MOTIVATION: Large-scale genome projects generate a rapidly increasing number of sequences, most of them biochemically uncharacterized. Research in bioinformatics contributes to the development of methods for the computational characterization of these sequences. However, the installation and application of these methods require experience and are time consuming. RESULTS: We present here an automatic system for preliminary functional annotation of protein sequences that has been applied to the analysis of sets of sequences from complete genomes, both to refine overall performance and to make new discoveries comparable to those made by human experts. The GeneQuiz system includes a Web-based browser that allows examination of the evidence leading to an automatic annotation and offers additional information, views of the results, and links to biological databases that complement the automatic analysis. System structure and operating principles concerning the use of multiple sequence databases, underlying sequence analysis tools, lexical analyses of database annotations and decision criteria for functional assignments are detailed. The system makes automatic quality assessments of results based on prior experience with the underlying sequence analysis tools; overall error rates in functional assignment are estimated at 2.5-5% for cases annotated with highest reliability ('clear' cases). Sources of over-interpretation of results are discussed with proposals for improvement. A conservative definition for reporting 'new findings' that takes account of database maturity is presented along with examples of possible kinds of discoveries (new function, family and superfamily) made by the system. System performance in relation to sequence database coverage, database dynamics and database search methods is analysed, demonstrating the inherent advantages of an integrated automatic approach using multiple databases and search methods applied in an objective and repeatable manner. AVAILABILITY: The GeneQuiz system is publicly available for analysis of protein sequences through a Web server at http://www.sander.ebi.ac. uk/gqsrv/submit

MOTIVATION: The underlying error rate for genomic sequencing sometimes results in the introduction of artificial frameshifts and in-frame stop codons into putative protein encoding genes. Severe errors are then introduced into the inferred transcripts through mis-translation or premature termination. RESULTS: We describe a system for screening segments of DNA for frameshift and in-frame stop errors in coding regions. The method is based on homology matching using blastx to compare all six reading frames of the query nucleotide sequence against selected protein sequence databases. Fragments of protein matching neighbouring regions of the query DNA are united and extended laterally to define candidate open reading frames, within which, frameshifts and stops are identified. Suitable targets include prokaryotic or other intron-free genomic sequence and complementary DNAs. As an example of its use, we report here two frameshifted ORFs that deviate from the original TIGR sequence annotations for the recently released Helicobacter pylori genome. AVAILABILITY: The tool is accessible via the URL http://www.sander.ebi.ac.uk/frame/. CONTACT: brown@ebi.ac.uk.

A thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs. These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE. Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.

BACKGROUND. The 1.83 Megabase (Mb) sequence of the Haemophilus influenzae chromosome, the first completed genome sequence of a cellular life form, has been recently reported. Approximately 75 % of the 4.7 Mb genome sequence of Escherichia coli is also available. The life styles of the two bacteria are very different - H. influenzae is an obligate parasite that lives in human upper respiratory mucosa and can be cultivated only on rich media, whereas E. coli is a saprophyte that can grow on minimal media. A detailed comparison of the protein products encoded by these two genomes is expected to provide valuable insights into bacterial cell physiology and genome evolution. RESULTS. We describe the results of computer analysis of the amino-acid sequences of 1703 putative proteins encoded by the complete genome of H. influenzae. We detected sequence similarity to proteins in current databases for 92 % of the H. influenzae protein sequences, and at least a general functional prediction was possible for 83 %. A comparison of the H. influenzae protein sequences with those of 3010 proteins encoded by the sequenced 75 % of the E. coli genome revealed 1128 pairs of apparent orthologs, with an average of 59 % identity. In contrast to the high similarity between orthologs, the genome organization and the functional repertoire of genes in the two bacteria were remarkably different. The smaller genome size of H. influenzae is explained, to a large extent, by a reduction in the number of paralogous genes. There was no long range colinearity between the E. coli and H. influenzae gene orders, but over 70 % of the orthologous genes were found in short conserved strings, only about half of which were operons in E. coli. Superposition of the H. influenzae enzyme repertoire upon the known E. coli metabolic pathways allowed us to reconstruct similar and alternative pathways in H. influenzae and provides an explanation for the known nutritional requirements. CONCLUSIONS. By comparing proteins encoded by the two bacterial genomes, we have shown that extensive gene shuffling and variation in the extent of gene paralogy are major trends in bacterial evolution; this comparison has also allowed us to deduce crucial aspects of the largely uncharacterized metabolism of H. influenzae.