Sequence motifs specific for cytosine methyltransferases

Using a new alignment method, the sequences of 13 m5C methyltransferases (MTases) have been examined. Five extremely well-conserved blocks of sequence have been detected and have been used as fixed points for the alignment of the 13 sequences. Following this initial alignment, five further blocks of similarity have been identified to give a total of ten recognizable blocks of sequence homology that are all arranged in a common order. The structures of these MTases consist of a variable-length N-terminal arm followed by eight well-conserved blocks each separated by small variable-length regions. A large variable-length segment of 90 to 270 amino acids (aa) then follows. After this are two blocks, and a variable-length C-terminal segment completes the sequence. Within the final alignment, 20 aa in the protein sequences, and 86 nucleotides in the nucleotide sequences are invariant. The strongest conservation is found in proximity to a suspected functional site that contains the dipeptide proline-cysteine. Consensus patterns can be defined for the five best conserved blocks and, when used as search motifs, are able to clearly distinguish between the m5C MTases and all other identified proteins in the PIR database. This suggests they may be of use in identifying putative MTases among protein sequences of unknown function.     

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).     

A method for prediction of conserved RNA secondary structures

The RNA secondary structure prediction is a classical problem in bioinformatics. The most efficient approach to this problem is based on the idea of a comparative analysis. In this approach the algorithms utilize multiple alignment of the RNA sequences and find common RNA structure. This paper describes a new algorithm for this task. This algorithm does not require predefined multiple alignment. The main idea of the algorithm is based on MEME-like iterative searching of abstract profile on different levels. On the first level the algorithm searches the common blocks in the RNA sequences and creates chain of this blocks. On the next step the algorithm refines the chain of common blocks. On the last stage the algorithm searches sets of common helices that have consistent locations relative to common blocks. The algorithm was tested on sets of tRNA with a subset of junk sequences and on RFN riboswitches. The algorithm is implemented as a web server (http://bioinf.fbb.msu.ru/RNAAlign/).     

A pairwise alignment algorithm which favors clusters of blocks.

Pairwise sequence alignments aim to decide whether two sequences are related and, if so, to exhibit their related domains. Recent works have pointed out that a significant number of true homologous sequences are missed when using classical comparison algorithms. This is the case when two homologous sequences share several little blocks of homology, too small to lead to a significant score. On the other hand, classical alignment algorithms, when detecting homologies, may fail to recognize all the significant biological signals. The aim of the paper is to give a solution to these two problems. We propose a new scoring method which tends to increase the score of an alignment when "blocks" are detected. This so-called Block-Scoring algorithm, which makes use of dynamic programming, is worth being used as a complementary tool to classical exact alignments methods. We validate our approach by applying it on a large set of biological data. Finally, we give a limit theorem for the score statistics of the algorithm.     

Comparative analysis of human and bovine papillomaviruses

A method is presented for the analysis and comparison of nucleic acid and protein sequences utilizing all identity blocks (the term "identity block" refers to a set of consecutive matches between two sequences) above a prescribed length. Moreover, such identity blocks are determined for various groupings of amino acids according to chemical, functional, charge, and hydrophobic classifications. Alignment maps based on these classifications and containing all statistically significant identity blocks between two or more sequences are constructed. New theoretical results for determining the expected length of the longest identity block between sequences are also presented and are used, along with permutation procedures, to ascertain the significance of sequence identity blocks. As an example of the type of information that can be obtained, comparison has been made of the complete DNA sequences and the E1, E2, L1, and L2 genes of human and bovine papillomaviruses based on the classification schemes described above.     

New method to predict conserved RNA structures

RNA secondary structure prediction is one of the classic problems of bioinformatics. The most efficient approaches to solving this problem are based on comparative analysis. As a rule, multiple RNA sequence alignment and subsequent determination of a common secondary structure are used. A new algorithm was developed to obviate the need for preliminary multiple sequence alignment. The algorithm is based on a multilevel MEME-like iterative search for a generalized profile. The search for common blocks in RNA sequences is carried out at the first level. Then the algorithm refines the chains consisting of these blocks. Finally, the search for sets of common helices, matched with alignment blocks, is carried out. The algorithm was tested with a tRNA set containing additional junk sequences and with RFN riboswitches. The algorithm is available at http://bioinf.fbb.msu.ru/RNAAlign.     

Isolation and Characterization of New Maize B Sequences from a Microdissected Library

Understanding the evolution of the maize B chromosome requires insight into the molecular organization of a large number of B clones, which can be potentially obtained by microdissection of the chromosome. Yet, the microdissection protocols currently available are ineffective for a large-scale isolation. In an attempt to improve its efficiency, a protocol was adopted to screen a microdissected B library with probes prepared from the degenerate oligonucleotide primed-PCR product of genomic DNA. This protocol resulted in 59 new B clones, most of which were highly repetitive sequences located in various B regions but mostly in the heterochromatic blocks of the long arm. They also appeared in A chromosomes. Twenty-four of these were retrotransposons, ten knob, 18 noncoding sequences, and seven unknown sequences. The implication of the new B sequences on the B evolution is discussed.     

The synthesis of blocked triplet-phosphoramidites and their use in mutagenesis.

A general approach for the synthesis of oligonucleotide-triplet phosphoramidites and the synthesis of four such blocks are described. A strategy was devised to minimize the number of dimer precursors needed for synthesis of a complete set of triplet-amidite blocks encoding all 20 amino acids. Whereas synthesis of 20 triplet-amidite blocks consisting of codon sequences requires 16 dimer blocks, just seven dimer blocks are required to synthesize all required antisense sequences. The antisense sequences are then converted to codons in template mediated replication. Using a mixture of four triplet-amidites and conventional automated solid-phase DNA synthesis, short (6mer) and medium length (30mer) oligonucleotide mixtures were synthesized and analyzed. The latter was replicated in vitro and used as a mutagenic cassette to produce four mutants of Asp 221 in the enzyme thymidylate synthase. The method establishes the direction and utility for the production and use of triplet-amidite blocks in DNA synthesis.     

织锦芋螺ο家族芋螺毒素的序列分析

为了从织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中尽可能多地分离出ο家族的毒素序列和研究其应用价值,在克隆了织锦芋螺α芋螺毒素的基础上进行了织锦芋螺ο家族芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍ ＲＮＡ,通过ＲＡＣＥ（ｒａｐｉｄ ａｍ ｐｌｉｆｉｃａｔｉｏｎ ｏｆｃＤＮＡ ｅｎｄｓ,ｃＤＮＡ 末端的快速扩增）－ＰＣＲ方法扩增获得ο家族芋螺毒素ｃＤＮＡ 片段,并进行克隆和序列分析．从织锦芋螺毒液中获得了６种新的芋螺毒素序列,且毒素序列的成熟肽部分均符合Ｃ－ Ｃ－ ＣＣ－ Ｃ－ Ｃ的保守半胱氨酸框架．这些是新的ο家族芋螺毒素序列,新序列的阐明为进一步研究其生物活性和应用打下了基础．     

Molecular and bioinformatic analysis of the FB-NOF transposable element

The Drosophila melanogaster transposable element FB-NOF is known to play a role in genome plasticity through the generation of all sort of genomic rearrangements. Moreover, several insertional mutants due to FB mobilizations have been reported. Its structure and sequence, however, have been poorly studied mainly as a consequence of the long, complex and repetitive sequence of FB inverted repeats. This repetitive region is composed of several 154 bp blocks, each with five almost identical repeats. In this paper, we report the sequencing process of 2 kb long FB inverted repeats of a complete FB-NOF element, with high precision and reliability. This achievement has been possible using a new map of the FB repetitive region, which identifies unambiguously each repeat with new features that can be used as landmarks. With this new vision of the element, a list of FB-NOF in the D. melanogaster genomic clones has been done, improving previous works that used only bioinformatic algorithms. The availability of many FB and FB-NOF sequences allowed an analysis of the FB insertion sequences that showed no sequence specificity, but a preference for A/T rich sequences. The position of NOF into FB is also studied, revealing that it is always located after a second repeat in a random block. With the results of this analysis, we propose a model of transposition in which NOF jumps from FB to FB, using an unidentified transposase enzyme that should specifically recognize the second repeat end of the FB blocks.     

Codon-based mutagenesis using dimer-phosphoramidites.

A new approach for the synthesis of randomized DNA sequences containing the 20 codons corresponding to all natural amino acids is described. The strategy is based on the use of dinucleotide phosphoramidite building blocks within a resin-splitting procedure. Through this protocol, a minimal number of seven dimers is sufficient to encode all 20 natural amino acids. This synthesis procedure is extremely flexible and allows codon usage from different hosts to be accommodated.     

Two human valyl-tRNA synthetase-encoding cDNA sequences deposited in GenBank display extensive differences

Five blocks of significant differences exist between two published sequences of the cDNA encoding human valyl-tRNA synthetase (GenBank X59303 and M98326). By comparison with the partial sequence of rat valyl-tRNA synthetase (GenBank M98327) the correct sequence can be deduced for two such blocks. The possible origin of the diversity for the two sequences is discussed.     

Space efficient computation of rare maximal exact matches between multiple sequences.

In this article, we propose a new method for computing rare maximal exact matches between multiple sequences. A rare match between k sequences S(1), ... , S(k) is a string that occurs at most t(i)-times in the sequence S(i), where the t(i) > 0 are user-defined thresholds. First, the suffix tree of one of the sequences (the reference sequence) is built, and then the other sequences are matched separately against this suffix tree. Second, the resulting pairwise exact matches are combined to multiple exact matches. A clever implementation of this method yields a very fast and space efficient program. This program can be applied in several comparative genomics tasks, such as the identification of synteny blocks between whole genomes.     

Random multi-recombinant PCR for the construction of combinatorial protein libraries

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries. For the construction of a ‘random shuffling library’, RM-PCR was used to shuffle six DNA fragments each encoding 25 amino acids; this affords many different fragment sequences whose every position has an equal probability to encode any of the six blocks. For the construction of the ‘alternative splicing library’, RM-PCR was used to perform different alternative splicings at the DNA level, which also yields different block sequences. DNA sequencing of the RM-PCR products in both libraries revealed that most of the sequences were quite different, and had a long open reading frame without a frame shift or stop codon. Furthermore, no distinct bias among blocks was observed. Here we describe how to use RM-PCR for the construction of combinatorial DNA libraries, which encode protein libraries that would be suitable for selection experiments in the global protein space.     

Exploring sequence space: profile analysis and protein-ligand docking to screen omega-aminotransferases with expanded substrate specificity

omega-Aminotransferase (omegaAT) is an interesting biocatalyst for the preparation of chiral amines, which are widely used as building blocks for pharmaceuticals, agrochemicals and fine chemicals. With the assumption that substrate and sequence spaces are in the process of co-evolution, we explored sequences space to screen the enzymes showing activity to new target compounds. Bacterial genome sequences (n=527) were analyzed by the profiles of subgroups in aminotransferase group II including ornithine aminotransferase (orAT), acetylornithine aminotransferase (aoAT), omegaAT, gamma-aminobutyrate aminotransferase (GABAAT) and 7,8-diaminopelargonate aminotransferase (DAPAAT). We selected the sequences having a Z score of 0-1.8 to guarantee the omegaAT reaction and to avoid the typical omegaAT sequences. Among the selected sequences, we filtered out the sequences with very low Z scores for the rest of four subgroups in aminotransferase group II to consider the diversity. For the selected sequences, we performed protein-ligand docking simulations to predict the docking pose of amino acceptor. Throughout all the analysis procedures, several candidate aminotransferase sequences for the asymmetric synthesis of chiral amines were obtained. An efficient procedure for virtual screening of novel enzymes was demonstrated.     

Structure of mitochondrial DNA control region of Fenneropenaeus chinensis and phylogenetic relationship among different populations

This paper deals with the structure of mitochondrial DNA control region of Fenneropenaeus chinensis. The termination-associated sequence (TAS), cTAS, CSB-D-CSB-F, and CSB-1 are detected in the species. The results indicate that the structures of these parts are similar to those of most marine organisms. Two conserved regions and many stable conserved boxes are found in the extended TAS area, central sequences blocks, and conserved sequences blocks (CSBs). This is the special character of F. chinensis. All the mtDNA control region sequences do not have CSB2 and CSB3 blocks, which is quite different from most vertebrates. In addition, the complete mtDNA control region sequences are used to analyze the phylogenetic relationships of F. chinensis. The phylogenetic trees show a lack of genetic structure among populations, which is similar to many previous studies.     

New hypothesis on the role of alternating sequences in calcium-alginate gels

The availability of mannuronan and mannuronan C-5 epimerases allows the production of a strictly alternating mannuronate-guluronate (MG) polymer and the MG-enrichment of natural alginates, providing a powerful tool for the analysis of the role of such sequences in the calcium-alginate gel network. In view of the calcium binding properties of long alternating sequences revealed by circular dichroism studies which leads eventually to the formation of stable hydrogels, their direct involvement in the gel network is here suggested. In particular, 1H NMR results obtained from a mixed alginate sample containing three polymeric species, G blocks, M blocks, and MG blocks, without chemical linkages between the block structures, indicate for the first time the formation of mixed junctions between G and MG blocks. This is supported by the analysis of the Young's modulus of hydrogels from natural and epimerized samples obtained at low calcium concentrations. Furthermore, the "zipping" of long alternating sequences in secondary MG/MG junctions is suggested to account for the shrinking (syneresis) of alginate gels in view of its dependence on the length of the MG blocks. As a consequence, a partial network collapse, macroscopically revealed by a decrease in the Young's modulus, occurred as the calcium concentration in the gel was increased. The effect of such "secondary" junctions on the viscoelastic properties of alginate gels was evaluated measuring their creep compliance under uniaxial compression. The experimental curves, fitted by a model composed of a Maxwell and a Voigt element in series, revealed an increase in the frictional forces between network chains with increasing length of the alternating sequences. This suggests the presence of an ion mediated mechanism preventing the shear of the gel.     

Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences

Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a NotI restriction fragment of approximately 5.3 Mb and on an MluI fragment of approximately 5.5 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,XX males positive for the repeat sequences. The rearranged NotI fragments in the 46,XX males were 4.4 Mb and the MluI fragments were 2.0 Mb in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome.     

Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction

Interspersed DNA elements of the form (dC-dA)n.(dG-dT)n constitute one of the most abundant human repetitive DNA families. We report that specific human (dC-dA)n.(dG-dT)n blocks are polymorphic in length among individuals and therefore represent a vast new pool of potential genetic markers. Comparison of sequences from the literature for (dC-dA)n.(dG-dT)n blocks cloned two or more times revealed length polymorphisms in seven of eight cases. Variations in the lengths of 10 (dC-dA)n.(dG-dT)n blocks were directly demonstrated by amplifying the DNA within and immediately flanking the repeat blocks by using the polymerase chain reaction and then resolving the amplified DNA on polyacrylamide DNA sequencing gels. Use of the polymerase chain reaction to detect DNA polymorphisms offers improved sensitivity and speed compared with standard blotting and hybridization.