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H Lehrach

Publications and source records attributed to H Lehrach.

At least 19 recordsLinked to original sources
PubMed2006

High-throughput RNA interference in functional genomics.

RNA interference (RNAi) refers to post-transcriptional silencing of gene expression as a result of the introduction of double-stranded RNA into cells. The application of RNAi in experimental systems has significantly accelerated elucidation of gene functions. In order to facilitate large-scale functional genomics studies using RNAi, several high-throughput approaches have been developed based on microarray or microwell assays. The recent establishment of large libraries of RNAi reagents combined with a variety of detection assays has further improved the performance of functional genome-wide screens in mammalian cells.

Animals↗
PubMed2005

High-throughput subcellular protein localization using cell arrays.

Accomplishment of the human and mouse genome projects resulted in accumulation of extensive gene sequence information. However, the information about the biological functions of the identified genes remains a bottleneck of the post-genomic era. Hence, assays providing simple functional information, such as localization of the protein within the cell, can be very helpful in the elucidation of its function. Transfected cell arrays offer a robust platform for protein localization studies. Open reading frames of unknown genes can be linked to a His6-tag or GFP (green fluorescent protein) reporter in expression vectors and subsequently transfected using the cell array. Cellular localization of the transfected proteins is detected either by specific anti-His-tag antibodies or directly by fluorescence of the GFP fusion protein and by counterstaining with organelle-specific dyes. The high throughput of the method in terms of information provided for every single experiment makes this approach superior to classical immunohistological methods for protein localization.

Animals↗
PubMed2004

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Animals↗
PubMed2004

Establishment of T cell-specific and natural killer cell-specific unigene sets: towards high-throughput genomics of leukaemia.

We report the establishment of highly non-redundant unigene sets consisting of cDNA clones derived from T lymphocytes and natural killer cells. Each set consists of 10 506 and 13 409 clones, respectively, arrayed on nylon membranes in duplicate. The sets provide an excellent tool for genome-wide gene expression analysis studies in immunology research.

Blotting, Northern↗
PubMed2003

Generation of Arabidopsis protein chips for antibody and serum screening.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.

Animals↗
PubMed2002

The genome sequence of Schizosaccharomyces pombe.

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.

Base Sequence↗
PubMed2001

Statistical evaluation of differential expression on cDNA nylon arrays with replicated experiments.

In this paper we focus on the detection of differentially expressed genes according to changes in hybridization signals using statistical tests. These tests were applied to 14 208 zebrafish cDNA clones that were immobilized on a nylon support and hybridized with radioactively labeled target mRNA from wild-type and lithium-treated zebrafish embryos. The methods were evaluated with respect to 16 control clones that correspond to eight different genes which are known to be involved in dorso-ventral axis specification. Moreover, 4608 Arabidopsis thaliana clones on the same array were used to judge statistical significance of expression changes and to control the false positive rates of the test decisions. Utilizing this special array design we show that differential expression of a high proportion of cDNA clones (15/16) and the respective genes (7/8) were identified, with a false positive error of <5% using the constant control data. Furthermore, we investigated the influence of the number of repetitions of experiments on the accuracy of the procedures with experimental and simulated data. Our results suggest that the detection of differential expression with repeated hybridization experiments is an accurate and sensitive way of identifying even small expression changes (1:1.5) of a large number of genes in parallel.

Animals↗
PubMed2001

Distinct expression patterns of two zebrafish homologues of the human APP gene during embryonic development.

The human amyloid protein precursor (APP) gene correlates with early onset of Alzheimer's disease in humans. We have identified two APP homologues in zebrafish, which we call appa and appb. They show a high degree of identity to human APP particularly in the beta APP42 and the transmembrane domain. Widespread expression of both appa and appb was detected from mid-gastrulation until the bud stage. During segmentation, the two genes diverged in their pattern of expression: at 14 h post-fertilisation (hpf) and 18 hpf both genes were expressed rostrally in the prospective CNS, but only appa was found caudally in the paraxial segmental plate and presomitic mesoderm, excluding the midline. In contrast, appb was found caudally in the neural rod at 14 hpf and the developing spinal cord at 18 hpf. Later, at 24 hpf both genes shared common expression domains, namely the telencephalon, the ventral diencephalon, the trigeminal ganglia, and the posterior lateral line ganglia. Unique expression domains for appa were the lens, the otic vesicles and the somites, while appb was expressed in a serially repeated set of nuclei within the hindbrain, the ventral mesencephalon and the motoneurones of the developing spinal cord.

Amyloid beta-Protein Precursor↗
PubMed2001

UniGene cDNA array-based monitoring of transcriptome changes during mouse placental development.

The placenta is a highly specialized organ essential for embryonic growth and development. Here, we have applied cDNA subtraction between extraembryonic tissues of early- (day 7.5 of gestation) and late-stage embryos (day 17.5) to generate stage-specific cDNA pools that were used for screening of high-density mouse UniGene cDNA arrays containing 25,000 clones. A total of 638 clones were identified, 488 with the e7.5-specific probe and 150 with the e17.5-specific probe. Importantly, 363/638 (56.9%) of the hybridizing clones were not known to be expressed during placental development before. Differential regulation was confirmed by Northern blot and in situ hybridization for a total of 44/44 of positive clones. Thus, this combination of cDNA subtraction and array hybridization was highly successful for identification of genes expressed and regulated during placental development. These included growth factors and receptors, components of the transcriptional and translational machinery, cell cycle regulators, molecular chaperones, and cytoskeletal elements. The extensive in situ hybridization analysis revealed extraembryonic structures with a high density of differentially expressed genes, most strikingly the ectoplacental cone and the spongiotrophoblast. This large-scale identification of genes regulated during placentogenesis is extremely useful to further elucidate the molecular basis of extraembryonic development.

Animals↗
PubMed2001

The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis.

The huntingtin interacting protein (HIP1) is enriched in membrane-containing cell fractions and has been implicated in vesicle trafficking. It is a multidomain protein containing an N-terminal ENTH domain, a central coiled-coil forming region and a C-terminal actin-binding domain. In the present study we have identified three HIP1 associated proteins, clathrin heavy chain and alpha-adaptin A and C. In vitro binding studies revealed that the central coiled-coil domain is required for the interaction of HIP1 with clathrin, whereas DPF-like motifs located upstream to this domain are important for the binding of HIP1 to the C-terminal 'appendage' domain of alpha-adaptin A and C. Expression of full length HIP1 in mammalian cells resulted in a punctate cytoplasmic immunostaining characteristic of clathrin-coated vesicles. In contrast, when a truncated HIP1 protein containing both the DPF-like motifs and the coiled-coil domain was overexpressed, large perinuclear vesicle-like structures containing HIP1, huntingtin, clathrin and endocytosed transferrin were observed, indicating that HIP1 is an endocytic protein, the structural integrity of which is crucial for maintenance of normal vesicle size in vivo.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse.

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:

Animals↗
PubMed2001

Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder with no effective treatment. Geldanamycin is a benzoquinone ansamycin that binds to the heat shock protein Hsp90 and activates a heat shock response in mammalian cells. In this study, we show by using a filter retardation assay and immunofluorescence microscopy that treatment of mammalian cells with geldanamycin at nanomolar concentrations induces the expression of Hsp40, Hsp70 and Hsp90 and inhibits HD exon 1 protein aggregation in a dose-dependent manner. Similar results were obtained by overexpression of Hsp70 and Hsp40 in a separate cell culture model of HD. This is the first demonstration that huntingtin protein aggregation in cells can be suppressed by chemical compounds activating a specific heat shock response. These findings may provide the basis for the development of a novel pharmacotherapy for HD and related glutamine repeat disorders.

Amino Acid Sequence↗
PubMed2001

A CE-MALDI interface based on the use of prestructured sample supports.

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.

Brain Chemistry↗
PubMed2001

Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics.

We present a new MALD1 sample preparation technique for peptide analysis using the matrix alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and prestructured sample supports. The preparation integrates sample purification, based on the affinity of microcrystalline CHCA for peptides, thereby simplifying the analysis of crude peptide mixtures. Enzymatic digests can thus be prepared directly, without preceding purification. Prepared samples are homogeneous, facilitating automatic spectra acquisition. This method allows preparation of large numbers of samples with little effort and without the need for automation. These features make the described preparation suitable for cost-efficient high-throughput protein identification. Performance of the sample preparation is demonstrated with in situ proteolytic digests of human brain proteins separated by two-dimensional gel electrophoresis.

Brain Chemistry↗
PubMed2001

Development of a technology for automation and miniaturization of protein crystallization.

The usage of standard 96 well microplates for the screening of crystallization conditions of recombinant proteins offers several advantages when compared to commonly used crystallization plate formats. The adoption of robotic technology for plate and glass slide preparation within a "hanging drop" vapour diffusion crystallization experiment enables to work with an increased throughput at reduced costs. In addition to commercial pipetting devices with a 96-channel aspirator/dispenser, solenoid ink-jet technology was applied to form 250 nl droplets with a diameter of approximately 1 mm. This allows miniaturization of crystallization screening set-ups with an estimated ten-fold cost reduction when compared to commonly used 24 well plates.

Concanavalin A↗