Biomedical subjects

Stephanie Bechtel

Publications and source records attributed to Stephanie Bechtel.

12 recordsLinked to original sources

PubMed2006

High-content microscopy identifies new neurite outgrowth regulators.

Neurons, with their long axons and elaborate dendritic arbour, establish the complex circuitry that is essential for the proper functioning of the nervous system. Whereas a catalogue of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in early events of neuronal differentiation, such as neurite initiation and elongation, are less well understood, mainly because the key molecules involved remain elusive. Here we describe the establishment and application of a microscopy-based approach designed to identify novel proteins involved in neurite initiation and/or elongation. We identified 21 proteins that affected neurite outgrowth when ectopically expressed in cells. Complementary time-lapse microscopy allowed us to discriminate between early and late effector proteins. Localization experiments with GFP-tagged proteins in fixed and living cells revealed a further 14 proteins that associated with neurite tips either early or late during neurite outgrowth. Coexpression experiments of the new effector proteins provide a first glimpse on a possible functional relationship of these proteins during neurite outgrowth. Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation.

Animals↗

PubMed2006

An anthropoid-specific segmental duplication on human chromosome 1q22.

Segmental duplications (SDs) play a key role in genome evolution by providing material for gene diversification and creation of variant or novel functions. They also mediate recombinations, resulting in microdeletions, which have occasionally been associated with human genetic diseases. Here, we present a detailed analysis of a large genomic region (about 240 kb), located on human chromosome 1q22, that contains a tandem SD, SD1q22. This duplication occurred about 37 million years ago in a lineage leading to anthropoid primates, after their separation from prosimians but before the Old and New World monkey split. We reconstructed the hypothetical unduplicated ancestral locus and compared it with the extant SD1q22 region. Our data demonstrate that, as a consequence of the duplication, new anthropoid-specific genetic material has evolved in the resulting paralogous segments. We describe the emergence of two new genes, whose new functions could contribute to the speciation of anthropoid primates. Moreover, we provide detailed information regarding structure and evolution of the SD1q22 region that is a prerequisite for future studies of its anthropoid-specific functions and possible linkage to human genetic disorders.

Animals↗

PubMed2006

The systematic functional characterisation of Xq28 genes prioritises candidate disease genes.

BACKGROUND: Well known for its gene density and the large number of mapped diseases, the human sub-chromosomal region Xq28 has long been a focus of genome research. Over 40 of approximately 300 X-linked diseases map to this region, and systematic mapping, transcript identification, and mutation analysis has led to the identification of causative genes for 26 of these diseases, leaving another 17 diseases mapped to Xq28, where the causative gene is still unknown. To expedite disease gene identification, we have initiated the functional characterisation of all known Xq28 genes. RESULTS: By using a systematic approach, we describe the Xq28 genes by RNA in situ hybridisation and Northern blotting of the mouse orthologs, as well as subcellular localisation and data mining of the human genes. We have developed a relational web-accessible database with comprehensive query options integrating all experimental data. Using this database, we matched gene expression patterns with affected tissues for 16 of the 17 remaining Xq28 linked diseases, where the causative gene is unknown. CONCLUSION: By using this systematic approach, we have prioritised genes in linkage regions of Xq28-mapped diseases to an amenable number for mutational screens. Our database can be queried by any researcher performing highly specified searches including diseases not listed in OMIM or diseases that might be linked to Xq28 in the future.

Animals↗

PubMed2006

The LIFEdb database in 2006.

LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

Cell Proliferation↗

PubMed2005

Functional profiling: from microarrays via cell-based assays to novel tumor relevant modulators of the cell cycle.

Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G1-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies.

Animals↗

PubMed2005

Large-scale protein expression for proteome research.

Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.

Amino Acid Sequence↗

PubMed2004

From ORFeome to biology: a functional genomics pipeline.

As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

Animals↗

PubMed2003

Identifying transcribed sequences, and beyond.

A report on the 12th International Workshop 'Beyond the Identification of Transcribed Sequences (BITS): Functional, Expression and Evolutionary Analysis', Washington DC, USA, 25-28 October 2002.

Alternative Splicing↗

PubMed2003

The German cDNA network: cDNAs, functional genomics and proteomics.

Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport. Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.

Cloning, Molecular↗

PubMed2002

The effect of amino-acid substitutions I112P, D147E and K152N in CYP11B2 on the catalytic activities of the enzyme.

By replacing specific amino acids at positions 112, 147 and 152 of the human aldosterone synthase (CYP11B2) with the corresponding residues from human, mouse or rat 11beta-hydroxylase (CYP11B1), we have been able to investigate whether these residues belong to structural determinants of individual enzymatic activities. When incubated with 11-deoxycorticosterone (DOC), the 11beta-hydroxylation activity of the mutants was most effectively increased by combining D147E and I112P (sixfold increase). The two substitutions displayed an additive effect. The same tendency can be observed when using 11-deoxycortisol as a substrate, although the effect is less pronounced. The second step of the CYP11B2-dependent DOC conversion, the 18-hydroxylation activity, was not as strongly increased as the 11beta-hydroxylation potential. Activity was unaffected by D147E, whereas the single mutant I112P displayed the most pronounced activation (70% enhancement), thus causing different increasing effects on the first two enzymatic reaction steps. A slightly enhanced aldosterone synthesis from DOC could be measured due to increased levels of the intermediates. However, the 18-oxidation activity of all the mutants, except for I112S and D147E, was slightly reduced. The strongly enhanced 18-hydroxycorticosterone and aldosterone formation observed in the mutants provides important information on a possible role of such amino-acid replacements in the development of essential hypertension. Furthermore, the results indicate the possibility of a differential as well as independent modification of CYP11B2 reaction steps. The combination of functional data and computer modelling of CYP11B2 suggests an indirect involvement of residue 147 in the regulation of CYP11B isoform specific substrate conversion due to its location on the protein surface. In addition, the results indicate the functional significance of amino-acid 112 in the putative substrate access channel of human CYP11B2. Thus, we present the first example of substrate recognition and conversion being attributed to the N-terminal part of human CYP11B2.

Amino Acid Sequence↗

PubMed2002

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences.

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.

18-Hydroxycorticosterone↗

PubMedDate not supplied

CDNAs for functional genomics and proteomics: the German Consortium.

To functionally characterize numerous novel proteins encoded by cDNAs sequenced by the German Consortium, 800 were tagged with green fluorescent protein. The subcellular localizations of the fusion proteins were examined in living cells, enabling their classification in subcellular groups. Their activity in cell growth, cell death, and protein transport was screened in high throughput using robotic liquid handling and reading stations. The resulting information is integrated with functional genomics and proteomics data for further understanding of protein functions in the cellular context.

DNA, Complementary↗