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Biomedical subjects

H Lehrach

Publications and source records attributed to H Lehrach.

At least 55 records · Page 3Linked to original sources
PubMed2000

A sea urchin genome project: sequence scan, virtual map, and additional resources.

Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains approximately 27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10(5) clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12, 000 sequences) have been reported to GenBank and are accessible on public web sites.

Aging↗
PubMed2000

Prestructured MALDI-MS sample supports.

Prestructured MALDI-MS sample supports have been developed that simplify high-throughput analysis of biomolecules and improve the detection sensitivity. The mass spectrometric sample support is coated with a thin layer of hydrophobic Teflon that carries an array of 200-microm gold spots, which provide hydrophilic sample anchors. Each transferred sample droplet contacts one anchor, on top of which, after solvent evaporation, the sample is exclusively deposited due to the strongly water repellent nature of the Teflon surface. The initial matrix concentration is kept low, enabling sample up-concentration by more than 2 orders of magnitudes before crystallization commences. As a result, the detection sensitivity is improved as documented by mass spectra recorded from 100 amol of various peptides, 1 fmol of a DNA 20 mer, and 5 fmol of a 130 bp PCR product. Size and spacing of the hydrophilic anchors are optimized for MALDI-MS performance (sample spot size approximately = laser irradiation spot size), for short analysis times (predetermined sample coordinates), and for high throughput sample preparation (sample anchor array according to the 1536 microtiter plate format).

DNA↗
PubMed2000

Improvements in protein identification by MALDI-TOF-MS peptide mapping.

A new strategy for identifying proteins in sequence data-bases by MALDI-MS peptide mapping is reported. The strategy corrects for systematic deviations of determined peptide molecular masses using information contained in the opened database and thereby renders unnecessary internal spectrum calibration. As a result, data acquisition is simplified and less error prone. Performance of the new strategy is demonstrated by identification of a set of recombinant, human cDNA expression products as well as native proteins isolated from crude mouse brain extracts by 2-D electrophoresis. Using one set of calibration constants for the mass spectrometric analyses, 20 proteins were identified without applying any molecular weight restrictions, which was not possible without data correction. A sequence database search program has been written that performs all necessary calculations automatically, access to which will be provided to the scientific community in the Internet.

Animals↗
PubMed2000

An algorithm for clustering cDNA fingerprints.

Clustering large data sets is a central challenge in gene expression analysis. The hybridization of synthetic oligonucleotides to arrayed cDNAs yields a fingerprint for each cDNA clone. Cluster analysis of these fingerprints can identify clones corresponding to the same gene. We have developed a novel algorithm for cluster analysis that is based on graph theoretic techniques. Unlike other methods, it does not assume that the clusters are hierarchically structured and does not require prior knowledge on the number of clusters. In tests with simulated libraries the algorithm outperformed the Greedy method and demonstrated high speed and robustness to high error rate. Good solution quality was also obtained in a blind test on real cDNA fingerprints.

Algorithms↗
PubMed2000

Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy.

The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease (HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. On the other hand, potential inhibitors of amyloid-beta formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory effect on huntingtin aggregation in vitro. The results obtained by the filtration assay were confirmed by electron microscopy, SDS/PAGE, and MS. Furthermore, cell culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in transiently transfected COS cells. Together, these findings contribute to a better understanding of the mechanism of huntingtin fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD.

Animals↗
PubMed2000

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Base Sequence↗
PubMed2000

Normalization strategies for cDNA microarrays.

Multiple Arabidopsis thaliana clones from an experimental series of cDNA microarrays are evaluated in order to identify essential sources of noise in the spotting and hybridization process. Theoretical and experimental strategies for an improved quantitative evaluation of cDNA microarrays are proposed and tested on a series of differently diluted control clones. Several sources of noise are identified from the data. Systematic and stochastic fluctuations in the spotting process are reduced by control spots and statistical techniques. The reliability of slide to slide comparison is critically assessed within the statistical framework of pattern matching and classification.

Arabidopsis↗
PubMed2000

Hailey-Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca(2+) pump.

Hailey-Hailey disease (HHD) is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Previous genetic linkage studies localized the gene to a 5 cM interval on human chromosome 3q21. After reducing the disease critical region to <1 cM, we used a positional cloning strategy to identify the gene ATP2C1, which is mutated in HHD. ATP2C1 encodes a new class of P-type Ca(2+)-transport ATPase, which is the homologue for the rat SPLA and the yeast PMR1 medial Golgi Ca(2+)pumps and is related to the sarco(endo)plasmic calcium ATPase (SERCA) and plasma membrane calcium ATPase (PCMA) families of Ca(2+)pumps. The predicted protein has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. ATP2C1 produces two alternative splice variants of approximately 4.5 kb encoding predicted proteins of 903 and 923 amino acids. We identified 13 different mutations, including nonsense, frameshift insertion and deletions, splice-site mutations, and non-conservative missense mutations. This study demonstrates that defects in ATP2C1 cause HHD and together with the recent identification of ATP2A2 as the defective gene in Darier's disease, provide further evidence of the critical role of Ca(2+)signaling in maintaining epidermal integrity.

Amino Acid Sequence↗
PubMed2000

A human cDNA library for high-throughput protein expression screening.

We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.

Algorithms↗
PubMed2000

A novel procedure for efficient genotyping of single nucleotide polymorphisms.

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.

Amyloid beta-Protein Precursor↗
PubMed2000

Interspersed repetitive sequence (IRS)-PCR for typing of whole genome radiation hybrid panels.

The typing of a radiation hybrid (RH) panel is generally achieved using a unique primer pair for each marker. We here describe a complementing approach utilizing IRS-PCR. Advantages of this technology include the use of a single universal primer to specify any locus, the rapid typing of RH lines by hybridization, and the conservative use of hybrid DNA. The technology allows the mapping of a clone without the requirement for STS generation. To test the technique, we have mapped 48 BAC clones derived from mouse chromosome 12 which we mostly identified using complex probes. As mammalian genomes are repeat-rich, the technology can easily be adapted to species other than mouse.

Animals↗
PubMed2000

Incidental prenatal detection of an Xp deletion using an anonymous primer pair for fetal sexing.

We report on the incidental prenatal detection of an interstitial X-chromosomal deletion in a male fetus and his mother by fetal sexing with a primer pair recognizing an X-Y homologous locus (DXYS19), formerly unassigned on the X chromosome. The proband asked for prenatal diagnosis because of her elevated age and risk of Duchenne muscular dystrophy (DMD). Prior to molecular genetic testing for DMD, fetal sexing was carried out on DNA prepared from cultured amniocytes. PCR analysis revealed the expected Y-chromosomal product, but did not show the constitutive X-chromosomal fragment. The absence of the X-chromosomal fragment in the fetus and on one X chromosome of the mother was confirmed by Southern hybridization of HindIII restricted DNA with probe pJA1165 (DXYS19). DXYS19X was mapped to Xp22.3 by combining several approaches, including: (1) analysis of somatic cell hybrid lines containing different fragments of the human X chromosome; (2) Southern hybridization of a yeast artificial chromosome (YAC)-filter panel provided by the Resource Center/Primary Database (RZPD); (3) FISH analysis; and (4) re-evaluation of two patients with interstitial deletions in Xp22.3. The extent of the deletion in the fetus was estimated by further markers from Xp22.3 and found to include the STS gene. Mental retardation could not be excluded since some mentally retarded patients exhibit overlapping deletions.

Adult↗
PubMed2000

A system for dual protein expression in Pichia pastoris and Escherichia coli.

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.

Brain Chemistry↗
PubMed2000

Induction of putative tumor-suppressing genes in Rat-1 fibroblasts by oncogenic Raf-1 as evidenced by robot-assisted complex hybridization.

The growth factor receptor-dependent protein kinase Raf-1 is activated by GTP-bound Ras, thereby activating the mitogen-activated protein kinase pathway. To study the role of Raf in transformation we transduced Rat-1 cells with a tetracycline-regulatable retroviral vector encoding the constitutively active oncogenic C-terminal fragment of the human Raf-1 protein. Using subtractive hybridization of mRNAs from induced and noninduced cells and robot-assisted screening by complex hybridization, Raf-induced genes with various different characteristics of induction were investigated. Among the strongly induced genes were those involved in carcinogenesis such as metalloproteinases 3, 10 and 13, cathepsin L, ornithine decarboxylase, and putative tumor-suppressing genes such as monocyte chemoattracting protein 1, interferon-induced protein 10, a recently identified 2'-5' oligoadenylate synthetase-like protein, and plasminogen activator inhibitor type 2. Other components of the plasminogen activator system were not induced. Plasminogen activator inhibitor type 2 is a down-regulator of the proteolytic cascade consisting of various metalloproteinases, some of which are induced by a carboxy-terminal Raf mutant (RafCT). In conclusion, RafCT induces factors which act in a conflicting manner in respect of carcinogenesis, especially within the proteolytic system of the extracellular matrix.

Animals↗
PubMed2000

Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate.

mRNA injection into the ventral blastomeres of Xenopus embryos of mRNA encoding Wnt pathway genes induces a secondary axis with complete head structures. To identify target genes of the pre-MBT dorsalization pathway that might be responsible for head formation in zebrafish, we have cloned zebrafish dickkopf1 (dkk1), which is expressed in tissues implicated in head patterning. We found that dkk1 blocks the post-MBT Wnt signaling and dkk1 is a target of the pre-MBT Wnt signaling. Dkk1 overexpression in the prechordal plate suggests that Dkk1, secreted from the prechordal plate, expands the forebrain at the expense of the midbrain in the anterior neural plate. Furthermore, dkk1 acts in parallel to the homeobox gene bozozok and bozozok is required for the maintenance of dkk1 expression. The nodal gene squint is also required for the maintenance of dkk1 expression. Among the mutually dependent target genes of the pre-MBT Wnt signaling, dkk1 plays an important role in patterning the anterior head of zebrafish.

Amino Acid Sequence↗
PubMed2000

Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.

We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.

Animals↗
PubMed2000

Protein arrays for gene expression and molecular interaction screening.

The array format has revolutionised biomedical experimentation and diagnostics, enabling ordered high-throughput analysis. During the past decade, classic solid phase substrates, such as microtitre plates, membrane filters and microscopic slides, were turned into high-density, chip-like structures. The concept of the arrayed library was central to this development which now extends from DNA to protein. The new and versatile protein array technology allows high-throughput screening for gene expression and molecular interactions. As a major platform for functional genomics, it is already on its way into medical diagnostics.

Bacterial Proteins↗