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PubMed2006

DSTHO: database of siRNAs targeted at human oncogenes: a statistical analysis.

Existing treatments of human cancer, which is characterized by abnormal proliferation of cells often lead to fatal outcomes. Sequence selective silencing of oncogene expression using siRNA technology is emerging as a potential solution for cancer treatment. The exclusive selectivity and easy application to virtually any therapeutic target including intracellular factors and transcription factors renders siRNA oligonucleotide applications very promising. However, synthesis of siRNA having sufficient knockdown efficiency is laborious and cost intensive. The database is designed in order to aid the synthesis of siRNAs, which target human oncogenes (OsiRNAs). It provides OsiRNAs of known efficacy from previous experiments with links to published literature and theoretically pre-generated putative target sequences. In addition, links to available theoretical tools, databases and literature corresponding to siRNAs in general are also provided. The links to literature provide information about role of siRNA in therapeutics, chemical properties and transfection methods. Statistical analysis of mono-, di- and tri- mers located in OsiRNAs of known efficacies is performed to identify positional preferences and screen specific motifs. This analysis aids the design and synthesis of effective siRNAs, which particularly target human oncogenes. The database can be accessed at .

Base Composition↗
PubMed2004

Analysis of Sarcoptes scabiei finds no evidence of infection with Wolbachia.

The endosymbiont Wolbachia has been detected in a range of filarial nematodes and parasitic mites and is known to affect host reproductive compatibility and potentially evolutionary processes. PCR of Wolbachia surface protein (wsp), ftsZ and 16SrRNA genes from individual Sarcoptes scabiei mites obtained from a series of individual hosts, and database searches of an S. scabiei var. hominis EST library failed to detect Wolbachia genes. Therefore, Wolbachia appears not to be involved in the genetic subdivision observed between varieties of host-associated S. scabiei or, involved in the inflammatory disease pathogenesis of scabies unlike its activity in filarial infection.

Animals↗
PubMed2006

Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.

Animals↗
PubMed2006

Evolutionary relationships of Spirurina (Nematoda: Chromadorea: Rhabditida) with special emphasis on dracunculoid nematodes inferred from SSU rRNA gene sequences.

The analysis of 26 new small subunit rRNA sequences obtained from helminths that primarily parasitize fishes sampled from five continents provided well-supported trees, allowing us to study the phylogenetic relationships among spirurid nematodes. The analyses have shown that Dracunculoidea is a paraphyletic taxon and Anguillicolidae and Gnathostomatidae constitute the basal branch of the suborder Spirurina. The genera Philometra and Philometroides appear to be paraphyletic, while on the higher taxonomic level, good correlation between the morphology-based system and molecular data was observed. Neither co-evolution of the studied helminths with their hosts, nor phylogeographic pattern, are apparent in our dataset.

Animals↗
PubMed2005

Establishment of tyrosinase sequence database in normally pigmented Indians and Japanese for rapid determination of novel mutations.

BACKGROUND: Many mutations of the tyrosinase gene have been reported in oculocutaneous albinism type I (OCA1) patient. In the future, a greater number of novel mutations will be found as the search for pathological mutations in the tyrosinase genes of OCA patients from various ethnic origins. For rapid determination in future whether an observed mutation is a polymorphism or a novel pathological one, sequence databases of the gene of various ethnic people are needed. OBJECTIVE: We established a sequence database of the tyrosinase gene of Japanese as well as Indian people. METHOD: We collected DNA from 109 Japanese and 103 Indians with normal pigmentation and analyzed their tyrosinase gene using a direct sequencing method. RESULT: The database shows an apparent difference between the two ethnic groups in polymorphisms of the tyrosinase gene namely, Q402 allele, Y192 allele and IV2+24 insT were found in the Indian population, but not in the Japanese. On the other hand, some Japanese had IV2-21 insT but none of the Indians did. The database supports the notion that the tyrosinase gene evolved and extended separately in the two ethnic groups. And the developing database confirmed that the reported mutations causing Indian and Japanese OCA were not among the polymorphisms in the database, which conversely gives genetical proof of the "genuine" pathological mutations. CONCLUSION: Eventually, the sequence database we established will contribute to demonstrating novel mutations of albinism in Indians and Japanese.

Albinism, Ocular↗
PubMed2004

The MRJP/YELLOW protein family of Apis mellifera: identification of new members in the EST library.

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.

Animals↗
PubMed2004

Alternative splicing mechanisms for the modulation of protein function: conservation between human and other species.

Alternative splicing (AS) is an important process in eukaryotic organisms by which a given gene may express a set of different protein isoforms depending on the tissue, or the developmental stage of the individual. In the present work, we have compared AS among species, focusing on the conservation of AS mechanisms for the modulation of protein function. For this purpose, we first analysed the frequency with which different species, human, mouse, rat and fruitfly, utilise them. Second, we focused more directly on the conservation among species of the mechanisms themselves. To this end, we compared biologically equivalent AS events between human and mouse, or rat. Our results indicate only minor differences in the frequency of use of these mechanisms, as well as a high degree of conservation among the species studied.

Alternative Splicing↗
PubMed2004

Analysis of the human Alu Ya-lineage.

The Alu Ya-lineage is a group of related, short interspersed elements (SINEs) found in primates. This lineage includes subfamilies Ya1-Ya5, Ya5a2 and others. Some of these subfamilies are still actively mobilizing in the human genome. We have analyzed 2482 elements that reside in the human genome draft sequence and focused our analyses on the 2318 human autosomal Ya Alu elements. A total of 1470 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual Ya-lineage Alu elements. About 22% (313/1452) of the Ya-lineage Alu elements were polymorphic for the insertion presence on human autosomes. Less than 0.01% (5/1452) of the Ya-lineage loci analyzed displayed insertions in orthologous loci in non-human primate genomes. DNA sequence analysis of the orthologous inserts showed that the orthologous loci contained older pre-existing Y, Sc or Sq Alu subfamily elements that were the result of parallel forward insertions or involved in gene conversion events in the human lineage. This study is the largest analysis of a group of "young", evolutionarily related human subfamilies. The size, evolutionary age and variable allele insertion frequencies of several of these subfamilies makes members of the Ya-lineage useful tools for human population studies and primate phylogenetics.

Alu Elements↗
PubMed2005

Extracting stacking interaction parameters for RNA from the data set of native structures.

A crucial step in the determination of the three-dimensional native structures of RNA is the prediction of their secondary structures, which are stable independent of the tertiary fold. Accurate prediction of the secondary structure requires context-dependent estimates of the interaction parameters. We have exploited the growing database of natively folded RNA structures in the Protein Data Bank (PDB) to obtain stacking interaction parameters using a knowledge-based approach. Remarkably, the calculated values of the resulting statistical potentials (SPs) are in excellent agreement with the parameters determined using measurements in small oligonucleotides. We validate the SPs by predicting 74% of the base-pairs in a dataset of structures using the ViennaRNA package. Interestingly, this number is similar to that obtained using the measured thermodynamic parameters. We also tested the efficacy of the SP in predicting secondary structure by using gapless threading, which we advocate as an alternative method for rapidly predicting RNA structures. For RNA molecules with less than 700 nucleotides, about 70% of the native base-pairs are correctly predicted. As a further validation of the SPs we calculated Z-scores, which measure the relative stability of the native state with respect to a manifold of higher free energy states. The computed Z-scores agree with estimates made using calorimetric measurements for a few RNA molecules. Structural analysis was used to rationalize the success and failures of SP and experimentally determined parameters. First, from the near perfect linear relationship between the number of native base-pairs and sequence length, we show that nearly 46% of nucleotides are not in stacks. Second, by analyzing the suboptimal structures that are generated in gapless threading we show that the SPs and experimentally determined parameters are most successful in predicting stacks that end in hairpins. These results show that further improvement in secondary structure prediction requires reliable estimates of interaction parameters for loops, bulges, and stacks that do not end in hairpins.

Base Pairing↗
PubMed2006

In silico approaches reveal the potential for DNA sequence-dependent histone octamer affinity to influence chromatin structure in vivo.

Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.

Base Sequence↗
PubMed2006

QCRNA 1.0: a database of quantum calculations for RNA catalysis.

This work outlines a new on-line database of quantum calculations for RNA catalysis (QCRNA) available via the worldwide web at http://theory.chem.umn.edu/QCRNA. The database contains high-level density functional calculations for a large range of molecules, complexes and chemical mechanisms important to phosphoryl transfer reactions and RNA catalysis. Calculations are performed using a strict, consistent protocol such that a wealth of cross-comparisons can be made to elucidate meaningful trends in biological phosphate reactivity. Currently, around 2000 molecules have been collected in varying charge states in the gas phase and in solution. Solvation was treated with both the PCM and COSMO continuum solvation models. The data can be used to study important trends in reactivity of biological phosphates, or used as benchmark data for the design of new semiempirical quantum models for hybrid quantum mechanical/molecular mechanical simulations.

Computer Graphics↗
PubMed2006

Use of IGHJ and IGHD gene mutations in analysis of immunoglobulin sequences for the prognosis of chronic lymphocytic leukemia.

The level of somatic point mutation in immunoglobulin genes is an important prognostic indicator for patients with chronic lymphocytic leukemia (CLL). Mutation analysis presently focuses solely upon the heavy chain IGHV gene, however mutation is a stochastic process that also targets IGHD and IGHJ genes. Here, we evaluate the completeness and reliability of the reported IGHJ gene repertoire, and demonstrate the likely consequences of the inclusion of IGHD and IGHJ mutations in CLL analysis, using a dataset of 607 sequences. Inclusion of these mutations would lead to the re-classification of many sequences, which should significantly improve the prognostic value of mutation analysis.

Computational Biology↗
PubMed2003

A simple tandem repeat polymorphism is present in the eighth intron of FLJ12960, a possible queuine salvage enzyme gene.

The putative gene FLJ12960 was identified from sequence analysis of the human genome and predicted to be a member of the family of tRNA-guanine-transglycosylases for queuine biosynthesis by protein sequence similarity at the Zn-binding site (Locus Link #79691 at http://www.ncbi.nlm.gov/LocusLink/). FLJ12960 is immediately downstream of the Dopamine Receptor D3 (DRD3) gene. A Simple Tandem Repeat (STR) polymorphism was identified in intron 8 of the FLJ12960 gene. Primers designed to amplify the CA repeat detect 16 alleles from 121 to 151 base pairs, with a heterozygosity of 0.74.

Base Sequence↗