Genome Function and Nuclear Architecture: From Gene Expression to Nanoscience
The Jackson Laboratory, Bar Harbor, Maine 04609, USA. Genome Research
(Impact Factor: 14.63).
07/2003; 13(6A):1029-41. DOI: 10.1101/gr.946403
Biophysical, chemical, and nanoscience approaches to the study of nuclear structure and activity have been developing recently and hold considerable promise. A selection of fundamental problems in genome organization and function are reviewed and discussed in the context of these new perspectives and approaches. Advancing these concepts will require coordinated networks of physicists, chemists, and materials scientists collaborating with cell, developmental, and genome biologists.
Available from: Christoph Cremer
- "In recent years, nuclear genome structure has emerged as a central theme of cell biology (for reviews, see Lamond and Earnshaw 1998; Cremer and Cremer 2001; O'Brien et al. 2003; Misteli 2005, 2007; Rouquette et al. 2010). To study chromatin nanostructure , methods of ultrastructure analysis using ionising radiation, in particular electron microscopy, have proven to be a valuable tool for imaging beyond the conventional optical resolution of light microscopy , until recently thought to pose an absolute limit of about half a wavelength or 200 nm in the object plane and around 600 nm in the direction of the optical axis (Abbe 1873; Rayleigh 1896). "
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ABSTRACT: The optical resolution of conventional far field fluorescence light microscopy is restricted to about 200 nm laterally and 600 nm axially and has been thought for many decades to be an insurmountable barrier for the quantitative spatial analysis of cellular and hence also nuclear constituents. Novel approaches in light microscopy have now overcome this barrier. Here, we report on a special method of localisation microscopy, spectral precision distance/position determination microscopy and its combination with fluorescence in situ hybridization to analyse the spatial distribution of specific DNA sequences in human cell nuclei at the macromolecular optical resolution level. As an example, repetitive DNA sequence DYZ2 located within the heterochromatin region on human chromosome Yq12 was labelled with clone pHY2.1. Between 300 and 700 single-probe molecules were resolved in individual chromatin domains, corresponding to a detected molecule density around 500/μm(2), i.e., many times higher than resolvable by conventional fluorescence microscopy. A mean localisation accuracy of about 20 nm indicated a mean optical resolution in the 50 nm range. Beyond new perspectives for light microscopic studies of specific chromatin nanostructures, this may open a new avenue towards the general analysis of copy number of specific DNA sequences in small regions of individual interphase nuclei.
Chromosome Research 01/2011; 19(1):5-23. DOI:10.1007/s10577-010-9171-6 · 2.48 Impact Factor
Available from: Daisuke Miyoshi
- "The nucleus is also one of the most important organelles because the determination of genome organization within the nucleus is a key to understanding the regulation mechanisms of genome functions. Eukaryotic nuclei are heterogeneous; they contain a variety of subnuclear structures such as the nucleolus , splicing-factor compartments, Cajal bodies, promyelocytic leukemia bodies, replication factors, and transcription factors (Fig. 1C)  . In contrast to the cytoplasmic organelles , which are largely discrete, membrane-bound structures, the subnuclear structures lack membrane boundaries. "
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ABSTRACT: Living cells contain a variety of biomolecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These biomolecules occupy a significant fraction (20-40%) of the cellular volume. The total concentration of biomolecules reaches 400gL(-1), leading to a crowded intracellular environment referred to as molecular crowding. Therefore, an understanding of the effects of molecular crowding conditions on biomolecules is important to broad research fields such as biochemical, medical, and pharmaceutical sciences. In this review, we describe molecular conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochemical and biophysical consequences of molecular crowding. Finally, we discuss the effect of molecular crowding on the structure, stability, and function of nucleic acids and the significance of molecular crowding in biotechnology and nanotechnology.
Biochimie 08/2008; 90(7):1040-51. DOI:10.1016/j.biochi.2008.02.009 · 2.96 Impact Factor
Available from: Dominique Ploton
- "The nucleus is a highly organized cell compartment where numerous functions take place, such as modulation of chromatin compaction, synthesis of DNA, transcription, and maturation of RNA (reviewed in Cremer and Cremer, 2001; O'Brien et al., 2003; Thiry and Goessens, 1996). Very recent work has demonstrated that nuclear proteins diVuse rapidly throughout the volume of the nucleus, and that their local concentration is very high within the sub-volumes where they are active (Bubulya and Spector, 2004; Gorski and Misteli, 2005; Misteli, 2005). "
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ABSTRACT: Fluoronanogold (FNG) contains a fluorochrome molecule (FITC, Alexa, and so on) and a 1.4-nm cluster of gold atoms; these permit visualization both by photon microscopy (direct visualization) and by electron microscopy (EM) (after silver or gold enhancement). Classical applications of FNG concern immunolabeling of any antigen and its visualization with cellular imaging in two dimensions (2D), that is, using wide-field optical microscopy and EM (ultrathin sections). In the present chapter, we show that FNG can be localized in three dimensions (3D), both by confocal light microscopy and electron tomography. These approaches are useful for investigating the volumetric organization of proteins within well-preserved organelles at different levels of resolution (200 nm in confocal microscopy and 10 nm in electron tomography), as shown with two different nucleolar proteins.
Methods in cell biology 02/2007; 79(79):559-74. DOI:10.1016/S0091-679X(06)79022-7 · 1.42 Impact Factor
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