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H Voss

Publications and source records attributed to H Voss.

At least 37 records · Page 2Linked to original sources
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

"Doublex" fluorescent DNA sequencing: two independent sequences obtained simultaneously in one reaction with internal labeling and unlabeled primers.

The novel "doublex" DNA sequencing technique that makes it possible to obtain simultaneously two independent sequences from one sequencing reaction with the use of unlabeled primers and internal labeling is described. The different sequencing products are labeled in parallel with fluorescein-15-dATP and Texas red-5-dCTP present in the same tube. The characteristics of T7 DNA polymerase are exploited to ensure that only either of the labeled dNTPs is incorporated into the corresponding sequencing products. Specificity of labeling is ensured by the selection of primers. One of the unlabeled primers is chosen to be followed by an "A," the other by a "C" to be incorporated immediately downstream from the primer binding site. The doublex sequencing technique is applicable to the simultaneous sequencing of either the same DNA template/strand or a mixture of different templates. Combinations of unlabeled and labeled primers in the same sequencing reaction are also possible. The two sequences can be determined in parallel and on-line in the same lanes of a gel with a novel automated DNA sequencer, which was previously described for use with labeled primers.

Autoanalysis↗
PubMed1996

[Accidental coincidence in cause of death?].

Upon the discovery of two deceased persons in close proximity within a short period of time, a death by other than natural causes is first assumed, and only after the elimination of such a cause of death through police investigations as well as an autopsy can a death by natural causes be assumed. The existence of two different natural causes of death in close proximity within a short period of time is extremely rare. In the subject of our autopsy we made two such observations within a short period of time which are presented as casuistry.

Adult↗
PubMed1996

Sequencing 39,350 bp of Saccharomyces cerevisiae chromosome XII utilizing ordered shotgun libraries.

A 39,350 bp cosmid containing DNA of Saccharomyces cerevisiae chromosome XII was sequenced by making use of ordered sub-clones of 1 kb insert-length selected from a physical clone map. In a first analysis, 96 clones were sequenced from both ends (10 gels) with two standard sequencing primers covering 91% of the total sequence (49% double-stranded). After selection of another eight clones six gaps of a total of 1.8 kb and several single-stranded stretches remained. These gaps were closed by 86 primer walks leading to an overall redundancy of 4.4 per base and a total of 292 sequencing reactions. The number of walking primers can be reduced significantly by more uniform clone lengths and longer sequencing reads, thus, the total amount of sequencing reactions can approach the minimum value achieved with primer walking strategies, with only very few walking primers needed for gap closure.

Chromosomes, Fungal↗
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↗
PubMed1995

A day-care pain clinic--its possibilities and limitations in the treatment of cancer patients.

A day-care unit was established to extend facilities of the pain clinic. During its first 2 years of operation 79 patients with cancer-related pain have been treated. Most patients were admitted because of intractable pain but also suffered other symptoms mainly concerning the gastrointestinal and respiratory tract. For more than half of the patients and their families, psychosocial problems were also an important issue. The central aim of the pain management programme is to improve the quality of life of patients with cancer-related pain through appropriate drug based or anesthetic interventions. Control of other symptoms and counseling for both patient and family are an integral part of the holistic approach. The first initial clinical experiences show that the day-care unit provides a noticeable improvement in patient care and constitutes an excellent link between the out- and inpatient therapeutic possibilities.

Adult↗
PubMed1994

The human gene (CSNK2A1) coding for the casein kinase II subunit alpha is located on chromosome 20 and contains tandemly arranged Alu repeats.

We have isolated and characterized a 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit alpha gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII alpha. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII alpha gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats.

Animals↗
PubMed1994

Direct shape determination of ribosomal proteins in solution and within the ribosome by means of neutron scattering.

Following the 'strategy of the glassy ribosome' single protonated ribosomal proteins (r-proteins) were reconstituted into deuterated 50S subunits of Escherichia coli. The deuteration of both rRNA and r-proteins were individually adjusted to such a degree that the ribosomal matrix appeared nearly homogeneous with respect to coherent neutron scattering and had a scattering density equivalent to a D2O solution of about 90%. Neutron scattering of ribosomal subunits was recorded in reconstitution buffer containing three different concentrations of D2O around 90% D2O (contrast variation). The signal-to-noise ratio achieved allowed us to make a direct determination of the radii of gyration of r-proteins within the 50S subunit and thus provides the first information relating to the shape of these proteins in situ. We present the radii of gyration of 11 r-proteins incorporated into 50S subunits and of 9 isolated r-proteins in solution. In addition, the data concerning the overall dimensions of the r-proteins we report on indicate that conformational changes of at least two individual r-proteins occur during the assembly process of the ribosome.

Escherichia coli↗
PubMed1994

Human protein kinase CK2 genes.

We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome.

Amino Acid Sequence↗
PubMed1994

Nonradioactive, solid-phase DNase I footprints analyzed on an A.L.F. DNA Sequencer.

Solid-phase DNase I footprinting provides a powerful tool for analyzing the sequence-specific interactions of DNA binding proteins. Classically this type of assay requires radioactively labeled DNA molecules. Substitution of the isotope by fluorescein labeling of the DNA fragments enables the analysis of footprint patterns on a standard automated laser fluorescent (A.L.F.) DNA Sequencer. The combination of solid-phase footprinting technology and fluorescence-based nonradioactive detection of fragments has unique advantages over established footprinting technologies.

DNA-Binding Proteins↗
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

Human casein kinase II. The subunit alpha protein activates transcription of the subunit beta gene.

Casein kinase II (CKII), an ubiquitous serine/threonine protein kinase in control of a variety of crucial cellular functions, is composed of catalytic subunits (alpha and alpha') and regulatory subunits (beta). The adjusted activity of CKII is determined by the actual conformational state of CKII beta and the stoichiometry of the CKII subunits. Thus, the expression control of CKII beta is of particular concern. Carrying out gel shifts and footprints with affinity-purified proteins and cellular extracts in combination with mutational analysis we find that aside NF1 and Sp1, two out of the many factors predicted to bind to the upstream promoter region of the human CKII beta gene (Voss, H., Wirkner, U., Jakobi, R., Hewitt, N. A., Schwager, C., Zimmermann, J., Ansorge, W., and Pyerin, W. (1991) J. Biol. Chem. 266, 13706-13711), CKII alpha protein is able to complex with the CKII beta gene promoter. The complex of CKII beta-DNA/CKII alpha-protein is shown to occur within the 170-239-base pair (bp) segment upstream of the first transcription start site of the gene. The DNA motif contains, in a distance of 44 bp, two GC-rich boxes, 5'-GGGGCCC and 5'-CCCCTGGGC, and represents a novel cis-acting element; the binding of the CKII alpha protein activates the CKII beta gene promoter. This is manifested by driving the expression of the indicator gene luciferase or of CKII alpha-cDNA in HeLa cells. The binding of the CKII alpha protein is inhibited due to CKII beta protein addition or by mimicking the corresponding situation in vivo by overexpression of the CKII subunits. The data suggest that cells may maintain a certain CKII subunit stoichiometry via transcriptional control; excess of nuclear CKII alpha protein could activate the CKII beta gene transcription causing CKII beta protein to increase which, in turn, could feed back to abolish the action of CKII alpha at the CKII beta gene promoter.

Base Sequence↗
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↗