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The structure of chromatin at the level of the 30 nm fibre has been studied in considerable detail, but little is known about how this fibre is arranged within the interphase chromosome territory. Over the years, various polymer models were developed to simulate chromosome structure, for example the random-walk/giant-loop (RWGL) model, the multi-loop subcompartment (MLS) model, and the interconnected-fibre-loop model (Friedland et al., 1999). These models differ mainly in the size and arrangement of the chromatin loops and, correspondingly, in the predicted distribution of chromatin density within the nucleus. It occurred to us that densely ionising radiation can be used to probe the actual distribution of chromatin density in human interphase cells. In contrast to sparsely ionising radiation (e.g. X-rays), which induces DNA double-strand breaks (DSB) that are distributed randomly within the nucleus, irradiation with densely ionising accelerated ions leads to spatial clustering of DSB. This inhomogeneity in DSB localisation, together with an inhomogeneity of DNA density within the nucleus, causes an over-dispersion in the resulting distribution of DNA fragment sizes that can be detected by pulsed-field gel electrophoresis. Using the above-mentioned chromosome models, we performed computer simulations to predict the DNA fragment size distributions resulting from irradiation with accelerated ions, and compared the predicted distributions with those obtained experimentally. We found that simulations based on the MLS model, in which local variations in chromatin density are higher than in the other models, resulted in the best agreement between calculation and experiment.

Antibodies are currently deployed as the recognition component of sensors that detect biological threat agents. Antibodies that detect simulants of bio-threat agents are also currently incorporated into detection platforms for testing and evaluating new devices and materials. Previously, we developed an anti-botulinum toxin antibody using a powerful genetic technology known as phage display, in which a very large library of immunoglobulin (antibody) genes are expressed on the surface of bacteriophage (bacterial virus) particles. However, antibodies expressed in bacteria lack molecular modifications made post-translationally by animal (eukaryotic) cells. It was therefore desirable to express Fab fragment antibody genes in insect cell lines and larvae. The use of insects as gene expression "bioreactors" poses far fewer ethical concerns than the use of mammalian systems. In this study we improved an existing baculovirus expression vector by inserting the reporter gene DsRed, then modified and inserted the heavy and light chain genes encoding an anti-botulinum toxin-binding Fab antibody. The structures of all plasmids constructed were verified by restriction analysis and sequencing. Preliminary data demonstrate that the reporter gene DsRed is strongly expressed in larvae of Trichoplusia ni, suggesting that this system may be an economical manufacturing process for recombinant antibodies., Original contains color plates: All DTIC reproductions will be in black and white.

To approach by virtual microscopy the three-dimensional organization of the human cell nucleus, the structural-, scaling- and dynamic properties of interphase chromosomes and cell nuclei were simulated with Monte Carlo and Brownian Dynamics methods. The 30 nm chromatin fiber was folded according to the Multi-Loop- Subcompartment (MLS) model, in which ~100 kbp loops form rosettes, connected by a linker, and the Random- Walk/Giant-Loop (RW/GL) topology, in which 1-5 Mbp loops are attached to a flexible backbone. Both the MLS and the RW/GL model form chromosome territories but only the MLS rosettes result in distinct subcompartments visible with light microscopy and low overlap of chromosomes, -arms and subcompartments. This morphology and the size of subcompartments agree with the morphology found by expression of histone autofluorescent protein fusions and fluorescernce in situ hybridization (FISH) experiments. Even small changes of the model parameters induced significant rearrangements of the chromatin morphology. Thus, pathological diagnoses based on this morphology, are closely related to structural changes on the chromatin level. The position of interphase chromosomes depends on their metaphase location, and suggests a possible origin of current experimental findings. The chromatin density distribution agrees with the MLS model and recent experiments. The scaling behaviour of the chromatin fiber topology and morphology of CLSM stacks revealed fine-structured multi-scaling behaviour in agreement with the model prediction. Review and comparison of experimental to simulated spatial distance measurements between genomic markers as function of their genomic separation also favour an MLS model with loop and linker sizes of 63 to 126 kbp. Visual inspection of the morphology reveals also big spaces allowing high accessibility to nearly every spatial location, due to the chromatin occupancy <30% and a mean mesh spacing of 29 to 82 nm for nuclei of 6 to 12 µm diameter. Th

The largely unresolved sequential organization, i.e. the relations within DNA sequences, and its connection to the three-dimensional organization of genomes was investigated by correlation analyses of completely sequenced chromosomes from Viroids, Archaea, Bacteria, Arabidopsis thaliana, Saccharomyces cerevisae, Schizosaccharomyces pombe, Encephalitozoon cunniculi, Drosophila melangoster, Homo sapiens, chloroplasts and mitochondria. All sequences revealed long-range power-law correlations almost on the entire observable scale. The local correlation coefficient shows close to random correlations on the scale of a few base pairs, a first maximum from 40-3400 bp, and often a region of one or more second maxima from 10^5-3x10^5 bp. This multi-scaling behaviour is species specific and can be explained by a block organization of genomes. Within this multi-scaling behaviour an additional fine-structure is present and attributable to the codon usage in all except the human sequences. Here it is connected to nucleosomal binding. Computer generated random sequences assuming a block organization, the codon usage and nucleosomal binding agree with these results. Mutation by simulated sequence reshuffling destroyed all correlations, thus their stability seems evolutionary tightly controlled and connected to the spatial genome organization. On large scales the sequence correlations agree very well with the three-dimensional folding of the 30 nm chromatin fibre into the Multi-Loop-Subcompartment (MLS) model, in which ~100 kbp loops form rosettes, connected by a linker, within chromosomes.

To approach the three-dimensional organization of the human cell nucleus, the structural-, scaling- and dynamic properties of interphase chromosomes and cell nuclei were simulated with Monte Carlo and Brownian Dynamics methods. The 30 nm chromatin fibre was folded according to the Multi-Loop-Subcompartment (MLS) model, in which ~100 kbp loops form rosettes, connected by a linker, and the Random-Walk/Giant-Loop (RW/GL) topology, in which 1-5 Mbp loops are attached to a flexible backbone. Both the MLS and the RW/GL model form chromosome territories but only the MLS rosettes result in distinct subcompartments visible with light microscopy and low overlap of chromosomes, -arms and subcompartments. This morphology and the size of subcompartments agree with the morphology found by expression of histone auto-fluorescent protein fusions and fluorescence in situ hybridization (FISH) experiments. Even small changes of the model parameters induced significant rearrangements of the chromatin morphology. Thus, pathological diagnoses based on this morphology, are closely related to structural changes on the chromatin level. The position of interphase chromosomes depends on their metaphase location, and suggests a possible origin of current experimental findings. The chromatin density distribution of simulated confocal (CLSM) images agrees with the MLS model and with recent experiments. The scaling behaviour of the chromatin fiber topology and morphology of CLSM stacks revealed fine-structured multi-scaling behaviour in agreement with the model prediction. Review and comparison of experimental to simulated spatial distance measurements between genomic markers as function of their genomic separation also favour an MLS model with loop and linker sizes of 63 to 126 kbp. Visual inspection of the morphology reveals also big spaces allowing high accessibility to nearly every spatial location, due to the chromatin occupancy <30% and a mean mesh spacing of 29 to 82 nm for nuclei of 6 to 1

The aim of these studies is the quantitative investigation of protein-protein interactions in the AP1 system in vivo. First results of FCS measurements show an exchange in the nucleus of the proteins Fos-CFP and Jun-YFP in the stably mono-transfected HeLa-Cells. This is also shown by fitting the bleaching curves measured in the nucleus with an appropriate model. We obtained dissociation times between 10 and 20 seconds in the nucleus. In the autocorrelation function a free and an obstructed component of diffusion are shown. For further studies doubly transfected cells with both proteins, Fos-CFP and Jun-YFP, were prepared. These cells will now be characterized with FCCS to investigate the protein-protein interactions. In order to obtain the dissociation rates of the complex in the cell nucleus bleaching curves will be recorded on these cell lines. We also overexpressed and purified Jun-YFP and Fos-CFP for in vitro studies.

Despite the succesful linear sequencing of the human genome the three-dimensional arrangement of chromatin, functional, and structural components is still largely unknown. Molecular transport and diffusion are important for processes like gene regulation, replication, or repair and are vitally influenced by the structure. With a comparison between fluorescence correlation spectroscopy (FCS) experiments and simulations we show here an interdisciplinary approach for the understanding of transport and diffusion properties in the human interphase cell nucleus. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fibre within these territories are under debate: In the Random-Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop-Subcompartment (MLS) model loops of around 120 kbp are forming rosettes which are also interconnected by the chromatin fibre. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as pre

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fiber within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes, which are also interconnected by the chromatin fiber. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescernce in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labeling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distrib

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fiber within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes, which are also interconnected by the chromatin fiber. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescernce in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labeling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distrib

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fiber within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes, which are also interconnected by the chromatin fiber. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescernce in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labeling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distrib

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fibre within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes which are also interconnected by the chromatin fibre. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distribu

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fibre within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes which are also interconnected by the chromatin fibre. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distribu

To study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models, which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Carlo and Brownian dynamics simulations we managed to model the chromatin fiber as a wormlike-chain polymer. A typical chromosome consists of 20.000 and a nucleus with all 46 chromosomes of 1.200.000 polymer chain segments. The parallel simulations are performed on a SP2512 and a Cray T3E. With fluorescent in situ hybridization and confocal microscopy we determined genomic marker distributions and chromosome arm overlap. Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model (126 kbp loops connected to rosettes connected by a 126 kbp chromatin linker). A fractal analysis of simulations leads to multi-fractal behaveour in good agreement with porous network research. The formation of chromosome territories was shown as predicted and low overlap of chromosomes and their arms was also reached in contrast to other models.

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown. However, the regulation of genes - their transcription and replication - has been shown to be closely connected to the three-dimensional organization of the genome and the cell nucleus. On the bases of polymer physics we have recently developed detailed and quantitative structural models for the folding of the 30 nm chromatin fiber within the human interphase cell nucleus. A quantitative test of several plausible theories resulted in a best agreement between computer simulations of chromosomes, cell nuclei and experiments for the so-called Multi-Loop-Subcompartment (MLS) model. Results concern the following properties: overlap of chromosome territories, -arms, -bands, 3D spatial distances between genomic markers as function of their genomic separation in base pairs, fractal analysis of simulations, mass distribution of chromatin in cell nuclei and the fragmentation distribution of cellular DNA after irradiation with carbon ions. Thus in an analogy to the Bauhaus principle that „form follows function“, analyzing in which form DNA is organized might help us to understand genomic function.

The synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has been shown to be connected closely to the three-dimensional organization of the genome in the cell nucleus. Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown.

To study the three-dimensional organization of chromosome territories and the human interphase cell nucleus we developed models which could be compared to experiments. Despite the successful linear sequencing of the human genome its 3D-organization is widely unknown. Using Monte Carlo and Brownian dynamics simulations we managed to model the chromatin fiber as a wormlike-chain polymer. A typical chromosome consists of 20.000 and a nucleus with all 46 chromosomes of 1.200.000 polymer chain segments. The parallel simulations are performed on a SP2512 and a Cray T3E. With fluorescent in situ hybridization and confocal microscopy we determined genomic marker distributions and chromosome arm overlap. Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model (126 kbp loops connected to rosettes connected by a 126 kbp chromatin linker). A fractal analysis of simulations leads to multi-fractal behaveour in good agreement with porous network research. The formation of chromosome territories was shown as predicted and low overlap of chromosomes and their arms was also reached in contrast to other models. Thus, the human interphase cell nucleus shows a higher degree of determinism than previously thought.

The synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has been shown to be connected closely to the threedimensional organization of the genome in the cell nucleus. Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown. The nucleus of the cell has for a long time been viewed as a 'spaghetti soup' of DNA bound to various proteins without much internal structure, except during cell division when chromosomes are condensed into separate entities. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in the interphase, i.e. between cell divisions. In an analogy of the Bauhaus principle that "form follows function" we believe that analyzing in which form DNA is organized in these territories will help us to understand genomic function. We use computer models - Monte Carlo and Brownian dynamics simulations - to develop plausible proposals for the structure of the interphase genome and compare them to experimental data. In the work presented here, we simulate interphase chromosomes for different folding morphologies of the chromatin fiber which is organized into loops of 100kbp to 3 Mbp that can be interconnected in various ways. The backbone of the fiber is described by a wormlike-chain polymer whose diameter and stiffness can be estimated from independent measurements. The implementation describes this polymer as a segmented chain with 3000 to 20000 segments for chromosome 15 depending on the phase of the simulation. The modeling is performed on a parallel computer (IBM SP2 with 80 nodes). We also determine genomic marker distributions within the Prader- Willi-Region on chromosome 15q11.2-13.3. For these measurements we use a fluorescence in situ hybridisatio

The objective of this work is development of two modules. These modules implement two discipline of Active Queue Management - DSRED and SDRED.