted in the formation of numerous clusters of high-molecular-mass fibrils with a different, ribbon-like morphology. These structures were reminiscent of prion rods and beta-amyloid fibrils in Alzheimer's disease. In agreement with our previous results with full-length GST-HD exon 1, the truncated fusion proteins GST-HD20 delta P and GST-HD30 delta P did not show any tendency to form more highly ordered structures, either with or without protease treatment.

Cloning, Molecular↗
PubMed1999

Protein microarrays for gene expression and antibody screening.

Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.

Antibody Specificity↗
PubMed1999

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.

Animals↗
PubMed1999

Sequence analysis of an amphioxus cosmid containing a gene homologous to members of the aldo-keto reductase gene superfamily.

To gain an insight into vertebrate genome evolution, we have analysed the organization of an approximately 40-kb genomic clone of an amphioxus (Branchiostoma floridae) cosmid library. Amphioxus is considered as being the last non-vertebrate relative to vertebrates. Sequencing and analysis of the above clone using three different exon prediction programs (Grail, GenScan, Mzef) have led to the identification of a gene of the aldo-keto reductase family as well as further exons that gave a significant database match to known genes.

Alcohol Oxidoreductases↗
PubMed1999

Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.

Amino Acid Sequence↗
PubMed1999

Analysis of the spermine synthase gene region in Fugu rubripes, Tetraodon fluviatilis, and Danio rerio.

A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22. 1. We show that whereas the human gene spans a genomic region of 54 kb, the Fugu rubripes gene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalent F. rubripes region. A cosmid clone containing the F. rubripes gene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of the F. rubripes, Tetraodon fluviatilis, mouse, human, and Danio rerio 5'UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compact F. rubripes genome. Analysis of a D. rerio PAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.

Alternative Splicing↗
PubMed1999

Transcription mapping in a medulloblastoma breakpoint interval and Smith-Magenis syndrome candidate region: identification of 53 transcriptional units and new candidate genes.

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith-Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith-Magenis syndrome.

Brain Neoplasms↗
PubMed1999

A method of one-step enzyme labelling of short oligonucleotide probes for filter hybridisation.

Here we describe a method of labelling short oligonucleotide probes with enzyme without purification or chemical modifications. Biotinylated oligonucleotides as short as 10 nt are coupled with streptavidin-conjugated enzyme, hybridised and detected with enzyme-triggered chemiluminescence. The detection of hybridisation signal is linear for two orders of magnitude of target dilution. It is shown to be comparable in sensitivity with standard procedures and with radioactive detection. The method is quick, simple and has potential for automation of large-scale oligo-nucleotide hybridisation and multiplex sequencing.

Alkaline Phosphatase↗
PubMed1999

IXDB, an X chromosome integrated database (update).

Chromosome specific databases are an important research tool as they integrate data from different directions, such as genetic and physical mapping data, expression data, sequences etc. They supplement the genome-wide repositories in molecular biology, such as GenBank, Swiss-Prot or OMIM, which usually concentrate on one type of information. The Integrated X Chromosome Database (IXDB, http://ixdb.mpimg-berlin-dahlem.mpg.de/) is a repository for physical mapping data of the human X chromosome and aims at providing a global view of genomic data at a chromosomal level. We present here an update of IXDB which includes schema extensions for storing submaps and sequence information, additional links to external databases, and the integration of an increasing number of physical and transcript mapping data. The gene data was completely updated according to the approved gene symbols of the HUGO Nomenclature Committee. IXDB receives over 1000 queries per month, an indication that its content is valuable to researchers seeking mapping data of the human X chromosome.

Chromosome Mapping↗
PubMed1999

Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation.

The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large-gel two-dimensional electrophoresis. Two-dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer-assisted image analysis and treatment with our spot detection program. The resulting two-dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/ humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) after in-gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two-dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post-translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the interesting finding that the size of spot families, i.e., the extent of modification or degradation of a protein, can be genetically determined. The investigation presented is a first step towards a systematic analysis of the proteome of the mouse. Proteome analysis was shown to become more efficient, and, at the same time, linked to the genome, by combining protein analytical and genetic methods.

Animals↗