EMBL Data Bank Research Articles

Isolation and characterization of a cDNA encoding rat liver cytosolic epoxide hydrolase and its functional expression in Escherichia coli

A cDNA of 1992 base pairs encoding the complete rat liver cytosolic epoxide hydrolase has been isolated using a polymerase chain reaction-derived DNA fragment (Arand, M., Knehr, M., Thomas, H., Zeller, H. D., and Oesch, F. (1991) FEBS Lett. 294, 19-22) known to represent the 3'-end of the cytosolic epoxide hydrolase mRNA. Sequence analysis revealed an open reading frame of 1662 nucleotides corresponding to 554 amino acids (M(r) = 62,268). The DNA sequence obtained did not display significant homology to the sequences of microsomal epoxide hydrolase or leukotriene A4 hydrolase or to any other DNA included in the EMBL Data Bank (release 32). On Northern blotting of rat liver RNA, a single mRNA species was detected that was strongly induced on treatment of the animal with fenofibrate, a potent peroxisome proliferator. The most significant structure of the deduced protein is a modified peroxisomal targeting signal (Ser-Lys-Ile) at the carboxyl terminus that is regarded to be responsible for the unusual dual localization of the cytosolic epoxide hydrolase in peroxisomes as well as in the cytosol. In addition, a leucine zipper-like motif was identified at the amino terminus. Its possible implication for the observed dimeric structure of cytosolic epoxide hydrolase is discussed. The isolated cDNA was expressed in bacteria to yield a catalytically active enzyme. Specific activity of the crude lysate obtained exceeded that of rat liver cytosols from maximally induced animals by a factor of 8.

Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction

The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

A comprehensive compilation of 1001 nucleotide sequences coding for proteins from the yeast Saccharomyces cerevisiae (= ListA2)

The amount of nucleotide sequence data is increasing exponentially. We therefore continued our effort to make a comprehensive database for the yeast Saccharomyces cerevisiae. In this database (ListA2) we have compiled 1001 protein coding sequences from this organism. Each sequence has been attributed a single genetic name and in the case of allelic duplicated sequences, synonyms are given, if necessary. For the nomenclature we have introduced a standard principle for naming gene sequences based on priority rules. We have also applied a simple method to distinguish duplicated sequences of one and the same gene from non-allelic sequences of duplicated genes. By using these principles we have sorted out a lot of confusion in the literature and databanks. Along with the genetic name, the mnemonic from the EMBL databank, the codon bias, reference of the publication of the sequence and the EMBL accession numbers are included for each entry. The database is available on request.

LISTA, a comprehensive compilation of nucleotide sequences encoding proteins from the yeast Saccharomyces.

The amount of nucleotide sequence data is increasing exponentially. We therefore made an effort to make a comprehensive database (LISTA) for the yeast Saccharomyces cerevisiae. Each sequence has been attributed a single genetic name and in the case of allelic duplicated sequences, synonyms are given, if necessary. For the nomenclature we have introduced a standard principle for naming gene sequences based on priority rules. We have also applied a simple method to distinguish duplicated sequences of one and the same gene from non-allelic sequences of duplicated genes. By using these principles we have sorted out a lot of confusion in the literature and databanks. Along with the genetic name, the mnemonic from the EMBL databank, the codon bias, reference of the publication of the sequence and the EMBL accession numbers are included in each entry.

Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of Streptococcus suis type 2.

We cloned and sequenced the gene encoding the muramidase-released protein (MRP) of a pathogenic Streptococcus suis type 2 strain to determine whether its amino acid sequence resembles that of proteins with known functions and to determine its function in virulence. The complete nucleotide sequence composing the gene and the regions flanking it was determined. The deduced amino acid sequence revealed the presence of a signal peptide at the N terminus and a cell envelope anchor at the C terminus, both of which resembled similar regions in several other surface proteins from gram-positive bacteria. The processed form of MRP has a length of 1,209 amino acids and a calculated molecular weight of 131,094. A highly repetitive region preceded the envelope anchor. The repeated units were preceded by a proline-rich stretch of amino acids (26 of 86). No overall homologies were observed between the amino acid sequence of MRP and protein sequences in the EMBL data bank. A particular region within the amino acid sequence, however, showed some similarity with the fibronectin-binding protein of Staphylococcus aureus. Binding of MRP to human fibronectin, however, could not be confirmed.

Identification of a new motif on nucleic acid sequence data using Kohonen's self-organizing map

Here we present a performance test of a Kohonen features map applied to the fast extraction of uncommon sequences from the coding region of the human insulin receptor gene. We used a network with 30 neurons and with a variable input window. The program was aimed at detecting unique or uncommon DNA regions present in crude sequence data and was able to automatically detect the signal peptide coding regions of a set of human insulin receptor gene data. The testing of this program with HSIRPR cDNA release (EMBL data bank) indicated the presence of unique features in the signal peptide coding region. On the basis of our results this program can automatically detect 'singularity' from crude sequencing data and it does not require knowledge of the features to be found.

Neural network detects errors in the assignment of mRNA splice sites

The use of databanks in genetic research assumes reliability of the information they contain. Currently, error-detection in the manually or electronically entered data contained in the nucleotide sequence databanks at EMBL, Heidelberg and GenBank at Los Alamos is limited. We have used a subset of sequences from these databanks to train neural networks to recognize pre-mRNA splicing signals in human genes. During the training on 33 human genes from the EMBL databank seven genes appeared to disturb the learning process. Subsequent investigation revealed discrepancies from the original published papers, for three genes. In four genes, we found wrongly assigned splicing frames of introns. We believe this to be a reflection of the fact that splicing frames cannot always be unambiguously assigned on the basis of experimental data. Thus incorrect assignment appear both due to mere typographical misprints as well as erroneous interpretation of experiments. Training on 241 human sequences from GenBank revealed nine new errors. We propose that such errors could be detected by computer algorithms designed to check the consistency of data prior to their incorporation in databanks.

Cloning and partial sequencing of a new rat brain specific cDNA

In order to find brain specific transcripts in a pUC13 cDNA library prepared from rat brain cytoplasmic poly(A)+ RNAs, the following steps were observed: 1. Randomly chosen cDNA clones from the brain library were screened by radiolabelled single stranded cDNAs (sscDNAs) prepared from liver, spleen, kidney and intestine mRNAs. 2. The brain clones containing genetic information shared with the peripheral organs were discarded. 3. After hybridization of the remaining clones with radiolabelled sscDNAs prepared from rat brain poly(A)+ RNAs, 210 possibly brain specific clones were selected. 4. Plasmids containing cDNA of each of these clones were purified and after estimation of their cDNA sizes, clones containing inserts of at least 500 base pairs (bp) were used as radiolabelled probes against rat brain, liver, spleen, kidney and intestine poly(A)+ RNAs spotted onto nitrocellulose filters (dot blot analysis). 5. By using this screening procedure, several brain specific clones were obtained. One of them (clone III 25) is presently subjected to sequencing in M13 phage. 6. A computer analysis of the partial sequence so far obtained gives no significant homology to the 15.000 genes contained in Genebank and EMBL data bank. It seems therefore that we have isolated a new brain specific cDNA coding for a not yet described peptide.