Comparison of human chromosome 21 conserved nongenic sequences (CNGs) with the mouse and dog genomes shows that their selective constraint is independent of their genic environment.

The analysis of conservation between the human and mouse genomes resulted in the identification of a large number of conserved nongenic sequences (CNGs). The functional significance of this nongenic conservation remains unknown, however. The availability of the sequence of a third mammalian genome, the dog, allows for a large-scale analysis of evolutionary attributes of CNGs in mammals. We have aligned 1638 previously identified CNGs and 976 conserved exons (CODs) from human chromosome 21 (Hsa21) with their orthologous sequences in mouse and dog. Attributes of selective constraint, such as sequence conservation, clustering, and direction of substitutions were compared between CNGs and CODs, showing a clear distinction between the two classes. We subsequently performed a chromosome-wide analysis of CNGs by correlating selective constraint metrics with their position on the chromosome and relative to their distance from genes. We found that CNGs appear to be randomly arranged in intergenic regions, with no bias to be closer or farther from genes. Moreover, conservation and clustering of substitutions of CNGs appear to be completely independent of their distance from genes. These results suggest that the majority of CNGs are not typical of previously described regulatory elements in terms of their location. We propose models for a global role of CNGs in genome function and regulation, through long-distance cis or trans chromosomal interactions.

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases.

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.

Direct visualization of supercoiled DNA molecules in solution.

The shape of supercoiled DNA molecules in solution is directly visualized by cryo-electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules.

Electrically assisted ocular gene therapy.

Electrotransfer and iontophoresis are being developed as innovative non-viral gene delivery systems for the treatment of eye diseases. These two techniques rely on the use of electric current to allow for higher transfection yield of various ocular cell types in vivo. Short pulses of relatively high-intensity electric fields are used for electrotransfer delivery, whereas the iontophoresis technique is based on the application of low voltage electric current. The basic principles of these techniques and their potential therapeutic application for diseases of the anterior and posterior segments of the eye are reviewed. Iontophoresis has been found most efficient for the delivery of small nucleic acid fragments such as antisense oligonucleotides, siRNA, or ribozymes. Electrotransfer, on the other hand, is being developed for the delivery of oligonucleotides or custom designed plasmids. The wide range of strategies already validated and the potential for targeting specific types of cells confirm the promising early observations made using electrotransfer and iontophoresis. These two nonviral delivery systems are safe and can be used efficiently for targeted gene delivery to ocular tissues in vivo. At the present, their application for the treatment of ocular human diseases is nearing its final stages of adaptation and practical implementation at the bedside.

DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region.

The three subtypes of the peroxisome proliferator-activated receptors (PPARalpha, beta/delta, and gamma) form heterodimers with the 9-cis-retinoic acid receptor (RXR) and bind to a common consensus response element, which consists of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1). As a first step toward understanding the molecular mechanisms determining PPAR subtype specificity, we evaluated by electrophoretic mobility shift assays the binding properties of the three PPAR subtypes, in association with either RXRalpha or RXRgamma, on 16 natural PPAR response elements (PPREs). The main results are as follows. (i) PPARgamma in combination with either RXRalpha or RXRgamma binds more strongly than PPARalpha or PPARbeta to all natural PPREs tested. (ii) The binding of PPAR to strong elements is reinforced if the heterodimerization partner is RXRgamma. In contrast, weak elements favor RXRalpha as heterodimerization partner. (iii) The ordering of the 16 natural PPREs from strong to weak elements does not depend on the core DR1 sequence, which has a relatively uniform degree of conservation, but correlates with the number of identities of the 5'-flanking nucleotides with respect to a consensus element. This 5'-flanking sequence is essential for PPARalpha binding and thus contributes to subtype specificity. As a demonstration of this, the PPARgamma-specific element ARE6 PPRE is able to bind PPARalpha only if its 5'-flanking region is exchanged with that of the more promiscuous HMG PPRE.

Nanopore detection of single molecule RNAP-DNA transcription complex.

In the past decade, a number of single-molecule methods have been developed with the aim of investigating single protein and nucleic acid interactions. For the first time we use solid-state nanopore sensing to detect a single E. coli RNAP-DNA transcription complex and single E. coli RNAP enzyme. On the basis of their specific conductance translocation signature, we can discriminate and identify between those two types of molecular translocations and translocations of bare DNA. This opens up a new perspectives for investigating transcription processes at the single-molecule level.

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

How do thyroid hormone receptors bind to structurally diverse response elements?

Three classes of thyroid hormone response elements have been described. They are composed of two half-sites arranged either as a palindromic, a direct repeat or as an inverted palindromic array. Receptor homodimers as well as heterodimers can bind to all three types of response element. While the ligand binding domain of the receptors provides the major dimerization surface, asymmetric contacts between the DNA binding domains are necessary for binding to a direct repeat. Moreover, some recent findings suggest that in TR, compared to RXR, the ligand binding domain has a 180 degrees rotation with respect to the DNA binding domain. This feature could explain the preferential binding of the RXR-TR heterodimer to the direct repeat response element, in which RXR exclusively binds the 5' half-site, and of the TR homodimer to the inverted palindrome response element.

Brownian dynamics simulation of DNA condensation.

DNA condensation observed in vitro with the addition of polyvalent counterions is due to intermolecular attractive forces. We introduce a quantitative model of these forces in a Brownian dynamics simulation in addition to a standard mean-field Poisson-Boltzmann repulsion. The comparison of a theoretical value of the effective diameter calculated from the second virial coefficient in cylindrical geometry with some experimental results allows a quantitative evaluation of the one-parameter attractive potential. We show afterward that with a sufficient concentration of divalent salt (typically approximately 20 mM MgCl(2)), supercoiled DNA adopts a collapsed form where opposing segments of interwound regions present zones of lateral contact. However, under the same conditions the same plasmid without torsional stress does not collapse. The condensed molecules present coexisting open and collapsed plectonemic regions. Furthermore, simulations show that circular DNA in 50% methanol solutions with 20 mM MgCl(2) aggregates without the requirement of torsional energy. This confirms known experimental results. Finally, a simulated DNA molecule confined in a box of variable size also presents some local collapsed zones in 20 mM MgCl(2) above a critical concentration of the DNA. Conformational entropy reduction obtained either by supercoiling or by confinement seems thus to play a crucial role in all forms of condensation of DNA.

Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver.

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

Leishmania RNA virus: when the host pays the toll.

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic "metastatic factors" or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically for the maintenance of LRV infection in Leishmania parasites, which is often at the expense of its human host.

DNA-DNA interactions in bacteriophage capsids are responsible for the observed DNA knotting.

Recent experiments showed that the linear double-stranded DNA in bacteriophage capsids is both highly knotted and neatly structured. What is the physical basis of this organization? Here we show evidence from stochastic simulation techniques that suggests that a key element is the tendency of contacting DNA strands to order, as in cholesteric liquid crystals. This interaction favors their preferential juxtaposition at a small twist angle, thus promoting an approximately nematic (and apolar) local order. The ordering effect dramatically impacts the geometry and topology of DNA inside phages. Accounting for this local potential allows us to reproduce the main experimental data on DNA organization in phages, including the cryo-EM observations and detailed features of the spectrum of DNA knots formed inside viral capsids. The DNA knots we observe are strongly delocalized and, intriguingly, this is shown not to interfere with genome ejection out of the phage.

The murine interleukin-2 receptor gamma chain gene: organization, chromosomal localization and expression in the adult thymus.

Defects in the interleukin-2 receptor gamma (IL-2R gamma) chain in the man result in an X-linked severe combined immunodeficiency, SCIDX1, characterized by an absence of T-cell differentiation. This phenotype may result from pertubations in IL-2, IL-4-, IL-7- or IL-15-mediated signaling, as the IL-2R gamma chain forms an integral component of these receptor systems. We have isolated and characterized cDNA and genomic clones for the murine IL-2R gamma. The gene (Il2rg) is well conserved between mouse and man with respect to overall structure and size, and contains regions of high conservation in the promoter region as well. Il2rg maps to mouse X chromosome region 40, in a region of synteny with human Xq12-13.1. We have also explored the expression of the IL-2R gamma during thymocyte development. IL-2R gamma transcripts are detected in the earliest thymocyte precursor cells and persist throughout intrathymic development into the mature peripheral compartment. Genomic clones for the murine IL-2R gamma will allow for further studies on the regulation and function of this gene in vivo.

Staphylococcus aureus clfB and spa alleles of the repeat regions are segregated into major phylogenetic lineages.

To reliably differentiate among Staphylococcus aureus isolates we recently developed the Double Locus Sequence Typing (DLST) based on the analysis of partial sequences of clfB and spa genes. This method is highly discriminatory and gives unambiguous definition of types. The highly clonal population structure of S. aureus suggests that isolates with identical clfB or spa alleles belong to the same clonal complex (CC) defined by Multi-Locus Sequence Typing (MLST). To test this hypothesis as well as to investigate putative intra-CC genetic structure, we analyzed a total of 289 isolates (186 MSSA and 103 MRSA) with DLST-, spa- and MLST-typing. Among the 289 strains, 242 were clustered into 7 major MLST CCs, 40 into minor CCs and 7 were not grouped into CCs. A total of 205 DLST- and 129 spa-types were observed. With one exception, all DLST-clfB, DLST-spa and spa-type alleles were segregated into CCs. DLST-types sharing an identical allele (clfB or spa) were clustered using eBURST. Except for one strain, all isolates from each DLST cluster belonged to the same CC. However, using both DLST- and spa-typing we were not able to disclose a clear intra-CC structure. Nevertheless, the high diversity of these loci confirmed that they are good markers for local epidemiological investigations.