Unbiased estimation of evolutionary distance between nucleotide sequences

A new algorithm for estimating the number of nucleotide substitutions per site (i.e., the evolutionary distance) between two nucleotide sequences is presented. This algorithm can be applied to many estimation methods, such as Jukes and Cantor's method, Kimura's transition/transversion method, and Tajima and Nei's method. Unlike ordinary methods, this algorithm is always applicable. Numerical computations and computer simulations indicate that this algorithm gives an almost unbiased estimate of the evolutionary distance, unless the evolutionary distance is very large. This algorithm should be useful especially when we analyze short nucleotide sequences. It can also be applied to amino acid sequences, for estimating the number of amino acid replacements.      

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.     

Tree and rate estimation by local evaluation of heterochronous nucleotide data

MOTIVATION: Heterochronous gene sequence data is important for characterizing the evolutionary processes of fast-evolving organisms such as RNA viruses. A limited set of algorithms exists for estimating the rate of nucleotide substitution and inferring phylogenetic trees from such data. The authors here present a new method, Tree and Rate Estimation by Local Evaluation (TREBLE) that robustly calculates the rate of nucleotide substitution and phylogeny with several orders of magnitude improvement in computational time. METHODS: For the basis of its rate estimation TREBLE novelly utilizes a geometric interpretation of the molecular clock assumption to deduce a local estimate of the rate of nucleotide substitution for triplets of dated sequences. Averaging the triplet estimates via a variance weighting yields a global estimate of the rate. From this value, an iterative refinement procedure relying on statistical properties of the triplets then generates a final estimate of the global rate of nucleotide substitution. The estimated global rate is then utilized to find the tree from the pairwise distance matrix via an UPGMA-like algorithm. RESULTS: Simulation studies show that TREBLE estimates the rate of nucleotide substitution with point estimates comparable with the best of available methods. Confidence intervals are comparable with that of BEAST. TREBLE's phylogenetic reconstruction is significantly improved over the other distance matrix method but not as accurate as the Bayesian algorithm. Compared with three other algorithms, TREBLE reduces computational time by a minimum factor of 3000. Relative to the algorithm with the most accurate estimates for the rate of nucleotide substitution (i.e. BEAST), TREBLE is over 10,000 times more computationally efficient. AVAILABILITY: jdobrien.bol.ucla.edu/TREBLE.html     

Resampling Nucleotide Sequences with Closest-Neighbor Trimming and Its Comparison to Other Methods

A large number of nucleotide sequences of various pathogens are available in public databases. The growth of the datasets has resulted in an enormous increase in computational costs. Moreover, due to differences in surveillance activities, the number of sequences found in databases varies from one country to another and from year to year. Therefore, it is important to study resampling methods to reduce the sampling bias. A novel algorithm–called the closest-neighbor trimming method–that resamples a given number of sequences from a large nucleotide sequence dataset was proposed. The performance of the proposed algorithm was compared with other algorithms by using the nucleotide sequences of human H3N2 influenza viruses. We compared the closest-neighbor trimming method with the naive hierarchical clustering algorithm and -medoids clustering algorithm. Genetic information accumulated in public databases contains sampling bias. The closest-neighbor trimming method can thin out densely sampled sequences from a given dataset. Since nucleotide sequences are among the most widely used materials for life sciences, we anticipate that our algorithm to various datasets will result in reducing sampling bias.     

Isolation of mRNA from protoplasts of spore-forming bacteria and study of its translation products

Total RNA was isolated from the protoplasts of Bacillus intermedius 7P which actively produced alkaline exocellular RNAse. A fraction of polyadenylated RNA was isolated from this RNA using chromatography on poly(U) Sepharose. The total RNA and poly(A) +RNA were translated in Xenopus laevis oocytes. Bacterial exocellular RNAse was identified among the products of translation by means of electrophoretic and immunological analyses. The mRNA of RNAse secreted by B. intermedius 7P was concluded to be polyadenylated.     

Sequence analysis and in vitro maturation of five precursor 5S RNAs from Bacillus Q.

Bacillus Q, which is closely related to B. subtilis, contains at least six different precursors of 5S rRNA. The complete nucleotide sequences of four of these precursors, as well as the major part of the sequence of a fifth one, have been determined. They all contain the same 5'-terminal non-conserved segment which is to a large degree homologous with the corresponding segment of the B. subtilis p5S RNAs (Sogin, M.L., Pace, N.R., Rosenberg, M., Weissman, S.M. (1976) J. Biol. Chem. 251, 3480-3488). On the other hand the 3'-terminal non-conserved sequences of the various Bacillus Q precursors show considerable differences both in length and in nucleotide sequence, while there is also little or no homology with the 3'-terminal non-conserved sequence of the B. subtilis precursors. Bacillus Q p5S RNAs do not possess tetranucleotide repeats around the sites which are cleaved during maturation, as does B. subtilis p5S RNA. Like in B. subtilis, however, the cleavage sites are contained within a double-helical region of the precursor molecules. Crude RNAse M5 isolated from various Bacillus strains can maturate the Bacillus Q p5S RNAs with high efficiency. Despite considerable differences in primary structure between the precursors from the various strains, each RNAs M5 preparation can maturate all these precursors with about the same efficiency.     

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Explicit analytic approximations for time-dependent solutions of the generalized integrated Michaelis-Menten equation

A single nucleotide polymorphism (SNP) is a common genetic variation when a single nucleotide differs between members of a species or paired chromosome. Due to its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we present two quantitative SNP detection methods for a single-base mismatch in RNA, based on nick-joining and nick-generating activities of T4 RNA ligase and DNAzyme, respectively. T4 RNA ligase successfully discriminated a one-base mismatch in the ligation junction, and the designed DNAzyme cleaved RNA by discerning a single-base mismatch in the cleaving site.     

The SL1 stem-loop structure at the 5'-end of potato virus X RNA is required for efficient binding to host proteins and for viral infectivity

The 5'-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.     

Mean free energy topology for nucleotide sequences of varying composition based on secondary structure calculations.

The mean free energy generated from the secondary structure of RNA sequences of varying length and composition has been studied by way of probability theory. The expected boundaries or maximal and minimal values of a given distribution are explored and a method for estimating error as a function of the number of shuffled sequences is also examined. For typical nucleotide sequences found in biologically active organisms, the mean free energy, free energy distributions and errors appear to be scalable in terms of a fixed set of algorithm-dependent parameters and the nucleotide composition of the particular sequence under evaluation. In addition, a general semi-analytical formula for predicting the mean free energy is proposed which, at least to first-order approximation, can be used to rapidly predict the mean free energy of any sequence length and composition of RNA. The general methodology appears to be algorithm independent. The results are expected to provide a reference point for certain types of analysis related to structure of RNA or DNA sequences and to assist in measuring the somewhat related matter of complexity in algorithm development. Some related applications are discussed.     

Electrophoretic transfer of DNA, RNA and protein onto diazobenzyloxymethyl (DBM) - paper.

A method has been developed for the electrophoretic transfer of DNA, RNA, protein and ribonucleoprotein particles from a variety of gels onto diazobenzyloxymethyl (DBM) - paper. Conditions for the electrophoretic transfer of these macromolecules have been optimized to allow for nearly quantitative transfer and covalent coupling. DNA and RNA electrophoretically transferred to DBM-paper retain their ability to hybridize with specific probes. The high efficiency of transfer and the high capacity of DBM-paper for nucleic acids makes possible the sensitive detection of specific nucleotide sequences. Similar efficiency is achieved in electrophoretic transfer and covalent coupling of proteins to DBM-paper. Macromolecules can also be electrophoretically transferred and bound to DBM-paper incapable of covalent bond formation. Their elution from the paper in high salt provides a new and useful preparative method for isolation of DNA, RNA and protein.     

Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena acts as a sequence-specific endoribonuclease. Specificity of cleavage is determined by Watson-Crick base pairing between the active site of the RNA enzyme (ribozyme) and its RNA substrate [Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature (London) 324, 429-433]. Surprisingly, single-base changes in the substrate RNA 3 nucleotides preceding the cleavage site, giving a mismatched substrate-ribozyme complex, enhance the rate of cleavage. Mismatched substrates show up to a 100-fold increase in kcat and, in some cases, in kcat/Km. A mismatch introduced by changing a nucleotide in the active site of the ribozyme has a similar effect. Addition of 2.5 M urea or 3.8 M formamide or decreasing the divalent metal ion concentration from 10 to 2 mM reverses the substrate specificity, allowing the ribozyme to discriminate against the mismatched substrate. The effect of urea is to decrease kcat and kcat/Km for cleavage of the mismatched substrate; Km is not significantly affected at 0-2.5 M urea. Thus, progressive destabilization of ribozyme-substrate pairing by mismatches or by addition of a denaturant such as urea first increases the rate of cleavage to an optimum value and then decreases the rate.     

S1 nuclease as a probe of yeast ribosomal 5 S RNA conformation.

5 S RNA was isolated from Saccharomyces cerevisiae grown in the presence of 32P-phosphate and digested with nuclease S1, a single-strand specific nuclease. Two different procedures were employed to determine the sites of attack on the RNA. First, 5 S RNA was isolated from nuclease S1 digests, digested to completion with ribonuclease T1, and then 'fingerprinted' by two-dimensional electrophoresis. Quantitation of each of the characteristic RNAase T1-derived oligonucleotides was employed to determine the relative susceptibility of various regions of the molecule to nuclease S1. A second procedure to define nuclease S1-susceptible sites in the molecule employed polyacrylamide gel electrophoretic fractionation of nuclease S1 digests followed by identification of the nucleotide sequences of the released RNA fragments. Both procedures showed that the region of the molecule between residues 9 and 60 was most susceptible to nuclease S1, with preferential cleavage occurring between residues 12-25 and 50-60. These results are discussed in relation to a proposed model for the secondary structure of yeast 5 S RNA.     

Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P

The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.