Biomedical subjects

C Grunau

Publications and source records attributed to C Grunau.

8 recordsLinked to original sources

PubMed2001

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters.

Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55 degrees C (4-18 h) and 95 degrees C (1 h). Under these conditions at least 84-96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (>/=97%) and 5-methylcytosine (>/=94%) were found to be identical for both templates.

5-Methylcytosine↗

PubMed2001

MethDB--a public database for DNA methylation data.

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Base Sequence↗

PubMed2000

Large-scale methylation analysis of human genomic DNA reveals tissue-specific differences between the methylation profiles of genes and pseudogenes.

Cytosine in CpG dinucleotides is frequently found to be methylated in the DNA of higher eukaryotes and differential methylation has been proposed to be a key element in the organization of gene expression in man. To address this question systematically, we used bisulfite genomic sequencing to study the methylation patterns of three X-linked genes and one autosomal pseudogene in two adult individuals and across nine different tissues. Two of the genes, SLC6A8 and MSSK1, are tissue-specifically expressed. CDM is expressed ubiquitously. The pseudogene, psi SLC6A8, is exclusively expressed in the testis. The promoter regions of the SLC6A8, MSSK1 and CDM genes were found to be essentially unmethylated in all tissues, regardless of their relative expression level. In contrast, the pseudogene psi SLC6A8 shows high methylation of the CpG islands in all somatic tissues but complete demethylation in testis. Methylation profiles in different tissues are similar in shape but not identical. The data for the two investigated individuals suggest that methylation profiles of individual genes are tissue specific. Taken together, our findings support a model in which the bodies of the genes are predominantly methylated and thus insulated from the interaction with DNA-binding proteins. Only unmethylated promoter regions are accessible for binding and interaction. Based on this model we propose to use DNA methylation studies in conjunction with large-scale sequencing approaches as a tool for the prediction of cis-acting genomic regions, for the identification of cryptic and potentially active CpG islands and for the preliminary distinction of genes and pseudogenes.

Aged↗

PubMed2000

MethTools--a toolbox to visualize and analyze DNA methylation data.

The Bisulfite Genomic Sequencing technique has found wide acceptance for the generation of DNA-methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil (and subsequent conversion to thymine via PCR), whereas 5-methylcytosine residues remain unchanged. Methylation maps are created by the comparison of bisulfite converted sequences with the untreated genomic sequence. 'MethTools' is a collection of software tools that replaces the time-consuming manual comparison process, generates graphical outputs of methylation patterns and methyl-ation density, estimates the systematic error of the experiment and searches for conserved methylated nucleotide patterns. The programs are written in Perl 5 and C, and the source code can be downloaded. All tools run independently but the programs are interfaced. Thus, a script can perform the entire analysis procedure automatically. In addition, a web-based remote analysis service is offered. Both the source code and the remote analysis are available at http://genome.imb-jena.de/methtools/

Animals↗

PubMed1999

Carpel, a new Arabidopsis epi-mutant of the SUPERMAN gene: phenotypic analysis and DNA methylation status.

The carpel (car) mutation affects the morphology of reproductive organs in Arabidopsis thaliana. car flowers have an increased number of carpels, on average 2.7 +/- 0.8 instead of two in the wild type. Through allelism test with fon1-3 and analysis of the methylation state of the SUPERMAN (SUP) gene in car mutants, we show that car is an epi-mutation of SUP. The methylation pattern of car is clearly distinct from that of fon1-3, another epi-mutation of the SUP gene. Methylation was found predominantly in Cp(A/T)p(A/G) triplets and in CpG pairs. We suggest that the extensive SUP methylation in car has arisen from an abundant methylation of a single CpG site that was already present in abscisic acid-insensitive (abi3-4) mutants, from which car was segregating.

Arabidopsis↗

PubMed1995

Bovine adrenodoxin--a mitochondrial iron-sulphur protein--binds to chaperonin GroEL.

The interaction of bovine adrenodoxin with the chaperonin GroEL was investigated using sucrose density centrifugation and analytical ultracentrifugation. It could be clearly established that denatured mature adrenodoxin comigrated in a sucrose density gradient with the GroEL oligomer, indicating that a complex had been formed. Up to 2 moles of adrenodoxin/mol GroEL can be bound. From the partial concentrations, association constants of 4.3 x 10(5) M-1 for the first adrenodoxin molecule and of 1.08 x 10(5) M-1 for the second molecule to the complex, respectively, were calculated. Upon addition of the cochaperonin GroES and Mg-ATP to the adrenodoxin-GroEL complex, adrenodoxin was released, indicating a specific binding between GroEL and adrenodoxin.

Adrenodoxin↗

PubMed1994

Immunocytochemical localization of heterologously expressed adrenodoxin and its electron acceptor cytochrome P45011B1 in Escherichia coli.

Adrenal steroid hydroxylase P45011B1 and its electron donor adrenodoxin were localized in the cortex of bovine adrenals by immunogold-silver staining. In order to test recently developed heterologous expression systems for both enzymes to enable structure-function studies, immunocytochemical marker methods were applied. Adrenodoxin, the ferredoxin of the adrenal gland, was successfully expressed and for the first time localized in Escherichia coli. By use of ultrathin cryosections and the protein A-gold technique, adrenodoxin was detectable in large amounts in the cytoplasm of the bacterial cells, and, following the insertion of the outer membrane protein A leader sequence of E. coli, also in the periplasmic space. A fusion protein between mature adrenodoxin and human P45011B1 was constructed and clearly localized in E. coli by antibodies against both proteins.

Adrenal Cortex↗

PubMedDate not supplied

Phylogenetic identification of the symbiotic hypermastigote Trichonympha agilis in the hindgut of the termite Reticulitermes speratus based on small-subunit rRNA sequence.

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a larger hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Animals↗