Regulation of ribonuclease III processing by double-helical sequence antideterminants

The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay. Escherichia coli ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs. The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops. The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity. An alignment of RNase III substrates revealed an exclusion of specific Watson–Crick bp sequences at defined positions relative to the cleavage site. Inclusion of these “disfavored” sequences in a model substrate strongly inhibited cleavage in vitro by interfering with RNase III binding. Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNASec, which acts as an antideterminant of EF-Tu binding to tRNASec. The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA. Base pair antideterminants also may protect double-helical elements in other RNA molecules with essential functions.

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

Characterization of BseMII, a new type IV restriction–modification system, which recognizes the pentanucleotide sequence 5′-CTCAG(N)10/8↓

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5′-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3′-protruding end. Magnesium ions and S-adenosyl-l-methionine (AdoMet) are required for cleavage. S-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5′-CTCAG-3′/5′-CTGAG-3′. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5′-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-l-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.

Diversity Arrays: a solid state technology for sequence information independent genotyping

Here we present the successful application of the microarray technology platform to the analysis of DNA polymorphisms. Using the rice genome as a model, we demonstrate the potential of a high-throughput genome analysis method called Diversity Array Technology, DArT‘. In the format presented here the technology is assaying for the presence (or amount) of a specific DNA fragment in a representation derived from the total genomic DNA of an organism or a population of organisms. Two different approaches are presented: the first involves contrasting two representations on a single array while the second involves contrasting a representation with a reference DNA fragment common to all elements of the array. The Diversity Panels created using this method allow genetic fingerprinting of any organism or group of organisms belonging to the gene pool from which the panel was developed. Diversity Arrays enable rapid and economical application of a highly parallel, solid-state genotyping technology to any genome or complex genomic mixtures.

Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria

The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared. In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined. Sequences similar to the σ70 promoters P1, P4 and P5, to the σE promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae. In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like σ70 promoter and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. Sequences similar to the regulatory boxes were not identified in these bacteria. This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal σ70 promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced σE-like promoter and a σ70 promoter. A second proximal σ70 promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli.

Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones

This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62.2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.

Gene2EST: a BLAST2 server for searching expressed sequence tag (EST) databases with eukaryotic gene-sized queries

Expressed sequence tags (ESTs) are randomly sequenced cDNA clones. Currently, nearly 3 million human and 2 million mouse ESTs provide valuable resources that enable researchers to investigate the products of gene expression. The EST databases have proven to be useful tools for detecting homologous genes, for exon mapping, revealing differential splicing, etc. With the increasing availability of large amounts of poorly characterised eukaryotic (notably human) genomic sequence, ESTs have now become a vital tool for gene identification, sometimes yielding the only unambiguous evidence for the existence of a gene expression product. However, BLAST-based Web servers available to the general user have not kept pace with these developments and do not provide appropriate tools for querying EST databases with large highly spliced genes, often spanning 50 000–100 000 bases or more. Here we describe Gene2EST (http://woody.embl-heidelberg.de/gene2est/), a server that brings together a set of tools enabling efficient retrieval of ESTs matching large DNA queries and their subsequent analysis. RepeatMasker is used to mask dispersed repetitive sequences (such as Alu elements) in the query, BLAST2 for searching EST databases and Artemis for graphical display of the findings. Gene2EST combines these components into a Web resource targeted at the researcher who wishes to study one or a few genes to a high level of detail.

OliI, a unique restriction endonuclease that recognizes the discontinuous sequence 5′-CACNN↓NNGTG-3′

A new type II restriction endonuclease designated OliI has been partially purified from the halophilic bacterium Oceanospirillum linum 4-5D. OliI recognizes the interrupted hexanucleotide palindrome 5′-CACNN↓NNGTG-3′ and cleaves it in the center generating blunt-ended DNA fragments.

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ∼2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ∼60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

The EMBL nucleotide sequence database

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) is maintained at the European Bioinformatics Institute (EBI) in an international collaboration with the DNA Data Bank of Japan (DDBJ) and GenBank at the NCBI (USA). Data is exchanged amongst the collaborating databases on a daily basis. The major contributors to the EMBL database are individual authors and genome project groups. Webin is the preferred web-based submission system for individual submitters, whilst automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via ftp, email and World Wide Web interfaces. EBI’s Sequence Retrieval System (SRS), a network browser for databanks in molecular biology, integrates and links the main nucleotide and protein databases plus many specialized databases. For sequence similarity searching a variety of tools (e.g. Blitz, Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT.

IMGT/HLA Database—a sequence database for the human major histocompatibility complex

The IMGT/HLA Database (www.ebi.ac.uk/imgt/hla/) specialises in sequences of polymorphic genes of the HLA system, the human major histocompatibility complex (MHC). The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. Many of the genes encode proteins of the immune system and these include the 21 highly polymorphic HLA genes, which influence the outcome of clinical transplantation and confer susceptibility to a wide range of non-infectious diseases. The database contains sequences for all HLA alleles officially recognised by the WHO Nomenclature Committee for Factors of the HLA System and provides users with online tools and facilities for their retrieval and analysis. These include allele reports, alignment tools and detailed descriptions of the source cells. The online IMGT/HLA submission tool allows both new and confirmatory sequences to be submitted directly to the WHO Nomenclature Committee. The latest version (release 1.7.0 July 2000) contains 1220 HLA alleles derived from over 2700 component sequences from the EMBL/GenBank/DDBJ databases. The HLA database provides a model which will be extended to provide specialist databases for polymorphic MHC genes of other species.

Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs

The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.

ACTIVITY: a database on DNA/RNA sites activity adapted to apply sequence-activity relationships from one system to another

ACTIVITY is a database on DNA/RNA site sequences with known activity magnitudes, measurement systems, sequence-activity relationships under fixed experimental conditions and procedures to adapt these relationships from one measurement system to another. This database deposits information on DNA/RNA affinities to proteins and cell nuclear extracts, cutting efficiencies, gene transcription activity, mRNA translation efficiencies, mutability and other biological activities of natural sites occurring within promoters, mRNA leaders, and other regulatory regions in pro- and eukaryotic genomes, their mutant forms and synthetic analogues. Since activity magnitudes are heavily system-dependent, the current version of ACTIVITY is supplemented by three novel sub-databases: (i) SYSTEM, measurement systems; (ii) KNOWLEDGE, sequence-activity relationships under fixed experimental conditions; and (iii) CROSS_TEST, procedures adapting a relationship from one measurement system to another. These databases are useful in molecular biology, pharmacogenetics, metabolic engineering, drug design and biotechnology. The databases can be queried using SRS and are available through the Web, http://wwwmgs.bionet.nsc.ru/systems/Activity/.

STACK: Sequence Tag Alignment and Consensus Knowledgebase

STACK is a tool for detection and visualisation of expressed transcript variation in the context of developmental and pathological states. The datasystem organises and reconstructs human transcripts from available public data in the context of expression state. The expression state of a transcript can include developmental state, pathological association, site of expression and isoform of expressed transcript. STACK consensus transcripts are reconstructed from clusters that capture and reflect the growing evidence of transcript diversity. The comprehensive capture of transcript variants is achieved by the use of a novel clustering approach that is tolerant of sub-sequence diversity and does not rely on pairwise alignment. This is in contrast with other gene indexing projects. STACK is generated at least four times a year and represents the exhaustive processing of all publicly available human EST data extracted from GenBank. This processed information can be explored through 15 tissue-specific categories, a disease-related category and a whole-body index and is accessible via WWW at http://www.sanbi.ac.za/Dbases.html. STACK represents a broadly applicable resource, as it is the only reconstructed transcript database for which the tools for its generation are also broadly available (http://www.sanbi.ac.za/CODES).