Molecular Probe Data Base: a database on synthetic oligonucleotides.

The Molecular Probe Data Base (MPDB) was designed to collect and make information on synthetic oligonucleotides available on-line. This paper briefly describes its purpose, contents and structure, forms and mode of data distribution. Particular emphasis is given to recent data extension and system enhancements that have been carried out in order to simplify access to MPDB for unskilled users.     

Molecular Probe Data Base (MPDB).

This paper provides an update on the contents and structure, forms and mode of data distribution of the Molecular Probe Data Base (MPDB), a database that collects and provides on-line information on the sequence, target gene, applications and bibliographic references of synthetic oligonucleotides. The recent data extension and the new means of accessing the database are discussed.     

Molecular Probe Data Base (MPDB).

In this paper, the current status of the Molecular Probe Data Base (http://www.biotech.ist.unige.it/interlab/ mpdb.html ) is briefly presented together with a short analysis of its activity during 1997. This has been performed by statistically evaluating the 'logs' of the Internet servers that are used for its distribution with reference to the geographical origin of the requests, the words that were utilized to carry out of the searches and the oligonucleotides that were retrieved. Planned enhancements of this database are also described. They include a revision of its data structure and, even more relevant, of its data management procedures.     

Molecular probe data base (MPDB).

The molecular probe data base (MPDB) contains detailed information on synthetic oligonucleotides, including their identification, target genes, applications and bibliographic references. It is available on-line through Internet and can be searched by using Network Information Retrieval tools. In this article the most recent enhancements of MPDB, both in terms of data contents and new ways of access, are described. These include a recently established collaboration with EMBL Data Library, in the sphere of SRSWWW network browser, in view of a better integration of MPDB with other molecular biology databases.     

Animal Viruses Probe Dataset (AVPDS) for Microarray-Based Diagnosis and Identification of Viruses

AVPDS (Animal Viruses Probe dataset) is a dataset of virus-specific and conserve oligonucleotides for identification and diagnosis of viruses infecting animals. The current dataset contain 20,619 virus specific probes for 833 viruses and their subtypes and 3,988 conserved probes for 146 viral genera. Dataset of virus specific probe has been divided into two fields namely virus name and probe sequence. Similarly conserved probes for virus genera table have genus, and subgroup within genus name and probe sequence. The subgroup within genus is artificially divided subgroups with no taxonomic significance and contains probes which identifies viruses in that specific subgroup of the genus. Using this dataset we have successfully diagnosed the first case of Newcastle disease virus in sheep and reported a mixed infection of Bovine viral diarrhea and Bovine herpesvirus in cattle. These dataset also contains probes which cross reacts across species experimentally though computationally they meet specifications. These probes have been marked. We hope that this dataset will be useful in microarray-based detection of viruses. The dataset can be accessed through the link https://dl.dropboxusercontent.com/u/94060831/avpds/HOME.html.     

A comparative view at comprehensive information resources on three-dimensional structures of biological macro-molecules.

The rapidly increasing amount of information on three-dimensional (3D) structures of biological macro-molecules has still an insufficient impact on genome analysis, functional genomics and proteomics as well as on many other fields in biomedicine including disease-related research. There are, however, attempts to make structural data more easily accessible to the bench biologist. As members of the world-wide Protein Data Bank (wwPDB), the RCSB Protein Data Bank (PDB), the Protein Data Bank Japan and the Macromolecular Structure Database are the primary information resources for 3D structures of proteins, nucleic acids, carbohydrates and complexes thereof. In addition, a number of secondary resources have been set up that also provide information on all currently known structures in a relatively comprehensive manner and not focusing on specific features only. They include PDBsum, the OCA browser-database for protein structure/function, the Molecular Modeling Database and the Jena Library of Biological Macromolecules--JenaLib. Both the primary and secondary resources often merge the information in the PDB files with data from other resources and offer additional analysis tools thereby adding value to the original PDB data. Here, we briefly describe these resources from a user's point of view and from a comparative perspective. It is our aim to guide researchers outside the structure biology field in getting the most out of the 3D structure resources.     

Novel fluorophore for labelling of oligonucleotides.

A novel fluorophore viz. 4-dansylamido-1,8-naphthalimido-N-pentanol has been designed, prepared and characterised. The comparative fluorescence has been studied in different solvents, solvent gradient, aqueous solutions of inorganic ions and buffers. This can be used for covalent tagging of oligonucleotides having potential application in Molecular Biology.     

PathogenMip assay: a multiplex pathogen detection assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.     

An analysis of the Gene-Tox Carcinogen Data Base

The Gene-Tox Carcinogen Data Base is an evaluated source of cancer data on 506 chemicals selected in part for their previous assessment in genetic toxicology bioassays. This data base has been analyzed for the distribution of these chemicals into chemical classes. The major chemical classes (6% or greater of the total data base) are: acyl-, alkyl-, and aryl-halides; alcohols and phenols; aliphatic and aromatic amines, amides, and sulfonamides; benzene-ring-containing chemicals; organo-lead, -mercury, -phosphorous compounds, metals and derivatives, phosphoric acid esters, and phosphoramides; and polycyclic aromatic hydrocarbons. Cancer studies representing a subset of the Gene-Tox Carcinogen Data Base, 199 chemicals which were rated as Sufficient Positive/Negative or Limited Positive/Negative, were examined for distribution of those studies by animal species, gender, route of chemical administration, duration of study, major tumor sites, and major tumor types. These analyses revealed that the Gene-Tox Carcinogen Data Base contains a large number of lifetime studies involving the rat and mouse treated by oral routes of administration. The major organs that were targets were: liver, lung, skin, forestomach, bladder, and mammary gland, while the major tumor types were: carcinoma, sarcoma, papilloma, and adenoma. Chemicals in the data base have been assessed for species-specific carcinogenic effects, and these results indicate that for mice and rats there is a high correspondence (85%). This number is higher than that (71%) reported by Tennant et al. (1986) based on the recent results of 72 chronic cancer bioassays performed by the National Toxicology Program. This difference is probably based on the nature of the chemicals selected for inclusion in both data bases. Although the absolute value of this correspondence is unknown, it would seem to be within this range. When chemicals in the Gene-Tox Carcinogen Data Base were examined for their previous evaluation in 73 genetic toxicology bioassays, only 26 of these bioassays had 30 or more chemicals. In these 26 bioassays, the prevalence of positive chemicals was generally greater than 80-90%. This suggests that a thorough evaluation of genetic toxicology bioassays in regard to their ability to predict carcinogenic effects in animals is premature at this time.     

Multiplex Ligation-dependent Probe Amplification (MLPA) et sondes « à façon » entièrement synthétiques. Guide pratique,recommandations et expérience au CHU de Grenoble

Multiplex Ligation-dependent Probe Amplification (MLPA) is a powerful technique for gene dosage quantification. This technique is based on the ligation followed by PCR amplification of two adjacently hybridising oligonucleotides. Its reliability and effectiveness have been proven and is now well established. Besides the commercial kits are distributed by the patentee, MRC-Holland?, it makes it possible to utilise custom-designed MLPA probes for a targeted use of this technique. This strategy has opened the field of MLPA for various applications ranging from diagnostic of rarest pathologies to confirmation of a CGH-detected abnormalities. However, it may be difficult to develop without a solid experience. Here, we detail the guidelines and recommendations for designing custom MLPA probes and illustrate their use in our laboratory.     

Synthesis of DNA coding for human proinsulin

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.     

Topography of rRNA in ribosomes. Effect of pancreatic RNAse on small ribosomal subunits]

The treatment of E. coli 30S ribosome subunits with pancreatic RNase under certain conditions resulted in the release of rRNA (about 15%). The subunit retained as a whole structure: sedimentation coefficient was unchanged and no protein release was observed. The released RNA is a set of oligonucleotides from 1 to 9 bases, except hepta- and octanenucleotides. Base composition of this RNA fraction is similar to 16S RNA, a slight increase of purines content being due to the specificity of nuclease. Analysis of isoplit content has revealed that a spliting of long oligonucleotides in stechiometric amount from 30S subunits takes place: one nonanucleotide, one hexanucleotide and two pentanucleotides.     

Cloning of genomic sequences of human prointerleukin 1 beta

The human brain library carried in the EMBL3 vector was employed for isolating prointerleukin 1 beta genomic sequences using three synthetic 20-member oligonucleotides. Oligonucleotides were homologous to the following mRNA regions: 3'-nontranslated region/C1/, 3'-translated region of mRNA/C2/ and the sequence coding N-terminal of mature protein/N1/. The oligonucleotide labeling utilized the terminal nucleotidyltransferase and [alpha-32P] dATP and specific activity of labeled oligonucleotides reached 1.6.10(10) cpm. The sizes of the synthesized labeled sequences (tails) were about 10 b.p. Hybridization probe C1 was used for the first screening and 24 hybridization positive clones were detected. For the next screening probe C2 was used and only 2 hybridization positive clones with different level of hybridization were detected from 24 clones. Probe N1 was used for the third screening and allowed to identify the only positive clone. The characterization by restriction mapping and Southern blot analyses have shown that recombinant phage DNA contains all three exons, coding the mature interleukin 1 beta. Some fragments were recloned to tg130 vector phage and nucleotide sequences of exon 5 (completely) and exon 7, intron 4 and 5 (partially) were determined.     

Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting

Carrier-mediated delivery holds great promise for significantly improving the cellular uptake and therefore the therapeutic efficacy of antisense oligonucleotides in vivo. A multivalent carbohydrate recognition motif for the asialoglycoprotein receptor has been designed for tissue- and cell-specific delivery of antisense drugs to parenchymal liver cells. To combine low molecular weight with high receptor affinity, the synthetic ligand contains three galactosyl residues attached to a cholane scaffold via epsilon-aminocapramide linkers. Three-dimensional structural calculations indicate that this unique design provides proper spacing and orientation of the three galactosyl residues to accomplish high affinity binding to the receptor. Covalent conjugation of the bulky carbohydrate cluster to oligonucleotides has been achieved by solid-phase synthesis using low-loaded macroporous resins and optimized synthesis protocols.     

Optimal sequencing by hybridization in rounds.

Sequencing by hybridization (SBH) is a method for sequencing DNA. The Watson-Crick complementarity of DNA can be used to determine whether the DNA contains an oligonucleotide substring. A large number of oligonucleotides can be arranged on an array (SBH chip). A combinatorial method is used to construct the sequence from the collection of probes that occur in it. We develop an idea of Margaritis and Skiena and propose an algorithm that uses a series of small SBH chips to sequence long strings. The total number of probes used by our method matches the information theoretical lower bound up to a constant factor.     

Mirrors in the PDB: left-handed α-turns guide design with D-amino acids

Background  

Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids.     

Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing

A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.