In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution

BACKGROUND: It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites. RESULTS: We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA 'lectins' that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection. CONCLUSIONS: Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these 'low molecular-weight effectors' may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.     

Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions

Enthalpy-entropy compensation is a general feature of many chemical reactions and processes in biological systems, but its origin has remained obscure. A simple thermodynamic argument suggests that enthalpy-entropy compensation is a general property of weak intermolecular interactions, and that the two contributions to the free energy should nearly balance out for a hydrogen bond at 300 K.     

In vitro selection analysis of trans-acting HDV ribozyme

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.     

Ionic interactions and the global conformations of the hammerhead ribozyme

Here we investigate the global conformation of the hammerhead ribozyme. Electrophoretic studies demonstrate that the structure is folded in response to the concentration and type of ions present. Folding based on colinear alignment of arms II and III is suggested, with a variable angle subtended by the remaining helix I. In the probable active conformation, a small angle is subtended between helices I and II. Using uranyl photocleavage, an ion binding site has been detected in the long single-stranded region. The folded conformation could generate a preactivation of the scissile bond to permit in-line attack of the 2'-hydroxyl group, with a bound metal ion playing an integral role in the chemistry.     

Social evolution and the emergence of leadership.

Used a cross-generational procedure to examine E. R. Service's (1975) redistribution theory of social evolution. 432 university students participated. The procedure involved 3 4-person groups that produced different products that could be retained or traded and that could be eventually exchanged for money. One group was advantaged in the diversity of its products, the time it took to make the products, and the value of its products. All trade was channeled through this central group. At the end of each time period, 1 member of each group was removed ("deceased") and a new member added. The monetary results indicate that the central group made the most money but that all groups made more money across generations, partially due to increased trade and partially due to increased production. Consistent with Service's prediction, the central group was perceived by the "deceased" group members as the overall leader. Ratings by observers, ratings by "deceased" members, and the division of money at the end of each generation all indicated that each of the 3 groups developed a seniority rule for within-group leadership. Ratings by "deceased" members indicated that this rule was stronger for the central than for the peripheral groups. (39 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Iron-manganese interactions in the evolution of iron deficiency

Since the publication of prior reviews on this topic, substantial clinical experience with a variety of operative strategies to prevent ischaemic cord complications has been reported. The available data on angiographic localisation of critical intercostal vessels, and, in particular, the evoked potential response to cross-clamping in patients indicates that risk of paraplegia varies considerably even among patients with equivalent TAA extent. Factors such as individual development of the ASA, patent critical intercostals, and the particulars of collateral circulation when intercostal aortic ostia are already occluded likely account for this variability. Information available from SSEP monitoring relative to the dynamic course of cord ischaemia with cross-clamping, and the parallel, if not, frustrating experience with angiographic localisation and intercostal vessel reconstruction indicates that a narrow temporal threshold of cord ischaemia with clamping is present in many patients. This reinforces the importance of both expeditious clamp intervals, critical intercostal re-anastomoses, and the desirability of neuroprotective manoeuvres during cross-clamp induced cord ischemia. As suggested in compelling experimental work our contemporary clinical experience, and predicted by prior reviewers, regional cord hypothermia provides significant promise for limiting or eliminating, in particular, immediate perioperative deficits. Avoidance of postoperative hypotension, spinal cord oedema, and preservation of critical intercostal vessels are additional strategies necessary to impact the development of delayed deficits favourably.     

Molecular evolution of RNA in vitro

Experimental studies of RNA evolution in vitro are reviewed in the context of Eigen's 1971 theory and its subsequent extensions. Current research activity and future prospects for using automated molecular biology techniques for in vitro evolution experiments are surveyed.     

Experimental evolution of parasites

Serial passage experiments are a form of experimental evolution that is frequently used in applied sciences; for example, in vaccine development. During these experiments, molecular and phenotypic evolution can be monitored in real time, providing insights into the causes and consequences of parasite evolution. Within-host competition generally drives an increase in a parasite's virulence in a new host, whereas the parasite becomes avirulent to its former host, indicating a trade-off between parasite fitnesses on different hosts. Understanding why parasite virulence seldom escalates similarly in natural populations could help us to manage virulence and deal with emerging diseases.     

Structural variability and new intermolecular interactions of Z-DNA in crystals of d(pCpGpCpGpCpG)

We have determined the single crystal x-ray structure of the synthetic DNA hexamer d(pCpGpCpGpCpG) in two different crystal forms. The hexamer pCGCGCG has the Z-DNA conformation and in both cases the asymmetric unit contains more than one Z-DNA duplex. Crystals belong to the space group C222(1) with a = 69.73, b = 52.63, and c = 26.21 A, and to the space group P2(1) with a = 49.87, b = 41.26, c = 21.91 A, and gamma = 97.12 degrees. Both crystals show new crystal packing modes. The molecules also show striking new features when compared with previously determined Z-DNA structures: 1) the bases in one duplex have a large inclination with respect to the helical axis, which alters the overall shape of the molecule. 2) Some cytosine nitrogens interact by hydrogen bonding with phosphates in neighbor molecules. Similar base-phosphate interactions had been previously detected in some B-DNA crystals. 3) Basepair stacking between the ends of neighbor molecules is variable and no helical continuity is maintained between contiguous hexamer duplexes.     

Eosinophil-Cryptococcus neoformans interactions in vivo and in vitro

Eosinophils are components of inflammatory responses to a variety of pathogens. Although a variety of beneficial and harmful functions have been ascribed to these cells, their role in protection against infectious agents remains uncertain. Previous studies have reported eosinophilic pneumonia in mice infected intratracheally with Cryptococcus neoformans. We confirmed this observation and studied the inflammatory response in the lung at day 14 by light and electron microscopy. Immunostaining for glucuronoxylomannan showed isolated cryptococci inside the eosinophilic cuffs. Eosinophils were found to be in close association with C. neoformans in vivo. Cryptococci were associated with eosinophils within eosinophilic perivascular cuffs, within granulomas, and lining the alveolar space. To further investigate this phenomenon in vitro, we isolated rat peritoneal eosinophils and studied cryptococcus-eosinophil interactions in the presence and absence of anti-capsular immunoglobulin G1 (IgG1) and IgE monoclonal antibody (MAb). Eosinophils phagocytosed C. neoformans only in the presence of specific antibody. Phagocytosis was rapid, and dense rings that appeared to consist of granule contents were formed around the organisms. Mast cells were observed to occasionally phagocytose C. neoformans in vitro in the presence of IgE MAb. Our observations suggest that eosinophils may be effector cells against C. neoformans.     

Random-priming in vitro recombination: an effective tool for directed evolution

A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.     

Characterization of recombinant CD45 cytoplasmic domain proteins. Evidence for intramolecular and intermolecular interactions

CD45 is a transmembrane two-domain tyrosine phosphatase required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain phosphatases, the role of the second phosphatase domain is unclear. In this study, recombinant CD45 cytoplasmic domain proteins purified from bacteria were used to evaluate the function of the individual phosphatase domains. A recombinant protein expressing the membrane-proximal region, first phosphatase domain, and spacer region of CD45 (rD1) was catalytically active and found to exist primarily as a dimer. In contrast to this, a recombinant protein expressing the spacer region, the second phosphatase domain and the carboxy tail of CD45 (rD2) existed as a monomer and had no catalytic activity against any of the substrates tested. Comparison of rD1 with the recombinant protein expressing the entire cytoplasmic domain of CD45 (rD1/D2) indicated that rD1/D2 was 2-3-fold more catalytically active, was more thermostable, and existed primarily as a monomer. Limited trypsin digestion of rD1/D2 provided evidence for a noncovalent association between an N-terminal 27-kDa fragment and a C-terminal 53-kDa fragment, suggesting an intramolecular interaction. Furthermore, rD1 was found to specifically associate with rD2 in an in vitro binding assay. Taken together, these data provide evidence for an intramolecular interaction occurring in the cytoplasmic domain of CD45. In the absence of the C-terminal region containing the second phosphatase domain, intermolecular interactions occur, resulting in dimer formation.     

Caged RNA: photo-control of a ribozyme reaction

We report here the first photo-chemical control of a ribozyme reaction by the site-specific modification of the 2'-hydroxyl nucleophile in the hammerhead system with a caging functionality. Rapid laser photolysis of the O-(2-nitrobenzyl) caging group initiates an efficient and accurate hammerhead-catalyzed cleavage of substrate RNA under native conditions. RNAs in which reactive functionalities or recognition elements are caged in this manner will be useful tools to probe RNA reactivity and dynamics.     

A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo

Gut endocrine cells possess the capacity to take up and decarboxylate biogenic amine precursors. Decarboxylation is mediated by aromatic L-amino acid decarboxylase (AADC) which is encoded by mRNAs differing in their 5' untranslated regions (UTR) depending on the usage of alternative first exons, 1a and 1b, each with its own acceptor site (-13 and -8 bases relative to the translation start site, respectively). We describe here a novel splice variant of exon 1a-AADC mRNA in rat antral mucosa. Both exon 1a and 1b mRNAs were expressed in rat antral mucosa, but the 1a form was spliced into the acceptor site usually associated with exon 1b (-8). An enteroendocrine cell line (STC-1) expressed exon 1a or exon 1b mRNAs spliced into the -8 acceptor site of exon 2. Transient transfection of a range of cell lines with reporter constructs revealed that all three 5' UTRs efficiently supported expression of the Luciferase reporter. There is therefore a novel, functional 5' UTR of AADC mRNA in rat antral mucosa; alternative AADC splice variants could provide the capacity for control at the level of mRNA translation.