Biomedical subjects

S Thalhammer

Publications and source records attributed to S Thalhammer.

9 recordsLinked to original sources

PubMed2004

Structural studies of oligonucleotides containing G-quadruplex motifs using AFM.

G-quadruplex DNAs are cyclic arrays of four guanine bases binding by Hoogsteen hydrogen bonds, found in the telomeric regions of chromosomes and in transcriptional regulatory regions of several important oncogenes. Here, we used high resolution atomic force microscopy (AFM) to observe a specific guanine (G) tetrad mediated complex formation of oligonucleotides containing a G-quadruplex motifs (G-ODN) combined with a palindromic sequence under physiological extracellular conditions. These oligonucleotides have been investigated in correlation to their immunostimulatory effects. We observed structural dependence on ion concentration and G-ODN concentration, where high concentration self-assembled DNA networks were formed.

Adjuvants, Immunologic↗

PubMed2004

LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

Bacterial Proteins↗

PubMed2003

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

Base Sequence↗

PubMed2001

Scaling-index method as an image processing tool in scanning-probe microscopy.

The scaling-index method (SIM) is a novel tool for image processing in scanning-probe microscopy. Originating from the theory of complex systems, the SIM can be used in order to extract structural information from arbitrary data sets. This method can readily be applied to the analysis of digital atomic-force microscopy (AFM) images. Especially for biomedical diagnostics, where genetic material is investigated by various microscopic methods, a reliable image segmentation based on the SIM algorithm is helpful. As a first application, AFM-images of GTG-banded human metaphase chromosomes (with G bands obtained by Trypsin using Giemsa) are compared with micrographs from conventional light microscopy by means of a scaling-index analysis. While the grey-level distributions of the optical and the AFM-images are largely different from each other, the scaling-index images are remarkably similar. Using this method, a fingerprint of an image can be produced which helps in the classification and interpretation of the measured data.

Algorithms↗

PubMed2001

GTG banding pattern on human metaphase chromosomes revealed by high resolution atomic-force microscopy.

Surface topography of human metaphase chromosomes following GTG banding was examined using high resolution atomic force microscopy (AFM). Although using a completely different imaging mechanism, which is based on the mechanical interaction of a probe tip with the chromosome, the observed banding pattern is comparable to results from light microscopy and a karyotype of the AFM imaged metaphase spread can be generated. The AFM imaging process was performed on a normal 2n = 46, XX karyotype and on a 2n = 46, XY, t(2;15)(q23;q15) karyotype as an example of a translocation of chromosomal bands.

Azure Stains↗

PubMed1999

Laser microdissection and laser pressure catapulting for the generation of chromosome-specific paint probes.

Chromosome-specific paint probes provide a powerful tool with wide applications in cytogenetic analysis. Here, we present a new approach using UV-laser microbeam microdissection in combination with laser-pressure catapulting, which allows the fast isolation of single chromosomes for the generation of chromosome-specific paint probes. To demonstrate the feasibility of this approach, single chromosomes were collected and amplified with degenerate oligonucleotide-primed PCR, hapten-labeled and hybridized onto normal metaphase spreads. Fluorescence in situ hybridization signals revealed specific painting of the respective chromosomes.

Animals↗

PubMed1997

The atomic force microscope as a new microdissecting tool for the generation of genetic probes.

The atomic force microscope (AFM) can be used to visualize and to manipulate biological material with relative case and high resolution. This study was carried out to investigate whether probe sets, specific for subregions of the human genome and useful for the painting of chromosome bands, can be established by PCR amplification of AFM-dissected chromosome regions. Compared to standard microdissection techniques, the AFM can be used with much higher precision for the dissection of the region of interest and subsequent nanoextraction of DNA material. After scanning the area of interest in noncontact mode AFM, chromosome bands were cut by the AFM tip at high force. The genetic material of a single cut attached itself to the tip and was extracted and amplified using degenerate oligonucleotide-primed-PCR. Subsequent to hapten labeling, fluorescence in situ hybridization was performed and chromosome band-specific probes were visualized by standard fluorescence microscopy.

Chromosomes, Human, Pair 2↗

PubMed1997

Cut out or poke in--the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease.

The optical micromanipulation systems UV(ultraviolet)-Laser Microbeam and Optical Tweezers Trap, already proven to be powerful tools for 'non-contact' micro-manipulation of gametes, cells and organelles, have now made their way into the nanocosmos of genes and molecules. Force measurements of DNA transcription have been performed and selective DNA molecule micromanipulation gives insight into single molecule behaviour. Retrievement of selected single cells without contamination is an import prerequisite for further processing with modern methods of molecular biology. Laser micro-dissection allows to precisely eliminate any unwanted material or to isolate pieces of chromosomes or single cells of interest with high accuracy and efficiency. This enables the cell or chromosome specific molecular analysis of genes and genetic defects underlying disease, such as cancer or infection. This review article gives an overview of current topics of laser microbeam application in biological or medical research and advanced molecular diagnosis.

Animals↗

PubMedDate not supplied

Correlative high-resolution morphologic analysis of the three-dimensional organization of human chromosomes.

A correlative morphologic analysis was carried out on isolated metaphase chromosomes by means of field emission in-lens scanning electron microscopy (FEISEM) and atomic force microscopy (AFM). Whereas FEISEM provides ultra-high resolution power and allows the surface analysis of biological structures free of any conductive coating, the AFM allows imaging of biological specimens in ambient as well as in physiologic conditions. The analysis of the same samples was made possible by the use of electrical conductive and light transparent ITO glass as specimen holder. Further preparation of the specimen specific for the instrumentation was not required. Both techniques show a high correlation of the respective morphologic information, improving their reciprocal biological significance. In particular, the biological coat represents a barrier for surface morphologic analysis of chromosome spreads and it is sensitive to protease treatment. The chemical removal of this layer permits high-resolution imaging of the chromatid fibers but at the same time alters the chromosomal dimension after rehydration. The high-resolution level, necessary to obtain a precise physical mapping of the genome that the new instruments such as FEISEM and AFM could offer, requires homogeneously cleaned samples with a high grade of reproducibility. A correlative microscopical approach that utilizes completely different physical probes provides complementary useful information for the understanding of the biological, chemical, and physical characteristics of the samples and can be applied to optimize the chromosome preparations for further improvement of the knowledge about spatial genome organization.

Chromosomes, Human↗