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T Werner

Publications and source records attributed to T Werner.

At least 91 records · Page 5Linked to original sources
PubMed1996

CONRAD: a method for identification of variable and conserved regions within proteins by scale-space filtering.

Advanced sequencing techniques allow rapid deduction of individual amino acid sequences of highly related proteins. Due to their quasi-species nature, viral genomes (e.g. HIV-1) represent one of the most common sources of related proteins. Another example of related proteins are immunoglobulins. Local differences in amino acid conservation are useful indicators of potential domain structures and immunological or functional epitopes prior to structural analysis of proteins. Although variability indices can be calculated by several methods, delineation of boundaries between sequence stretches with similar variability indices is left to the user. We use algorithmic scale-space filtering for delineation of conserved and variable sequence stretches within a protein which is performed on an algorithmic basis avoiding arbitrary assignments. Out method correctly identified variable regions for the human immunoglobulin lambda-chain V-regions (subgroup I). Prediction of the variable regions of the HIV-1 gp120 env protein was in agreement with empirical derived definitions. These examples indicate that our method is useful for the regional assignment of protein variability solely on the basis of amino acid sequences.

Algorithms↗
PubMed1996

GenomeInspector: a new approach to detect correlation patterns of elements on genomic sequences.

MOTIVATION: Most of the sequences determined in current genome sequencing projects remain at least partially unannotated. The available software for DNA sequence analysis is usually limited to the prediction of individual elements (level 1 methods), but does not assess the context of different motifs. However, the functionality of biological units like promoters depends on the correct spatial organization of multiple individual elements. RESULTS: Here, we present a second-level software package called GenomeInspector [[http:@www.gsf.de/biodv/genomeinspector.html ]], for further analysis of results obtained with level 1 methods (e.g. MatInspector [[http:@www.gsf.de/biodv/matinspector.html ]] or ConsInspector [[http:@www.gsf.de/biodv/consinspector.html++ +]]). One of the main features of this modular program is its ability to assess distance correlations between large sets of sequence elements which can be used for the identification and definition of basic patterns of functional units. The program provides an easy-to-use graphical user interface with direct comprehensive display of all results for megabase sequences. Sequence elements showing spatial correlations can be easily extracted and traced back to the nucleotide sequence with the program. GenomeInspector identified promoters of glycolytic enzymes in yeast [[http:@www.mips.biochem.mpg.de/mips/yeast/]] as members of a subgroup with unusual location of an ABF1 site. Solely on the basis of distance correlation analysis, the program correctly selected those transcription factors within these promoters already known to be involved in the regulation of glycolytic enzymes, demonstrating the power of this method.

Algorithms↗
PubMed1996

A new variant of B1 elements, vB1, identified upstream of a retroviral integration site.

A new variant of the murine B1 retroposon family, vB1, was identified in antisense orientation 165 bases upstream of the integration site of RFB MuLV in infected NIH3T3 fibroblasts. vB1 revealed a characteristic B1 structure, but contained two additional unique repeats. This variant facilitates identification of related vB1 elements which are considered to represent a minor subgroup of B1 elements.

3T3 Cells↗
PubMed1996

The Cincinnati Payer Initiative.

In 1991, four companies (Procter & Gamble, General Electric, Cincinnati Bell, and Kroger) located in Cincinnati, OH, decided to cooperate in a collaborative partnership with hospitals and a health care data consulting firm to get more information about the health care options available in their community. The Cincinnati Quality Initiative was formed with the goal of stimulating competition around quality and value in the health care market place. The 14 hospitals in the Cincinnati area were invited to participate in a 3-year program of investigation and analyses. The Initiative implemented the investigation in order to gather information that could be used to make health care choices for their employees. Hospitals that performed well over the 3-year period-those that improved over time-would then be selected by the Initiative. However, this selection process did not occur. Nevertheless, the 3-year evaluation proved useful because it generated valuable information about the health care options in Cincinnati and spurred competition among the hospitals. It also led to a dramatic reduction in health care costs for those hospitals that agreed to participate in the investigation.

Cost Control↗
PubMed1995

MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data.

The identification of potential regulatory motifs in new sequence data is increasingly important for experimental design. Those motifs are commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. However, sufficiently large libraries of pre-compiled matrices are a prerequisite for practical application of any matrix-based approach and are just beginning to emerge. Here we present a set of tools for molecular biologists that allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices. We also supply a large library (> 200) of transcription factor binding site matrices that has been compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. Our search method includes position weighting of the matrices based on the information content of individual positions and calculates a relative matrix similarity. We show several examples suggesting that this matrix similarity is useful in estimating the functional potential of matrix matches and thus provides a valuable basis for designing appropriate experiments.

Base Sequence↗
PubMed1995

Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reverse-transcription polymerase chain reaction using a general set of degenerate primers.

GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis. cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanus and Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilis and the higher plant Mucuna hassjo using primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique. A number of regions were found to be strictly conserved between unrelated eukaryotes. These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia coli enzyme.

Amino Acid Sequence↗
PubMed1995

Identification of two enhancer elements downstream of the human c-myc gene.

Expression of the proto-oncogene c-myc is tightly regulated in vivo. Transcription of c-myc is assumed to be controlled by a number of positive and negative cis-acting control elements located upstream or within exon 1 and intron 1. However, these regulatory elements are not sufficient for c-myc expression after stable transfection or in transgenic mice. Transcription of c-myc in vivo thus requires additional control elements located outside the tested HindIII-EcoRI gene fragment. In order to identify these putative additional control elements, we mapped DNase I hypersensitive sites around the human c-myc gene in nine different tumor cell lines and in primary lymphocytes. Within the coding and 5' region of the gene, an almost identical pattern of DNase I hypersensitive sites was detected in the various cells. In contrast, chromatin analysis of the c-myc 3' region revealed a complex pattern of constitutive and tissue-specific DNase I hypersensitive sites. In enhancer trap experiments we identified two cis-acting control elements, both co-localizing with DNase I hypersensitive sites, that stimulated c-myc transcription after transient transfection in Raji or HeLa cells. Both regulatory elements exerted their enhancer activity in either orientation and regardless of their location within the plasmids. Both elements also conferred activation on a heterologous promoter. The association of these enhancers with DNase I hypersensitive sites, indicating their functional activity in vivo, make them potential candidates for the postulated regulatory control element(s) required for c-myc expression in vivo.

Animals↗
PubMed1995

Distribution of HIV genomic DNA in brains of AIDS patients.

BACKGROUND: Data concerning the distribution of HIV in the brains of AIDS patients at different stages of viral infection might contribute towards: (1) understanding the route(s) of HIV entry into the brain and virus dissemination within the brain and (2) establishing a possible correlation between the extent of CNS damage and the distribution of virus in AIDS brains. OBJECTIVE: To determine the distribution of HIV-1 genomic DNA within the brains of three deceased AIDS patients by polymerase chain reaction (PCR). STUDY DESIGN: The brains of three deceased AIDS patients were examined. Two brains had limited neuropathologic findings (brains I and II), and one brain (brain III) showed primary HIV-specific neuropathologic damage. Tissues were taken from different locations within each brain, and high molecular weight DNA isolated from the tissues was assessed for HIV-1 genomic DNA by PCR. RESULTS: HIV-1 genomic DNA was found in all three brains, but the amount was low: order of magnitude of 1 viral genome per 1,000 cells. Multiple PCR analyses of DNA from brain I showed that the viral genomic DNA in this brain was non-uniformly distributed; only samples taken from the brainstem were clearly positive for HIV-1. HIV-1 genomic DNA in brain II was found in portions of the lower and upper hemispheres. All but one of the brain III samples were clearly positive for HIV-1, and they had been taken from locations spread throughout this brain. CONCLUSIONS: Our results suggest that in early or latent stages of HIV-infection of the brain, viral genomic DNA is localized at restricted regions. At later stages this DNA is distributed more uniformly throughout the brain. Our data are compatible with the concept of rare infection events followed by viral spreading within brain tissues.

Journal Article↗
PubMed1994

Molecular characterization of HPH-1: a mouse mutant deficient in GTP cyclohydrolase I activity.

GTP cyclohydrolase I catalyzes the initial and rate limiting step of the biosynthesis of tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylation. The mouse mutant HPH-1, previously generated by chemical mutagenesis, shows a phenylketonuria due to decreased hepatic GTP cyclohydrolase I activity. We show that both parameters GTP cyclohydrolase I activity and tetrahydrobiopterin synthesis significantly increase after weaning, but remain reduced during the lifetime. In the wild type mouse (C57BL/6), interferon-gamma and kit ligand induce GTP cyclohydrolase I activity in primed T-cells and in bone marrow-derived mast cells, respectively. The same is true for the HPH-1 mutant, but the absolute values remain lower throughout. The open reading frame of GTP cyclohydrolase I is not affected by the hph-1 mutation as shown by sequencing. Northern blot analysis demonstrates a marked decrease in the steady state mRNA level specific for GTP cyclohydrolase I.

Animals↗
PubMed1994

Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme.

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

Amino Acid Sequence↗
PubMed1994

Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.

The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.

Animals↗
PubMed1994

tRNA genes of Streptomyces lividans: new sequences and comparison of structure and organization with those of other bacteria.

Three closely linked Streptomyces lividans tRNA genes encoding two tRNA(Lys)s and a tRNA(Gly) were cloned and sequences. The structure of tRNA(Gly) is unusual for eubacterial tRNAs. Including those in previous reports (R. Sedlmeier and H. Schmieger, Nucleic Acids Res. 18:4027, 1990, and R. Sedlmeier, G. Linti, K. Gregor, and H. Schmieger, Gene 132:125-130, 1993), 18 S. lividans tRNA genes were physically mapped on the chromosome of the closely related strain Streptomyces coelicolor A3(2). The structure and organization of tRNA genes of S. lividans and S. coelicolor are compared with those of Escherichia coli and Bacillus subtilis.

Bacillus subtilis↗
PubMed1994

Transcription of HERV-K-related LTRs in human placenta and leukemic cells.

The human genome contains a family of endogenous retroviruses, HERV-K, with sequence homology to the B-type mouse mammary tumor virus (MMTV). We have detected HERV-K-LTR related cDNA clones by screening a human placenta cDNA library with a HERV-K LTR probe. Three of the isolated cDNA clones were characterized by nucleotide sequencing. The analyzed clones did not contain any retroviral sequences other than those related to HERV-K LTRs, but were found to be coexpressed with cellular sequences. Furthermore, transcripts containing HERV-K LTR sequences were demonstrated by Northern blotting and PCR in human leukemic and normal white blood cells, as well as in various tumor cell lines, indicating abundant transcription of solitary HERV-K LTRs in human tissues. In patients with lymphatic leukemias, a transcript of about 6 kb hybridizing with HERV-K LTR was detected that was not found in patients with myelogenous leukemias or in healthy persons.

Base Sequence↗
PubMed1993

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Amino Acid Sequence↗
PubMed1993

Computer-assisted prediction, classification, and delimitation of protein binding sites in nucleic acids.

We present a method to determine the location and extent of protein binding regions in nucleic acids by computer-assisted analysis of sequence data. The program ConsIndex establishes a library of consensus descriptions based on sequence sets containing known regulatory elements. These defined consensus descriptions are used by the program ConsInspector to predict binding sites in new sequences. We show the programs to correctly determine the significant regions involved in transcriptional control of seven sequence elements. The internal profile of relative variability of individual nucleotide positions within these regions paralleled experimental profiles of biological significance. Consensus descriptions are determined by employing an anchored alignment scheme, the results of which are then evaluated by a novel method which is superior to cluster algorithms. The alignment procedure is able to include several closely related sequences without biasing the consensus description. Moreover, the algorithm detects additional elements on the basis of a moderate distance correlation and is capable of discriminating between real binding sites and false positive matches. The software is well suited to cope with the frequent phenomenon of optional elements present in a subset of functionally similar sequences, while taking maximal advantage of the existing sequence data base. Since it requires only a minimum of seven sequences for a single element, it is applicable to a wide range of binding sites.

Algorithms↗