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H Lehrach

Publications and source records attributed to H Lehrach.

At least 91 records · Page 5Linked to original sources
PubMed1999

Characterization of the Hydra lamin and its gene: A molecular phylogeny of metazoan lamins.

We report sequences for nuclear lamins from the teleost fish Danio and six invertebrates. These include two cnidarians (Hydra and Tealia), one priapulid, two echinoderms, and the cephalochordate Branchiostoma. Combining these results with earlier data on Drosophila, Caenorhabditis elegans, and various vertebrates, the following conclusions on lamin evolution can be drawn. First, all invertebrate lamins resemble in size the vertebrate B-type lamin. Second, all lamins described previously for amphibia, birds and mammals as well as the first lamin of a fish, characterized here, show a cluster of 7 to 12 acidic residues in the tail domain. Since this acidic cluster is absent from all invertebrate lamins including that of the cephalochordate Branchiostoma, it was acquired with the vertebrate lineage. The larger A-type lamin of differentiated cells must have arisen subsequently by gene duplication and insertion of an extra exon. This extra exon of the vertebrate A-lamins is the only major change in domain organization in metazoan lamin evolution. Third, the three introns of the Hydra and Priapulus genes correspond in position to the last three introns of vertebrate B-type lamin genes. Thus the entirely different gene organization of the C. elegans and Drosophila Dmo genes seems to reflect evolutionary drift, which probably also accounts for the fact that C. elegans has the most diverse lamin sequence. Finally we discuss the possibility that two lamin types, a constitutively expressed one and a developmentally regulated one, arose independently on the arthropod and vertebrate lineages.

Amino Acid Sequence↗
PubMed1999

Linkage of the NKG2 and CD94 receptor genes to D12S77 in the human natural killer gene complex.

The human natural killer (NK) gene complex is located on the short arm of chromosome 12 and contains a number of genes encoding C-type lectin receptors important for natural killer cell function. Among these are CD94 and the five NKG2 genes. The CD94 protein associates with different NKG2 isoforms to heterodimeric receptors which function to inhibit or trigger cytotoxicity of NK cells depending on the NKG2 isoform. We selected two yeast artificial chromosome clones comprising approximately 1.5 Mb of the NK gene complex and established a contig of underlying P1-derived artificial chromosome clones containing all NKG2 and the CD94 genes. A detailed analysis shows that all six genes are found within a region of 100 to 200 kilobases proximal of the marker D12S77. The gene order established is D12S77 - CD94 - NKG2D - NKG2F - NKG2E - NKG2C - NKG2A. The NKG2 genes are of identical transcriptional orientation, whereas the CD94 gene is placed in opposite orientation. The tight genomic linkage of these genes and the identical orientation of the NKG2 genes suggest coordinate regulation of expression during the differentiation of natural killer cells.

Antigens, CD↗
PubMed1999

A 5x genome coverage bovine BAC library: production, characterization, and distribution.

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

Animals↗
PubMed1999

Construction and analysis of arrayed cDNA libraries.

For any attempt to understand the biology of an organism the incorporation of a cDNA-based approach is unavoidable, because it is a major approach to studying gene function. The complete sequence of the genome alone is not sufficient to understand any organism; its gene regulation, expression, splice variation, posttranslational modifications, and protein-protein interactions all need to be addressed. Because the majority of vertebrate genes have probably been identified as ESTs the next stage of the Human Genome Project is attributing functional information to these sequences. In most cases hybridization-based approaches on arrayed pieces of DNA represent the most efficient way to study the expression level and splicing of a gene in a given tissue. Similar technology, now being applied at the protein level using protein expression libraries, high-density protein membranes, and antibody screening, should allow studies of protein localization and modifications. Coupled to these approaches is the use of technologies, which although lacking the highly parallel nature of hybridization, can potentially characterize large numbers of samples individually and with high accuracy. Automated gel-based DNA sequencing is an example of such a technique; protein sequencing and mass fingerprinting are further examples. In the case of mass spectroscopic analysis, the speed and sensitivity are vastly superior to that of gel-based approaches; however, the preparation of samples is more tedious. Our laboratory is developing a system to characterize DNA samples by mass spectrometry, allowing more rapid genotyping than is currently possible using gel-based technologies ([symbol: see text]. Gut, [symbol: see text]. Berlin and H. Lehrach, personal communication, 1998). Such technology would make information on gene polymorphisms widely accessible. Data generated using all of these techniques at the DNA and protein level will be connected by both protein expression libraries and database comparisons; finally, two hybrid library screens will identify many of the protein-protein interactions, linking genes together. In this way we will start to understand the interplay between genes on a global scale, both at the level of molecular interaction and the biological processes they regulate.

Alternative Splicing↗
PubMed1999

A complete BAC-based physical map of the Arabidopsis thaliana genome.

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.

Arabidopsis↗
PubMed1999

Folding of oligoglutamines: a theoretical approach based upon thermodynamics and molecular mechanics.

Folding of oligoglutamine chains of different lengths is of crucial interest for exploring the molecular mechanisms of Huntington's disease. A simple oligoglutamine model based upon the Flory-Huggins theory of polymer solutions demonstrates a random coil instability in chains containing more than 40 glutamine residues with respect to beta-sheet formation. This is in striking quantitative agreement with biochemical results on the chain length dependence of polyglutamine aggregation in vivo and in vitro, as well as with clinical data on the polyglutamine chain length dependence of the onset of Huntington's disease. Furthermore, a detailed molecular-mechanical investigation of a polypeptide chain carrying 40 glutamine residues was performed. Two possible folding modes of such an oligoglutamine chain were revealed: a) a beta-hairpin and b) a highly compact random coil entity stabilized by a wealth of H-bonds among the glutamine side chains. A possible role of these folding modes in polyglutamine aggregation, as well as in the onset of Huntington's disease, is discussed.

Amino Acid Sequence↗
PubMed1999

AIRE encodes a nuclear protein co-localizing with cytoskeletal filaments: altered sub-cellular distribution of mutants lacking the PHD zinc fingers.

The gene responsible for autoimmune polyendocrino-pathy candidiasis ectodermal dystrophy (APECED) recently has been positionally cloned to 21q22.3. This novel gene, AIRE, encodes for a predicted 57.7 kDa protein featuring two PHD-type zinc fingers shared by other proteins involved in chromatin-mediated tran-scriptional regulation. APECED is an autosomal recessive condition characterized by multiple polyendocrinopathies, and the typical triad of APECED symptoms includes hypoparathyroidism, primary adrenocortical failure and chronic mucocutaneous candidiasis. The aetiology of APECED is linked directly to mutations within the coding region of AIRE. These mutations are predicted to lead to truncated forms of the protein lacking at least one of the PHD zinc fingers. In this study, we have investigated the sub-cellular localization of AIRE expressed transiently in COS cells and fibroblasts. We found that AIRE has a dual nuclear and cytoplasmic localization. The wild-type protein is directed to speckled domains in the nucleus and also shows co-localization with cytoskeletal filaments. N-terminal AIRE fragments deleted for the PHD domain show altered nuclear localization, suggesting that the APECED mutations may elicit their primary effects in the nucleus.

Animals↗
PubMed1999

Large-scale clustering of cDNA-fingerprinting data.

Clustering is one of the main mathematical challenges in large-scale gene expression analysis. We describe a clustering procedure based on a sequential k-means algorithm with additional refinements that is able to handle high-throughput data in the order of hundreds of thousands of data items measured on hundreds of variables. The practical motivation for our algorithm is oligonucleotide fingerprinting-a method for simultaneous determination of expression level for every active gene of a specific tissue-although the algorithm can be applied as well to other large-scale projects like EST clustering and qualitative clustering of DNA-chip data. As a pairwise similarity measure between two p-dimensional data points, x and y, we introduce mutual information that can be interpreted as the amount of information about x in y, and vice versa. We show that for our purposes this measure is superior to commonly used metric distances, for example, Euclidean distance. We also introduce a modified version of mutual information as a novel method for validating clustering results when the true clustering is known. The performance of our algorithm with respect to experimental noise is shown by extensive simulation studies. The algorithm is tested on a subset of 2029 cDNA clones coming from 15 different genes from a cDNA library derived from human dendritic cells. Furthermore, the clustering of these 2029 cDNA clones is demonstrated when the entire set of 76,032 cDNA clones is processed.

Algorithms↗
PubMed1999

Panel of microsatellite markers for whole-genome scans and radiation hybrid mapping and a mouse family tree.

To facilitate whole-genome scan experiments, we selected a panel of 128 microsatellite markers on the basis of spacing and polymorphism in the strains DBA/2, BALB/c, AKR, C57BL/6, C57BL/10, A/J, C3H, 129/J, SJL/J, JF1, and PWB. Many of the primer pairs were redesigned for better performance. The last four strains were not characterized previously using these markers. JF1 and PWB are particularly interesting for intersubspecific crosses offering high polymorphism. We provide allele size data for the markers on these strains and add them to the emerging radiation hybrid framework map, which is not continuous except for chromosome 17 and 13. Information on the interrelationships of strains is useful both because of the importance of polymorphism in designing crosses and the background in assessing phenotypes. Microsatellites offer a widely dispersed, selectively neutral set of characters that lends itself conceptually to parsimony methods of analysis. The microsatellite allele size data were recoded as binary discrete characters in such a way that adjacent sizes differ by one step. Trees were generated using a Wagner parsimony method. As expected, the non-Mus domesticus strains, PWB (musculus) and JF1 (molossinus), are excluded from the domesticus strains. Among the domesticus strains, C57BL/6 and C57BL/10 (derived from the same founding pair) form a strongly supported group, as do C3H, A/J, and BALB/c (derived from the Bagg albino stock). No unique branching order for SJL/J, AKR, and DBA/2 is strongly supported, which may reflect a complicated history. Strain 129/J is clearly placed as the most deeply diverged of the domesticus strains represented.

Alleles↗
PubMed1999

Zebrafish tbx-c functions during formation of midline structures.

Several genes containing the conserved T-box region in invertebrates and vertebrates have been reported recently. Here, we describe three novel members of the T-box gene family in zebrafish. One of these genes, tbx-c, is studied in detail. It is expressed in the axial mesoderm, notably, in the notochordal precursor cells immediately before formation of the notochord and in the chordoneural hinge of the tail bud, after the notochord is formed. In addition, its expression is detected in the ventral forebrain, sensory neurons, fin buds and excretory system. The expression pattern of tbx-c differs from that of the other two related genes, tbx-a and tbx-b. The developmental role of tbx-c has been analysed by overexpression of the full-length tbx-c mRNA and a truncated form of tbx-c mRNA, which encodes the dominant-negative Tbx-c. Overexpression of tbx-c causes expansion of the midline mesoderm and formation of ectopic midline structures at the expense of lateral mesodermal cells. In dominant-negative experiments, the midline mesoderm is reduced with the expansion of lateral mesoderm to the midline. These results suggest that tbx-c plays a role in formation of the midline mesoderm, particularly, the notochord. Moreover, modulation of tbx-c activity alters the development of primary motor neurons. Results of in vitro analysis in zebrafish animal caps suggest that tbx-c acts downstream of early mesodermal inducers (activin and ntl) and reveal an autoregulatory feedback loop between ntl and tbx-c. These data and analysis of midline (ntl-/- and flh-/-) and lateral mesoderm (spt-/-) mutants suggest that tbx-c may function during formation of the notochord.

Amino Acid Sequence↗
PubMed1999

The mouse Aire gene: comparative genomic sequencing, gene organization, and expression.

Mutations in the human AIRE gene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; OMIM 240300). Previously, we have cloned hAIRE and shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization of Aire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of the AIRE gene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT-PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.

Amino Acid Sequence↗
PubMed1999

A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2 based on high- throughput nonisotopic library screenings.

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.

Chromosome Mapping↗
PubMed1998

Characterization of the mouse Src homology 3 domain gene Sh3d2c on Chr 7 demonstrates coexpression with huntingtin in the brain and identifies the processed pseudogene Sh3d2c-ps1 on Chr 2.

Formation of intracellular protein complexes is often mediated by Src homology 3 domain-containing proteins interacting with proline-rich target sequences on other proteins. The Sh3d2c gene or its rat/human orthologs have been implicated in synaptic vesicle recycling due to interaction with dynamin I and synaptojanin in nerve terminals. In a yeast two-hybrid system, association with a huntingtin fragment containing an elongated stretch of polyglutamines was observed recently. By genetic mapping and fluorescence in situ hybridization we demonstrate the localization of Sh3d2c on mouse chromosome 7. A processed pseudogene of Sh3d2c, Sh3d2c-ps1, was identified and mapped to mouse chromosome 2. Using RNA in situ hybridization, we show that Sh3d2c is transcribed in various regions of the brain. The striatum, hippocampus, cortex, basal hypothalamus, brain stem, and cerebellum are the most prominent sites of expression. Because huntingtin and Sh3d2c are coexpressed in most regions of the brain, it can be speculated that there is a link between the association of huntingtin/Sh3d2c and the pathogenesis of Huntington disease.

Animals↗
PubMed1998

Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects.

Large-scale genomic sequencing projects generally rely on random sequencing of shotgun clones, followed by different gap closing strategies. To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select optimal shotgun clone sets prior to sequencing. Both computer simulations and experimental results, obtained from five PAC-derived shotgun libraries spanning 535 kb of the 17p11.2 region of the human genome, demonstrate that at least a 2-fold reduction in the number of sequence reads required to sequence an individual genomic clone (cosmid, PAC, etc.) can be achieved. Treatment of clone contigs with significant clone overlaps will allow an even greater reduction.

Chromosomes, Human, Pair 17↗
PubMed1998

A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.

Antibodies, Monoclonal↗