The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs

Background

Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields.

Results

We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems.

Conclusions

This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.     

The enzymatic basis of processivity in lambda exonuclease

λ Exonuclease is a highly processive 5′→3′ exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by λ exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of λ biology, λ exonuclease enzymology and the availability of the X-ray crystallographic structure of λ exonuclease make it a suitable model to dissect the mechanisms of processivity. λ Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in λ exonuclease involved in recognizing the 5′-phosphate at the ends of broken dsDNA. The preference of λ exonuclease for a phosphate moiety at 5′ dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5′-phosphate is due to the formation of inert enzyme–substrate complexes. By examining a λ exonuclease mutant impaired in 5′-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of λ exonuclease has been elucidated. We propose a model in which processivity of λ exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.     

Stem cells and the regulation of proliferation, differentiation and patterning in the intestinal epithelium: emerging insights from gene expression patterns, transgenic and gene ablation studies

Tissues that undergo self-renewal such as the skin, the haematopoeitic system and the intestine are all maintained and renewed by a small group of multipotent stem cells. The stem cells of the intestinal epithelium are located in the crypts and give rise to its four main lineages located mainly in the finger like projections- the villi. An increasing number of genes are now being identified as either being necessary for or involved in the maintenance of intestinal stem cells and regulating differentiation along the crypt-villus axis. These developmental regulatory genes include among others, Tcf-4, Cdx-1 Fkh6, HFH11 and Nkx2-3. Other genes such as the integrins, and Indian hedgehog (Ihh) also affect function of the progenitor cells of the intestinal epithelium. This mini-review will focus on the more recent data on expression patterns of genes in the intestinal epithelium and the direct or indirect effects of their ablation on proliferation and differentiation.     

A C-terminal region dictates the apical plasma membrane targeting of the human sodium-dependent vitamin C transporter-1 in polarized epithelia

The human sodium-dependent vitamin C transporter (hSVCT1) mediates sodium-dependent cellular uptake of the essential micronutrient l-ascorbic acid (vitamin C). However, the molecular determinants that control the cell surface expression, subcellular distribution, and dynamics of hSVCT1 remain undefined. To identify molecular determinants involved in hSVCT1 targeting in polarized epithelia, we used live cell imaging approaches to resolve the targeting and trafficking dynamics of hSVCT1 truncation mutants in renal and intestinal cells. Confocal imaging demonstrated that hSVCT1 was expressed at the apical cell surface and video rate measurements revealed hSVCT1 also resided in a heterogeneous population of intracellular organelles with discrete dynamic properties. By progressive truncation of the cytoplasmic C-terminal tail of hSVCT1, we delimited an essential role for an embedded ten amino acid sequence PICPVFKGFS (amino acids 563-572) in defining the physiological targeting of hSVCT1. Intriguingly, this sequence bears significant homology to recently identified apical targeting motifs in two other sodium-dependent transporters, and we suggest this conservation is reflected topologically through the adoption of a beta-turn confirmation in the cytoplasmic C-tail of each transporter. Our results provide the first direct resolution of functional hSVCT1 expression at the apical cell surface of polarized epithelia and define an apical targeting signal of relevance to transporters of diverse substrate specificity.     

Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures

Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.     

Effect of aspartame on circadian oscillations of calcium and inorganic phosphorus in rats

The effect of aspartame on circadian rhythms of calcium and inorganic phosphorus levels was studied in rats. Acrophase delays in calcium rhythms and advances in inorganic phosphorus rhythms and alteration in mesor values in both rhythms were observed in aspartame-treated rats. However, no change in amplitude values was observed. Oral administration of aspartame leads to increased levels of aspartate in the brain, which could alter the characteristics of calcium and inorganic phosphorus rhythms, possibly by modulating transmission in several areas/nuclei in brain, including retinohypothalamic tract (RHT) and suprachiasmatic nuclei (SCN).     

Artificial neural network as an alternative to multiple regression analysis in optimizing formulation parmaeters of cytarabine liposomes

The objective of the study was to optimize the formulation parameters of cytarabine liposomes by using artificial neural networks (ANN) and multiple regression analysis using 3³ factorial design (FD). As model formulations, 27 formulations were prepared. The formulation variables, drug (cytarabine)/lipid (phosphatidyl choline [PC] and cholesterol [Chol]) molar ratio (X ₁, PC/Chol in percentage ratio of total lipids (X ₂), and the volume of hydration medium, (X ₃) were selected as the independent variables; and the percentage drug entrapment (PDE) was selected as the dependent variable. A set of causal factors was used as tutorial data for ANN and fed into a computer. The optimization was performed by minimizing the generalized distance between the predicted values of each response and the optimized one that was obtained individually. In case of 3³ factorial design, a second-order full-model polynomial equation and a reduced model were established by subjecting the transformed values of independent variables to multiple regression analysis, and contour plots were drawn using the equation. The optimization methods developed by both ANN and FD were validated by preparing another 5 liposomal formulations. The predetermined PDE and the experimental data were compared with predicted data by pairedt test, no statistically significant difference was observed. ANN showed less error compared with multiple regression analysis. These findings demonstrate that ANN provides more accurate prediction and is quite useful in the optimization of pharmaceutical formulations when compared with the multiple regression analysis method.