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Biomedical subjects

C Schwager

Publications and source records attributed to C Schwager.

At least 19 recordsLinked to original sources
PubMed2006

Needlestick injuries and other occupational exposures to body fluids amongst employees and medical students of a German university: incidence and follow-up.

The aim of this study was to obtain data concerning the incidence, reporting and follow-up of occupational exposure to blood or other body fluids (OEB). A questionnaire was distributed to employees and medical students (N=787) and official reports of OEB during the year 2003 (N=203) and their consequent follow-up (N=100) were evaluated. The percentages of needlestick injuries were 29.5% for students and 22.5% for employees. Incidence rates per 1000 employee days were 0.61 for needlestick injuries or sharp object injuries and 0.27 for mucocutaneous exposure to body fluids. The mean rate of underreporting was approximately 45%. Contrary to expectations, only 4.3% of nurses and 3.9% of doctors officially reported an OEB in 2003. The number of persons who did not attend for a serological test increased during the follow-up period. Considering all documented test results, 35 out of 100 affected persons were lost to follow-up due to default of appearance. As a consequence, the employer should provide safety devices and enforce didactical interventions with practical training and incident reporting. Periodical occupational health medicals, including serological testing, should be mandatory for all employees, including medical students and student nurses. To increase compliance after OEB, a short follow-up period using improved laboratory tests requires further discussion.

Follow-Up Studies↗
PubMed2002

Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays.

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.

DNA Probes↗
PubMed2001

The genomic response of the Drosophila embryo to JNK signaling.

During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. We analyzed the genome-wide transcriptional response to activation of the JNK pathway in the Drosophila embryo by serial analysis of gene expression (SAGE) and identified loci encoding cell adhesion molecules and cytoskeletal regulators as JNK responsive genes. The role of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, in embryogenesis was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly.

Actins↗
PubMed2000

Using a neural network for lane-tracking of DNA sequencing slab gels.

High quality lane-tracking of gel images is the first task, and thus a prerequisite, for successful trace processing and base-calling of DNA sequencing slab gels. In most approaches, it is based on statistical calculations, for instance variance and co-variance analysis between neighboring pixel columns in the image. On the basis of these statistical calculations, Kohonen's self-organization neural network model was introduced. We have found that, using several well-structured input data, Kohonen's self-organization neural network model can be trained to fulfill our task of lane-tracking. Furthermore, the quality of lane-tracking could be improved compared to algorithmic approaches.

Algorithms↗
PubMed1998

Post-traumatic stress disorder in paediatric patients and their parents: an exploratory study.

OBJECTIVE: To determine the prevalence of post-traumatic stress disorders (PTSD) in a selected population of paediatric patients and their parents. METHODOLOGY: Twenty-three high-risk patients (serious accidents, newly diagnosed cancer) and 11 low-risk patients (simple planned surgery) and their parents were consecutively selected and assessed 6-8 weeks after the event with a DSM-IV based diagnostic interview and the PTSD Symptom Scale. RESULTS: There is a high prevalence of PTSD in children and parents of the high-risk group. In the low-risk group such disorders are almost nonexistent. Parents in both groups report more PTSD symptoms than their children. Sex and age of the child and duration of hospitalisation have no significant influence on the development of PTSD. CONCLUSIONS: The high prevalence of PTSD in high-risk paediatric patients and their parents offers support for the applicability of a post-traumatic stress model for understanding the psychological impact of accidents, severe illnesses and their medical treatment.

Adolescent↗
PubMed1997

DNA sequencing and analysis of 130 kb from yeast chromosome XV.

We have determined the nucleotide sequence of 129,524 bases of yeast (Saccharomyces cerevisiae) chromosome XV. Sequence analysis revealed the presence of 59 non-overlapping open reading frames (ORFs) of length > 300 bp, three tRNA genes, four delta elements and one Ty-element. Among the 21 previously known yeast genes (36% of all ORFs in this fragment) were nucleoporin (NUP1), ras protein (RAS1), RNA polymerase III (RPC1) and elongation factor 2 (EF2). Further, 31 ORFs (53% of the total) were found to be homologous to known protein or DNA sequences, or sequence patterns. For seven ORFs (11% of the total) no homology was found. Among the most interesting protein identification in this DNA fragment are an inositol polyphosphatase, the second gene of this type found in yeast (homologous to the human OCRL gene involved in Lowe's syndrome), a new ADP ribosylation factor of the arf6 subfamily, the first protein containing three C2 domains, and an ORF similar to a Bacillus subtilis cell-cycle related protein. For each ORF detailed sequence analysis was carried out, with a full consideration of its biological function and pointing out key regions of interest for further functional analysis.

ADP-Ribosylation Factors↗
PubMed1996

Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames.

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.

Base Sequence↗
PubMed1996

pDAP2: a vector for construction of alkaline phosphatase fusion-proteins.

BACKGROUND: Expression of enzymatically active protein fusions in Escherichia coli could facilitate the analysis of proteins and even replace some reagents frequently used in immunology such as chemically produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available. OBJECTIVES: The vector pDAP2 has been designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alkaline phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatography. STUDY DESIGN: Several different single-chain Fv genes as well as peptide coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purification properties and usefulness in ELISA and immunowestern blotting. RESULTS: The fusion proteins from pDAP2 can be prepared at levels of several milligrams per liter culture from the periplasma of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting. CONCLUSION: pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alkaline phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple analysis of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.

Alkaline Phosphatase↗
PubMed1995

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Animals↗
PubMed1995

Simultaneous on-line DNA sequencing on both strands with two fluorescent dyes.

We describe an automated DNA-sequencing technique which allows both the simultaneous sequencing from the two strands of double-stranded templates and the subsequent detection of the sequencing products online and in parallel. The technique is based on hardware technology also used in the ALF DNA sequencer (Pharmacia, Uppsala). A helium-neon laser was mounted into the sequencing device additionally to the standard argon laser. Two different primers, labeled with either fluorescein or Texas red, are used in a single sequencing reaction resulting in an output of two sequences. Both sequencing products are then analyzed on-line in the same lanes of a gel. This technique is especially useful for the complete sequencing of DNA fragments up to 1 kb. High accuracy sequencing of PCR products in double-stranded form can now be accomplished in only one sequencing reaction.

Fluorescent Dyes↗
PubMed1995

Nucleotide sequence and analysis of the centromeric region of yeast chromosome IX.

We have determined the nucleotide sequence of a cosmid (pIX338) containing the centromere region of yeast (Saccharomyces cerevisiae) chromosome IX. The complete nucleotide sequence of 33.8 kb was obtained by using an efficient directed sequencing strategy in combination with automated DNA sequencing on the A.L.F. DNA sequencer. Sequence analysis revealed the presence of 17 open reading frames (ORFs), four of them previously known yeast genes (sly12, pan1, sts1 and prl1), a tRNA gene and the centromere motif. Exhaustive database searches detected sequence homologues of known function for as many as 14 of the 17 ORFs. These include a mammalian tyrosine kinase substrate; the Escherichia coli cell cycle protein MinD; the human inositol polyphosphate-5-phosphatase (gene OCRL) involved in Lowe's syndrome, a developmental disorder; and helicases, for which the new yeast member defines a distinct DEAD/H-box subfamily. A surprisingly large fraction of the ORFs (at least six out of 17) in the centromeric region are apparently involved in RNA or DNA binding.

Adenosine Triphosphatases↗
PubMed1995

Primer design for automated DNA sequencing utilizing T7 DNA polymerase and internal labeling with fluorescein-15-dATP.

We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.

Binding Sites↗
PubMed1994

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Base Sequence↗
PubMed1994

Improved fluorescent cycle sequencing protocol allows reading nearly 1000 bases.

Recently available thermostable DNA polymerases result in enhanced resolution and accuracy compared to thermal enzymes used previously in fluorescent cycle sequencing. These new enzymes produce less variations in peak intensities, enhance gel resolution and are less sensitive to unspecific termination caused by either DNA structure or impurities in the DNA preparation. Optimization of nucleotide ratios and the usage of high concentrations of detergents in the sequencing reaction result in sequence readings up to 1000 bases and improve overall reliability of the sequencing protocol; this works successfully in about 90% of cases.

DNA-Directed DNA Polymerase↗
PubMed1993

Sequencing and analysis of 51.6 kilobases on the left arm of chromosome XI from Saccharomyces cerevisiae reveals 23 open reading frames including the FAS1 gene.

We have sequenced two segments containing a total of 51.6 kb of the left arm from chromosome XI of Saccharomyces cerevisiae. The first segment of 38.5 kb contains 18 open reading frames (ORFs) of more than 100 amino acid residues. Five ORFs encode known yeast genes, including the fatty acid synthase gene (FAS1). Three new yeast genes were discovered with homologies to non-yeast genes and ten new genes without homologies to any known sequences. The second segment of 13 kb contains five ORFs with two known yeast genes and three unknown genes. The sequences from cosmid pUKG041 were obtained entirely with the walking primer strategy resulting in a very low overall sequence redundancy of 2.8 and an average reading length of 443 bases.

Base Sequence↗
PubMed1993

Automated low-redundancy large-scale DNA sequencing by primer walking.

Low-redundancy automated DNA sequencing by primer walking is described. T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 bases (in the unedited raw data). The low error rate allows efficient sequencing with low (2-3 times) redundancy. Plasmid subclones covering 20 kb of a cosmid insert were sequenced with an overall redundancy of 2.7 in the course of the European community Saccharomyces cerevisiae genome sequencing project. Neighboring plasmid subclones were linked by direct cosmid sequencing. Sets of ten walking primers are synthesized on the EMBL multiple segmental DNA synthesizer at low costs and used for sequencing with greater than 95% efficiency. The accuracy of the directed approach is improved by simultaneous walking on both strands by designing two primers in opposite directions in the same starting region. One primer is used to confirm sequence data on the opposite strand, and the other primer to obtain new sequence data.

Autoanalysis↗