Positioning the genome within the nucleus

Higher eukaryotic genomes contain both housekeeping genes and genes of which the expression is restricted to a defined time and space. It is well established that a correlation exists between structural organization of the genome and gene expression control. The functional mechanisms underlying this correlation are still poorly understood. Here I describe several observations that are the basis of present concepts of genome organization and nuclear architecture related to functionality. Regarding the relationship between positioning and disturbed cell functionality, I describe observations showing that the proximity of selected gene loci is statistically correlated with their propensity for oncogenic translocations as well as observations of patterns occurring in neurodegenerative disorders where unstable repeats are translated into an expanded polyglutamine tract. Such observations underscore the importance to understand how genetic perturbations lead to the global reorganization of nuclear architecture, chromatin structure and widespread changes in gene expression.     

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.     

Turtle phylogeny: insights from a novel nuclear intron

Introns have gained considerable popularity as markers for molecular phylogenetics. However, no primers exist for a nuclear intron that amplifies across all turtles. Available data from morphology and mitochondrial DNA have not unambiguously resolved relationships within the superfamily Trionychoidea and the family Chelidae, which together form a large portion of extant turtle diversity. We tested the phylogenetic utility of a novel intron from the RNA fingerprint protein 35 (R35) as applied to these two areas of turtle systematics. We found the intron to be a single-copy locus that provides excellent resolving power for lineages among turtles, though problems with alignment made it impossible to infer deeper amniote relationships. Maximum parsimony and maximum likelihood both demonstrated the polyphyly of Trionychoidea and the reciprocal monophyly of Australian/New Guinea and South American chelid turtles. This is the first study to resolve such relationships with strong statistical support, and we suggest that R35 holds great promise for resolving additional persistent problems in the phylogeny of living turtles.     

First nuclear DNA C-values for 28 angiosperm genera

This paper reports first DNA C-values for 28 angiosperm genera. These include first DNA C-values for 25 families, of which 16 are monocots. Overall familial representation is 47.2 % for angiosperms, but is now much higher for monocots (75 %) and basal angiosperms (73.1 %) than for eudicots (38.7 %). Chromosome counts are reported for 22 taxa, including first records for six genera plus seven species. Unrepresented families will become increasingly enriched for monotypic taxa from obscure locations that are harder to access. Thus, completing familial representation for genome size for angiosperms may prove impossible in any short period, and progress towards this goal will become slower.     

Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy

The interaction of Ca(2+)-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40-44) between EF-1 and EF-2, linker-3 (residues 112-117) between EF-3 and EF-4, and the end of the alpha-helix H (residues 145-148) may be involved in the binding process. The dissociation constant (K(d)), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2 x 10(-5) to 8.8 x 10(-5) M. This result demonstrates that the interaction falls into the weak binding regime.     

In vitro measurement of nuclear permeability changes in apoptosis

In the eukaryotic cell, exchange of biomolecules between nucleus and cytoplasm is a highly regulated process which responds sensitively to changes of the environment. One well-known cellular response to environmental challenges is cell death by apoptosis. In fact, apoptosis has been shown to affect the nucleocytoplasmic transport machinery, in particular the nuclear pore, by modulating its size exclusion limit for passive diffusion. The underlying molecular factors are still unknown, mainly because of the lack of a suitable system to detect and quantitate the apoptotic effects on the nuclear pore. Here we present an assay that was designed to measure alterations of the permeability of the nuclear envelope under apoptotic conditions. The assay is based on the well-established technique of selective permeabilization of the plasma membrane with digitonin and allows assessment of permeability changes in nonfixed samples. It comprises a computer program, called Nuclear Permeability Assay, for the quantitation of the nuclear fluorescence signal, which may be generally employed for the evaluation of in vitro transport systems using semipermeabilized cells, such as assays for nuclear import and export.     

Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor

The retinoid-X receptor (RXR) is a ligand activated nuclear receptor that is the heterodimer partner for many class II nuclear receptors. Previously identified natural ligands for this receptor include 9-cis retinoic acid (9cRA), docosahexaenoic acid, and phytanic acid. Our studies were performed to determine if there are any unidentified, physiologically important RXR ligands. Agonists for RXR were purified from rat heart and testes lipid extracts with the use of a cell-based reporter assay to monitor RXR activation. Purified active fractions contained a variety of unsaturated fatty acids and components were quantified by gas-liquid chromatography of derivatized samples. The corresponding fatty acid standards elicited a similar response in the reporter cell assay. Competition binding analysis revealed that the active fatty acids compete with [3H]9cRA for binding to RXR. Non-esterified fatty acids were analyzed from lipid extracts of isolated heart and testes nuclei and endogenous concentrations were found to be within the range of their determined binding affinities. Our studies reveal tissue dependent profiles of RXR agonists and support the idea of unsaturated fatty acids as physiological ligands of RXR.     

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.