Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response.

BACKGROUND: Bovine milk contains extracellular vesicles (EVs) that play a role in cellular communication, acting in either an autocrine, paracrine, or an exocrine manner. The unique properties of the EVs protect the cargo against degradation. We profiled the ncRNAs (non-coding RNA) present in the EVs from seven dairy products - raw whole milk, heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, pasteurized heavy whipping cream, sweet cream buttermilk and cultured buttermilk with four replicates each, obtained at different processing steps from a commercial dairy plant. EVs and their cargo were extracted by using a validated commercial kit that has been shown to be efficient and specific for EVs. Further, to find the annotation of ncRNAs, we probed bovine and other organism repositories(such as miRBase, miRTarBase, Ensemble) to find homolog ncRNA annotation in case the annotations of ncRNA are not available in Bos Taurus database. RESULTS: Specifically, 30 microRNAs (miRNAs), were isolated throughout all the seven milk samples, which later when annotated with their corresponding 1546 putative gene targets have functions associated with immune response and growth and development. This indicates the potential for these ncRNAs to beneficially support mammary health and growth for the cow as well as neonatal gut maturation. The most abundant miRNAs were bta-miR-125a and human homolog miR-718 based on the abundance values of read count obtained from the milk samples.bta-miR-125a is involved in host bacterial and viral immune response, and human homolog miR-718 is involved in the regulation of p53, VEGF, and IGF signaling pathways, respectively. Sixty-two miRNAs were up-regulated and 121 miRNAs were down-regulated throughout all the milk samples when compared to raw whole milk. In addition, our study explored the putative roles of other ncRNAs which included 88 piRNAs (piwi-interacting RNA), 64 antisense RNAs, and 105 lincRNAs (long-intergenic ncRNAs) contained in the bovine exosomes. CONCLUSION: Together, the results indicate that bovine milk contains significant numbers of ncRNAs with putative regulatory targets associated with immune- and developmental-functions important for neonatal bovine health, and that processing significantly affects the ncRNA expression values; but statistical testing of overall abundance(read counts) of all miRNA samples suggests abundance values aren't much affected. This can be attributed to the breakage of exosomal vesicles during the processing stages. It is worth noting, however, that these gene regulatory targets are putative, and further evidence could be generated through experimental validation.

Structure-derived potentials and protein simulations.

There has recently been an explosion in the number of structure-derived potential functions that are based on the increasing number of high-resolution protein crystal structures. These functions differ principally in their reference states; the usual two classes correspond either to initial solvent exposure or to residue exposure of residues. Reference states are critically important for applications of these potentials functions. Inspection of the potential functions and their derivation can tell us not only about protein interaction strengths themselves, but can also provide suggestions for the design of better folding simulations. An appropriate goal in this field is achieving self-consistency between the details in the derivation of potentials and the applied simulations.

Distinct patterns in homooligomer tract sequence context in prokaryotic and eukaryotic DNA.

The distributions of the junction sequences of homooligomer tracts of various lengths have been examined in prokaryotic DNA sequences and compared with those of eukaryotes. The general trends in the nearest and next to nearest neighbors to the tracts are similar for both groups. In both prokaryotes and eukaryotes A/T runs are preferentially flanked on either the 5' or the 3' ends by A and/or T. G/C runs are preferentially flanked by G and/or C. There is discrimination against A/T runs flanked by G or C and G/C runs flanked by A or T. However, whereas the distribution of prokaryotic homooligomer tract junction sequences was quite homogeneous, large variations were observed in the 5-fold larger eukaryotic database, increasing in magnitude from tracts of length 2 to 3 to 4 base pairs long. Possible DNA conformational implications and in particular DNA curvature and packaging aspects of prokaryotes and eukaryotes are discussed.

Protein sequence entropy is closely related to packing density and hydrophobicity.

We investigated the correlation between the Shannon information entropy, 'sequence entropy', with respect to the local flexibility of native globular proteins as described by inverse packing density. These are determined at each residue position for a total set of 130 query proteins, where sequence entropies are calculated from each set of aligned residues. For the accompanying aggregate set of 130 alignments, a strong linear correlation is observed between the calculated sequence entropy and the corresponding inverse packing density determined at an associated residue position. This region of linearity spans the range of C(alpha) packing densities from 12 to 25 amino acids within a sphere of 9 angstrom radius. Three different hydrophobicity scales all mimic the behavior of the sequence entropies. This confirms the idea that the ability to accommodate mutations is strongly dependent on the available space and on the propensity for each amino acid type to be buried. Future applications of these types of methods may prove useful in identifying both core and flexible residues within a protein.

Inter-residue potentials in globular proteins and the dominance of highly specific hydrophilic interactions at close separation.

Residue-specific potentials between pairs of side-chains and pairs of side-chain-backbone interaction sites have been generated by collecting radial distribution data for 302 protein structures. Multiple atomic interactions have been utilized to enhance the specificity and smooth the distance-dependence of the potentials. The potentials are demonstrated to successfully discriminate correct sequences in inverse folding experiments. Many specific effects are observable in the non-bonded potentials; grouping of residue types is inappropriate, since each residue type manifests some unique behavior. Only a weak dependence is seen on protein size and composition. Effective contact potentials operating in three different environments (self, solvent-exposed and residue-exposed) and over any distance range are presented. The effective contact potentials obtained from the integration of radial distributions over the distance interval r < or = 6.4 A are in excellent agreement with published values. The hydrophobic interactions are verified to be dominantly strong in this range. Comparison of these with a newly derived set of effective contact potentials for closer inter-residue separations (r < or = 4.0 A) demonstrates drastic changes in the most favorable interactions. In the closer approach case, where the number of pairs with a given residue is approximately one, the highly specific interactions between charged and polar side-chains predominate. These closer approach values could be utilized to select successively the relative positions and directions of residue side-chains in protein simulations, following a hierarchical algorithm optimizing side-chain-side-chain interactions over the two successively closer distance ranges. The homogeneous contribution to stability is stronger than the specific contribution by about a factor of 5. Overall, the total non-bonded interaction energy calculated for individual proteins follows a dependence on the number of residues of the form of n1.28, indicating an enhanced stability for larger proteins.

Vibrational dynamics of transfer RNAs: comparison of the free and synthetase-bound forms.

The vibrational dynamics of transfer RNAs, both free, and complexed with the cognate synthetase, are analyzed using a model (Gaussian network model) which recently proved to satisfactorily describe the collective motions of folded proteins. The approach is similar to a normal mode analysis, with the major simplification that no residue specificity is taken into consideration, which permits us (i) to cast the problem into an analytical form applicable to biomolecular systems including about 10(3 )residues, and (ii) to acquire information on the essential dynamics of such large systems within computational times at least two orders of magnitude shorter than conventional simulations. On a local scale, the fluctuations calculated for yeast tRNAPhe and tRNAAsp in the free state, and for tRNAGln complexed with glutaminyl-tRNA synthetase (GlnRS) are in good agreement with the corresponding crystallographic B factors. On a global scale, a hinge-bending region comprising nucleotides U8 to C12 in the D arm, G20 to G22 in the D loop, and m7G46 to C48 in the variable loop (for tRNAPhe), is identified in the free tRNA, conforming with previous observations. The two regions subject to the largest amplitude anticorrelated fluctuations in the free form, i.e. the anticodon region and the acceptor arm are, at the same time, the regions that experience the most severe suppression in their flexibilities upon binding to synthetase, suggesting that their sampling of the conformational space facilitates their recognition by the synthetase. Likewise, examination of the global mode of motion of GlnRS in the complex indicates that residues 40 to 45, 260 to 270, 306 to 314, 320 to 327 and 478 to 485, all of which cluster near the ATP binding site, form a hinge-bending region controlling the cooperative motion, and thereby the catalytic function, of the enzyme. The distal beta-barrel and the tRNA acceptor binding domain, on the other hand, are distinguished by their high mobilities in the global modes of motion, a feature typical of recognition sites, also observed for other proteins. Most of the conserved bases and residues of tRNA and GlnRS are severely constrained in the global motions of the molecules, suggesting their having a role in stabilizing and modulating the global motion.

Residue-residue potentials with a favorable contact pair term and an unfavorable high packing density term, for simulation and threading.

Attractive inter-residue contact energies for proteins have been re-evaluated with the same assumptions and approximations used originally by us in 1985, but with a significantly larger set of protein crystal structures. An additional repulsive packing energy term, operative at higher densities to prevent overpacking, has also been estimated for all 20 amino acids as a function of the number of contacting residues, based on their observed distributions. The two terms of opposite sign are intended to be used together to provide an estimate of the overall energies of inter-residue interactions in simplified proteins without atomic details. To overcome the problem of how to utilize the many homologous proteins in the Protein Data Bank, a new scheme has been devised to assign different weights to each protein, based on similarities among amino acid sequences. A total of 1168 protein structures containing 1661 subunit sequences are actually used here. After the sequence weights have been applied, these correspond to an effective number of residue-residue contacts of 113,914, or about six times more than were used in the old analysis. Remarkably, the new attractive contact energies are nearly identical to the old ones, except for those with Leu and the rarer amino acids Trp and Met. The largest change found for Leu is surprising. The estimates of hydrophobicity from the contact energies for non-polar side-chains agree well with the experimental values. In an application of these contact energies, the sequences of 88 structurally distinct proteins in the Protein Data Bank are threaded at all possible positions without gaps into 189 different folds of proteins whose sequences differ from each other by at least 35% sequence identity. The native structures for 73 of 88 proteins, excluding 15 exceptional proteins such as membrane proteins, are all demonstrated to have the lowest alignment energies.

A role for CH...O interactions in protein-DNA recognition.

The concept of CH...O hydrogen bonds has recently gained much interest, with a number of reports indicating the significance of these non-classical hydrogen bonds in stabilizing nucleic acid and protein structures. Here, we analyze the CH...O interactions in the protein-DNA interface, based on 43 crystal structures of protein-DNA complexes. Surprisingly, we find that the number of close intermolecular CH...O contacts involving the thymine methyl group and position C5 of cytosine is comparable to the number of protein-DNA hydrogen bonds involving nitrogen and oxygen atoms as donors and acceptors. A comprehensive analysis of the geometries of these close contacts shows that they are similar to other CH...O interactions found in proteins and small molecules, as well as to classical NH...O hydrogen bonds. Thus, we suggest that C5 of cytosine and C5-Met of thymine form relatively weak CH...O hydrogen bonds with Asp, Asn, Glu, Gln, Ser, and Thr, contributing to the specificity of recognition. Including these interactions, in addition to the classical protein-DNA hydrogen bonds, enables the extraction of simple structural principles for amino acid-base recognition consistent with electrostatic considerations.

A parallel DNA triplex as a model for the intermediate in homologous recombination.

Parallel DNA triplexes considered to be putative intermediates in homologous recombination, are studied by means of theoretical conformational analysis. These triplexes are denoted as the R-form DNA. Two types of triplexes are analyzed: extended R-form DNA, modeling the triple standard structure, created transiently in the presence of recombination proteins (e.g. RecA); and collapsed R-form, obtained after deproteinization. These structures are stereochemically possible for any arbitrary sequence and have the following properties: (1) the third, R-strand, is parallel to the identical duplex strand and is located in the major groove of the duplex; (2) positions of all four bases in the R-strand are nearly isomorphic; (3) the proposed triplets are consistent with the chemical modification data for deproteinized DNA; we suggest, however, that they are the same in the RecA-DNA complex as well. Since the patterns of charges on each base of the R-strand are strictly complementary to the charges of the homologous Watson-Crick (WC) pair in the major groove, we propose that the selection of the homologous sequence may occur through these complementary electrostatic interactions (electrostatic recognition code). We demonstrate that in the collapsed triplex with a rise of about 3.4 A, the bases from the third R-strand can be inclined and interact with two WC base-pairs simultaneously, which could lead to recognition errors. These mispairings are unlikely in the extended triplex. Therefore, we speculate that a functional role of the extended and underwound DNA structure, transiently formed in the complex with RecA protein, is to obviate such errors and increase the stringency of recognition. In other words, RecA plays the role of a DNA chaperone facilitating the recognition of the single stranded DNA and the duplex. Finally, we show that the proposed isomorphic triplets are conformationally advantageous for strand exchange.

Collective motions in HIV-1 reverse transcriptase: examination of flexibility and enzyme function.

In order to study the inferences of structure for mechanism, the collective motions of the retroviral reverse transcriptase HIV-1 RT (RT) are examined using the Gaussian network model (GNM) of proteins. This model is particularly suitable for elucidating the global dynamic characteristics of large proteins such as the presently investigated heterodimeric RT comprising a total of 982 residues. Local packing density and coordination order of amino acid residues is inspected by the GNM to determine the type and range of motions, both at the residue level and on a global scale, such as the correlated movements of entire subdomains. Of the two subunits, p66 and p51, forming the RT, only p66 has a DNA-binding cleft and a functional polymerase active site. This difference in the structure of the two subunits is shown here to be reflected in their dynamic characteristics: only p66 has the potential to undergo large-scale cooperative motions in the heterodimer, while p51 is essentially rigid. Taken together, the global motion of the RT heterodimer is comprised of movements of the p66 thumb subdomain perpendicular to those of the p66 fingers, accompanied by anticorrelated fluctuations of the RNase H domain and p51 thumb, thus providing information about the details of one processivity mechanism. A few clusters of residues, generally distant in sequence but close in space, are identified in the p66 palm and connection subdomains, which form the hinge-bending regions that control the highly concerted motion of the subdomains. These regions include the catalytically active site and the non-nucleoside inhibitor binding pocket of p66 polymerase, as well as sites whose mutations have been shown to impair enzyme activity. It is easily conceivable that this hinge region, indicated by GNM analysis to play a critical role in modulating the global motion, is locked into an inactive conformation upon binding of an inhibitor. Comparative analysis of the dynamic characteristics of the unliganded and liganded dimers indicates severe repression of the mobility of the p66 thumb in RT's global mode, upon binding of non-nucleoside inhibitors.

Influence of fluctuations on DNA curvature. A comparison of flexible and static wedge models of intrinsically bent DNA.

Matrix-generator and Monte Carlo methods have been employed to study the influence of thermal fluctuations on the overall sizes and shapes of curved pieces of DNA. The DNA model involves the independent angular parameters relating successive base-pair steps: the sequence-dependent equilibrium values and fluctuations of the twist, tilt, and roll angles. The curved sequence under study is the (A5X5)n repeating polymer, the AA and XX steps having different equilibrium roll and twist values. Both planar circles and superhelices are analysed. Detailed comparison is made between the rigorous statistical mechanical representation of the DNA and simplified static models currently used in the literature. That is, a more realistic "flexible wedge" model is contrasted with the existing "static wedge" model of DNA curvature, which is demonstrated to be inadequate. The size of the coils is described by the unperturbed root-mean-square end-to-end distance and the shape by a ratio of the principal moments of the radius of gyration. The moment ratios indicate that when DNA is relatively short (e.g. its length is shorter than half a turn of the static superhelix), the flexible chains are more "short and thick" than the static structure. The end-to-end distances, however, are practically the same in the two models. For longer DNA fragments, the flexible chain is more extended in terms of the end-to-end distance and more globular in terms of the moment ratio. Thus, fluctuations "blur" the curvature of longer DNA fragments compared with static models. Furthermore, the overall average shape of slightly curved DNA subject to natural bending and twisting fluctuations is essentially indistinguishable from that of the corresponding "straight" DNA. Such configurational similarities are apparently responsible for the relative insensitivity of the polyacrylamide gel matrix to small degrees of DNA curvature. These findings raise serious questions regarding the quantitative estimation of wedge angles in DNA from electrophoretic experiments, based on static models. Comparison between planar circles and superhelices shows that when fluctuations are considered, the flexible circles are more spherical than the superhelices. The results imply that when DNA bending is exactly "in phase" with the helical repeat (i.e. when the DNA loop is exactly planar at 0 K), the DNA coil is packed more tightly than when bending and twisting are "out of phase" (and a superhelix is formed at 0 K). This finding is consistent with polyacrylamide gel electrophoresis data testifying to an increase in DNA retardation when twisting is more precisely "tuned".(ABSTRACT TRUNCATED AT 400 WORDS)

Probing the structure of a putative intermediate in homologous recombination: the third strand in the parallel DNA triplex is in contact with the major groove of the duplex.

A three-stranded DNA that is a putative intermediate of homologous recombination is a novel DNA triplex, R-form DNA. In R-form DNA the third strand includes both purines and pyrimidines and is parallel to the identical strand of the duplex. To test and refine our previously proposed R-form base triplets we have used two approaches: (1) dimethyl sulfate protection of R-form DNA; and (2) thermal dissociation of R-form DNAs in which the duplex strands were substituted in a strand-specific manner with either 7-deaza-guanine or 7-deaza-adenine. Together, the footprinting and isosteric substitution results demonstrate that the third strand in R-form DNA is in contact with the purines in the N7 position in the major groove of the Watson-Crick duplex in three ((GC):G, (AT):A and (TA):T) out of the four possible triplets. Furthermore, these results suggest that the N7 positions of the duplex play a significant role in stabilizing the DNA-DNA contacts during the homology recognition process.

Amino acid pair interchanges at spatially conserved locations.

Here we study the pattern of amino acid interchanges at spatially, locally conserved regions in globally dissimilar and unrelated proteins. By using a method which completely separates the amino acid sequence from its respective structure, this work addresses the question of which properties of the amino acids are the most crucial for the stability of conserved structural motifs. The proteins are taken from a structurally non-redundant dataset. The spatially conserved substructural motifs are defined as consisting of a "large enough" number of Calpha atoms found to provide a geometric match between two proteins, regardless of the order of the Calpha atoms in the sequence, or of the sequence composition of the substructures. This approach can apply to proteins with little or no sequence similarity but with sufficient structural similarity, and is unique in its ability to handle local, non-topological matches between pairs of dissimilar proteins. The method uses a computer-version based algorithm, the Geometric Hashing. Since the Geometric Hashing ignores sequence information it lends itself to answer the question posed above. The interchanges at geometrically similar positions that have been obtained with our method demonstrate the expected behaviour. Yet, a closer inspection reveals some distant characteristics, as compared with interchanges based upon sequence-order based techniques, or from energy-contact-based considerations. First, a pronounced division of the amino acids into two classes is displayed: Lys, Glu, Arg, Gln, Asp, Asn, Pro, Gly, Thr, Ser and His on the one hand, and Ile, Val, Leu, Phe, Met, Tyr, Trp, Cys and Ala on the other. These groups further cluster into subgroups: Lys, Glu, Arg, Gln; Asp Asn; Pro, Gly; Ile, Val, Leu, Phe. The other amino acids stand alone. Analysis of the conservation among amino acids indicates proline to be consistently, by far, the most conserved. Next are Asp, Glu, Lys and Gly. Cys is also highly conserved. Interestingly, oppositely charged amino acids are interchanged roughly as frequently as those of the same charge. These observations can be explained in terms of the three-dimensional structures of the proteins. Most of all, there is a clear distinction between residues which prefer to be on the protein surfaces, compared to those frequently buried in the interiors. Analysis of the interchanges indicates their low information content. This, together with the separation into two groups, suggest that the predictive value of the spatial positions of the Calpha+ atoms is not much greater than the sequence alone, aside from their hydrophobicity/hydrophillicity classification.

Ideal architecture of residue packing and its observation in protein structures.

A simple model of sphere packing has been investigated as an ideal model for long-range interactions for the packing of non-bonded residues in protein structures. By superposing all residues, the geometry of packing around a central residue is investigated. It is found that all residues conform almost perfectly to this lattice model for sphere packing when a radius of 6.5 A is used to define non-bonded (virtual) interacting residues. Side-chain positions with respect to sequential backbone segments are relatively regular as well. This lattice can readily be used in conformation simulations to reduce the conformational space.

A role for surface hydrophobicity in protein-protein recognition.

The role of hydrophobicity as a determinant of protein-protein interactions is examined. Surfaces of apo-protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol-binding protein, and their associated ligands with available X-ray structures for their complexed forms, are scanned to determine clusters of surface-accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co-crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one-third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well-defined loci for possible ligand attachment.

A preference-based free-energy parameterization of enzyme-inhibitor binding. Applications to HIV-1-protease inhibitor design.

The interface between protein receptor-ligand complexes has been studied with respect to their binary interatomic interactions. Crystal structure data have been used to catalogue surfaces buried by atoms from each member of a bound complex and determine a statistical preference for pairs of amino-acid atoms. A simple free energy model of the receptor-ligand system is constructed from these atom-atom preferences and used to assess the energetic importance of interfacial interactions. The free energy approximation of binding strength in this model has a reliability of about +/- 1.5 kcal/mol, despite limited knowledge of the unbound states. The main utility of such a scheme lies in the identification of important stabilizing atomic interactions across the receptor-ligand interface. Thus, apart from an overall hydrophobic attraction (Young L, Jernigan RL, Covell DG, 1994, Protein Sci 3:717-729), a rich variety of specific interactions is observed. An analysis of 10 HIV-1 protease inhibitor complexes is presented that reveals a common binding motif comprised of energetically important contacts with a rather limited set of atoms. Design improvements to existing HIV-1 protease inhibitors are explored based on a detailed analysis of this binding motif.

Identification of kinetically hot residues in proteins.

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.

Empirical solvent-mediated potentials hold for both intra-molecular and inter-molecular inter-residue interactions.

Whether knowledge-based intra-molecular inter-residue potentials are valid to represent inter-molecular interactions taking place at protein-protein interfaces has been questioned in several studies. Differences in the chain connectivity effect and in residue packing geometry between interfaces and single chain monomers have been pointed out as possible sources of distinct energetics for the two cases. In the present study, the interfacial regions of protein-protein complexes are examined to extract inter-molecular inter-residue potentials, using the same statistical methods as those previously adopted for intra-molecular residue pairs. Two sets of energy parameters are derived, corresponding to solvent-mediation and "average residue" mediation. The former set is shown to be highly correlated (correlation coefficient 0.89) with that previously obtained for inter-residue interactions within single chain monomers, while the latter exhibits a weaker correlation (0.69) with its intra-molecular counterpart. In addition to the close similarity of intra- and inter-molecular solvent-mediated potentials, they are shown to be significantly more residue-specific and thereby discriminative compared to the residue-mediated ones, indicating that solvent-mediation plays a major role in controlling the effective inter-residue interactions, either at interfaces, or within single monomers. Based on this observation, a reduced set of energy parameters comprising 20 one-body and 3 two-body terms is proposed (as opposed to the 20 x 20 tables of inter-residue potentials), which reproduces the conventional 20 x 20 tables with a correlation coefficient of 0.99.

Combining the GOR V algorithm with evolutionary information for protein secondary structure prediction from amino acid sequence.

We have modified and improved the GOR algorithm for the protein secondary structure prediction by using the evolutionary information provided by multiple sequence alignments, adding triplet statistics, and optimizing various parameters. We have expanded the database used to include the 513 non-redundant domains collected recently by Cuff and Barton (Proteins 1999;34:508-519; Proteins 2000;40:502-511). We have introduced a variable size window that allowed us to include sequences as short as 20-30 residues. A significant improvement over the previous versions of GOR algorithm was obtained by combining the PSI-BLAST multiple sequence alignments with the GOR method. The new algorithm will form the basis for the future GOR V release on an online prediction server. The average accuracy of the prediction of secondary structure with multiple sequence alignment and full jack-knife procedure was 73.5%. The accuracy of the prediction increases to 74.2% by limiting the prediction to 375 (of 513) sequences having at least 50 PSI-BLAST alignments. The average accuracy of the prediction of the new improved program without using multiple sequence alignments was 67.5%. This is approximately a 3% improvement over the preceding GOR IV algorithm (Garnier J, Gibrat JF, Robson B. Methods Enzymol 1996;266:540-553; Kloczkowski A, Ting K-L, Jernigan RL, Garnier J. Polymer 2002;43:441-449). We have discussed alternatives to the segment overlap (Sov) coefficient proposed by Zemla et al. (Proteins 1999;34:220-223).

A model of the strain-induced B-Z transition.

The B-to-Z transition in supercoiled circular DNA is modeled as a strain-induced nonlinear excitation process. Using a model, in which DNA is regarded as a chain of units with a bistable energy function along the twisting coordinate together with a harmonic inter-unit interaction, we show that a Z region and the accompanying two B-Z junctions of finite width appear naturally as a solution of nonlinear equations, when the strain exceeds a critical value. We examine the B-Z transition behaviour as a function of twist under various situations. We also analyse available experimental results on B-Z transition in supercoiled plasmid with G-C insertions by this mechanistic model in order to estimate the magnitude of model parameters. The energy barrier of the B-Z transition is estimated to be of the order of 1 kcal/mole per base pair. The analysis shows that if the length of the insertion is less than a certain value, the entire insertion converts to Z form at a transition point, but if the insertion is much longer, the B-Z transition exhibits a different behavior, in which part of the insertion flips to Z form and the Z region expands linearly upon changing linking number.