Prediction of human mRNA donor and acceptor sites from the DNA sequence

Artificial neural networks have been applied to the prediction of splice site location in human pre-mRNA. A joint prediction scheme where prediction of transition regions between introns and exons regulates a cutoff level for splice site assignment was able to predict splice site locations with confidence levels far better than previously reported in the literature. The problem of predicting donor and acceptor sites in human genes is hampered by the presence of numerous amounts of false positives: here, the distribution of these false splice sites is examined and linked to a possible scenario for the splicing mechanism in vivo. When the presented method detects 95% of the true donor and acceptor sites, it makes less than 0.1% false donor site assignments and less than 0.4% false acceptor site assignments. For the large data set used in this study, this means that on average there are one and a half false donor sites per true donor site and six false acceptor sites per true acceptor site. With the joint assignment method, more than a fifth of the true donor sites and around one fourth of the true acceptor sites could be detected without accompaniment of any false positive predictions. Highly confident splice sites could not be isolated with a widely used weight matrix method or by separate splice site networks. A complementary relation between the confidence levels of the coding/non-coding and the separate splice site networks was observed, with many weak splice sites having sharp transitions in the coding/non-coding signal and many stronger splice sites having more ill-defined transitions between coding and non-coding.     

Detection of multivalent interactions through two-tiered energy transfer

A method based on two-tiered fluorescence resonant energy transfer (FRET) has been developed for selective and sensitive detection of species involved in a multivalent interaction. Pentavalent binding between cholera toxin and ganglioside GM1 is used as a model system to demonstrate the advantage of the two-tiered FRET over one-stage FRET in both conventional fluorimeter and flow cytometer. In the system, three fluorescent probes (namely, fluorescence donor, acceptor, and intermediate) are covalently tagged to receptors, and the intermediate is used to bridge the energy transfer between the donor and acceptor even though the donor's fluorescence spectrum does not overlap with absorption spectrum of the acceptor. One of the most significant improvements of the scheme over one-stage FRET is a dramatic decrease in the background fluorescence of the acceptor fluorescence, which, theoretically and practically, increases the detection sensitivity.     

Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing

Alternative splicing of alpha-tropomyosin pre-mRNA involves mutually exclusive utilization of exons 2 and 3, exon 3 being preferentially selected in most cells. This mutually exclusive behavior is enforced by absolute incompatibility between the adjacent splice sites of the two exons, due to close proximity of the exon 3 branch point to exon 2. The branch point, with an associated polypyrimidine tract, is in an unusual location, 177 nt upstream of the acceptor, only 42 nt from the exon 2 splice donor site. Splicing of exon 2 to 3 is consequently blocked prior to formation of an active spliceosome complex. This block to splicing can be relieved by insertion of spacer elements that increase the donor site-branch point separation to 51-59 nt. The unconventional relative location of the constitutive cis splicing elements therefore provides a simple mechanistic basis for strict mutually exclusive splicing. These results not only demonstrate that the branch point is not specified by proximity to the splice acceptor site, but rather suggest that it is the acceptor site which is specified relative to the branch point.     

Concurrent Optimization of Organic Donor–Acceptor Pairs through Machine Learning

In this work an instance of the general problem occurring when optimizing multicomponent materials is treated: can components be optimized separately or the optimization should occur simultaneously? This problem is investigated from a computational perspective in the domain of donor–acceptor pairs for organic photovoltaics, since most experimental research reports optimization of each component separately. A collection of organic donors and acceptors recently analyzed is used to train nonlinear machine learning models of different families to predict the power conversion efficiency of donor–acceptor pairs, considering computed electronic and structural parameters of both components. The trained models are then used to predict photovoltaic performance for donor–acceptor combinations for which experimental data are not available in the data set. Data structure, and the usefulness of the trained models are critically assessed by predicting some donor–acceptor pairs that recently appeared in the literature, and the best combinations are proposed as worth investigating experimentally.     

High-throughput screening methods for selecting -threonine aldolases with improved activity

More and more, aldolases are being recognized as useful catalysts that carry out the reversible addition of a ketone donor to an aldehyde acceptor in achieving high stereoselectivity. Threonine aldolases catalyze the synthesis of variable β-hydroxy-α-amino acids, which are important structural units of various antibiotics and immunosuppressants. However, the enzymatic properties need to be improved to support a broader application to synthetic chemistry. Although directed-evolution is a powerful tool for improving enzymatic properties, the successful outcome depends on the efficiency of screening systems. We designed and proposed two high-throughput screening schemes for selecting -threonine aldolase mutants with improved properties. These schemes utilized the toxicity of aldehyde, which acts as an acceptor in the aldol condensation. In these schemes, the following occurs: (1) the higher -threonine aldolase activity reduces the toxic effect of aldehyde, which leads to the survival of the corresponding clone (the positive-selection scheme), and (2) the higher -threonine aldolase activity produces more toxic aldehyde, which causes the death of the corresponding clone (the negative-selection scheme). According to the positive-selection scheme, we successfully selected -threonine aldolase mutants with higher activities than the wild-type, from a randomly generated LTA library.     

Transitional endoplasmic reticulum membranes and vesicles isolated from animals and plants

Summary The process of formation from endoplasmic reticulum and transfer to Golgi apparatus of small 50–70 nm transition vesicles has been reconstituted in a cell-free system. Fractions enriched in transition elements derived from part-rough, part-smooth transitional regions of the endoplasmic reticulum were prepared from elongation zones of hypocotyls of etiolated seedlings of soybean and coleoptiles of maize and were compared with those from rat liver. When activated with nucleoside triphosphate, cytosol and an ATP regenerating system, time- and temperature-dependent transfer of membranes to Golgi apparatus acceptor was demonstrated. The fractions enriched in transition elements were radioiodinated with¹²⁵I by the Bolton-Hunter procedure. Acceptor Golgi apparatus stacks were immobilized to nitrocellulose strips to facilitate analysis. In heterologous transfer experiments, the plant and animal acceptors and donors could be interchanged. The transfer was limited primarily by the donor (rat liver > soybean hypocotyl > maize coleoptiles) and determined secondarily by the source of the acceptor. The acceptor fractions were most efficacious when prepared from the same source as the donor. Thus, 50–70 nm vesicles bud from transitional endoplasmic reticulum elements of plants function in a manner similar to those of animal cells to transfer membrane materials to the Golgi apparatus. The recognition signals that determine vesicle fusion appear to be conserved both among species and between the plant and animal kingdoms to the extent that donor and acceptor sources may be interchanged with only small reductions in overall efficiency of transfer.Abbrevations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid     

Distribution of exonic splicing enhancer elements in human genes

Ab initio prediction of functional exon splicing enhancer (ESE) elements based on RNA sequences present a challenge in the evaluation of the functional impacts of human genetic polymorphisms on splicing. To better understand the behavior of ESEs, we studied their distribution in human exons and introns for four known SR protein-binding motifs: SF2/SAF, SC35, SRp40, and SRp55. ESEs are enriched in regions in exons that are close to the splice sites, especially in the region 80 to 120 bases away from the ends of splice acceptor sites. Significant enrichment of ESEs is associated with weak splice acceptor sites but not weak donor sites. ESE density decreases at the 3 ends of long exons. ESEs are also enriched in introns with weak donor or acceptor sites. These characteristics of ESEs may help to predict functional ESE sites in RNA sequences.     

Determination of the mechanism and kinetic constants for hog kidney gamma-glutamyltransferase.

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.     

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.     

A clean data set of EST-confirmed splice sites from Homo sapiens and standards for clean-up procedures.

A clean data set of verified splice sites from Homo sapiens are reported as well as the standards used for the clean-up procedure. The sites were validated by: (i) standard cleaning procedures such as requiring consistency in the annotation of the gene structural elements, completeness of the coding regions and elimination of redundant sequences; (ii) clustering by decision trees coupled with analysis of ClustalW alignments of the translated protein sequence with homologous proteins from SWISS-PROT; (iii) matching against human EST sequences. The sites are categorised as: (i) donor sites, a set of 619 EST-confirmed donor sites, for which 138 are either the sites or the regions around the sites involved in alternative splice events; (ii) acceptor sites, a set of 623 EST-confirmed acceptor sites, for which 144 are either the sites or the regions around the sites are involved in alternative splice events; (iii) genuine splice sites, a set of 392 splice sites wherein both the donor and acceptor sites had EST confirmation and were not involved in any alternative splicing; (iv) alternative splice sites, a set of 209 splice sites wherein both the donor and acceptor sites had EST confirmation and the sites or the regions around them were involved in alternative splicing. A set of nucleotide regions that can be used to generate a control set of false splice sites that have a high confidence of being non-functional are also reported.     

Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes

A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a quencher. Different peptides bearing Rox at the amino terminus and Fam linked through different diamines to the carboxyl terminus were synthesized to examine the effects of the chain length and rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylenediamine-Fam versus free ROX with 488 nm excitation was approximately 14 and is similar to that obtained for optimized oligonucleotide primers bearing the same dyes [Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351].     

Charge Transport in Pure and Mixed Phases in Organic Solar Cells

In organic solar cells continuous donor and acceptor networks are considered necessary for charge extraction, whereas discontinuous neat phases and molecularly mixed donor–acceptor phases are generally regarded as detrimental. However, the impact of different levels of domain continuity, purity, and donor–acceptor mixing on charge transport remains only semiquantitatively described. Here, cosublimed donor–acceptor mixtures, where the distance between the donor sites is varied in a controlled manner from homogeneously diluted donor sites to a continuous donor network are studied. Using transient measurements, spanning from sub‐picoseconds to microseconds photogenerated charge motion is measured in complete photovoltaic devices, to show that even highly diluted donor sites (5.7%–10% molar) in a buckminsterfullerene matrix enable hole transport. Hopping between isolated donor sites can occur by long‐range hole tunneling through several buckminsterfullerene molecules, over distances of up to ≈4 nm. Hence, these results question the relevance of “pristine” phases and whether a continuous interpenetrating donor–acceptor network is the ideal morphology for charge transport.     

Remote control of alpha- or beta-stereoselectivity in (1-->3)-glucosylations in the presence of a C-2 ester capable of neighboring-group participation

In (1-->3)-glucosylation the glycosyl bond originally present in either donor or acceptor is shown to control the stereoselectivity of the forthcoming bond, i.e., the newly formed glycosidic linkage has the opposite anomeric configuration of that of either the donor or acceptor. Therefore, with alpha-(1-->3)-linked disaccharides with nonreducing ends that have the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with an alpha-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with 3-OH free as the acceptor, beta-linked trisaccharides were obtained. Meanwhile, with beta-(1-->3)-linked disaccharides that have nonreducing ends with the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with a beta-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with the 3-OH free as the acceptor, alpha-linked trisaccharides were obtained in spite of the C-2 neighboring group participation.     

Crystal structures of the T4 phage beta-glucosyltransferase and the D100A mutant in complex with UDP-glucose: glucose binding and identification of the catalytic base for a direct displacement mechanism

T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.     

Lipid transfer between vesicles: effect of high vesicle concentration

The problem of the desorption of a lipid molecule from a lipid vesicle (donor) and its incorporation into another vesicle (acceptor) at high acceptor concentrations, which has been investigated experimentally (Jones, J. D. and Thompson, T. E., 1990. Biochemistry, 29:1593-1600), is analyzed here from a theoretical point of view, formulated in terms of the diffusion equation with appropriate boundary conditions. The goal is to determine whether or not the observed acceleration of the off-rate from a donor is caused by interaction with an acceptor vesicle at short range, or is simply the result of statistical effects due the proximity of the acceptor and its influence on the probability of the test lipid returning to the donor. We establish a correspondence between the theoretical parameters and the experimental, thermodynamic and dynamic variables entering the problem. The solution shows that, because of the extremely high Gibbs activation energy for desorption of a phospholipid, the process would always be first-order, even at very high vesicle concentrations. This means that acceleration of the off-rate must be due to donor-acceptor interactions at short distances, as proposed in the experimental work.     

Electron tunneling pathways in proteins: influences on the transfer rate

A strategy for calculating the tunneling matrix element dependence on the medium intervening between donor and acceptor in specific proteins is described. The scheme is based on prior studies of small molecules and is general enough to allow inclusion of through bond and through space contributions to the electronic tunneling interaction. This strategy should allow the prediction of relative electron transfer rates in a number of proteins. It will therefore serve as a design tool and will be explicitly testable, in contrast with calculations on single molecules. As an example, the method is applied to ruthenated myoglobin and the tunneling matrix elements are estimated. Quantitative improvements of the model are described and effects due to motion of the bridging protein are discussed. The method should be of use for designing target proteins having tailored electron transfer rates for production with site directed mutagenesis. The relevance of the technique to understanding certain photosynthetic reaction center electron transfer rates is discussed.     

Concerning the mechanism of action of bovine liver phospholipid exchange protein: Exchange or net transfer

Bovine liver phospholipid exchange protein catalyzes the transfer of phosphatidylcholine between donor and acceptor populations of single bilayer phospholipid vesicles. In comparing egg and dimyristoylphosphatidylcholine vesicles, larger transfer rates are found for the unsaturated phospholipid. The bidirectional transfer rates measured from donor to acceptor and from acceptor to donor, are equivalent, suggesting that the protein facilitates an exchange rather than a net transfer of phosphatidylcholine.