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Microarray technology has become one of the most important functional genomics technologies. A proliferation of microarray databases has resulted. It can be difficult for researchers exploring this technology to know which bioinformatics systems best meet their requirements. In order to obtain a better understanding of the available systems, a survey and comparative analysis of microarray databases was undertaken. The survey included databases that are currently available, as well as databases that should become available in early 2001. Databases fall into three categories: (i) those that can be installed locally, (ii) those available for public data submission and (iii) those available for public query. Developers of microarray gene-expression databases were asked questions regarding the scope and availability of their database, its system requirements, its future compliance with MGED (Microarray Gene Expression Database) standards, and its associated analytical tools. Participants included AMAD (Stanford/Berkeley/UCSF), ArrayExpress (EBI), ChipDB (MIT/Whitehead), GeneX (NCGR), GeNet (Silicon Genetics), GeneDirector (BioDiscovery), GEO (NCBI), GXD (Jackson Laboratory), mAdb (NCI), maxdSQL (University of Manchester), NOMAD (UCSF), RAD (University of Pennsylvania) and SMD (Stanford University). Other database developers were contacted but data was not available at the time of manuscript preparation. Each database fulfils a different role, reflecting the widely varying needs of microarray users.

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MOTIVATION: Sequence databases represent an enormous resource of phylogenetic information, but there is a lack of tools for accessing that information in order to assess the amount of evolutionary information in these databases that may be suitable for phylogenetic reconstruction and for identifying areas of the taxonomy that are under-represented for specific gene sequences. RESULTS: We have developed TreeGeneBrowser which allows inspection and evaluation of gene sequence data for phylogenetic reconstruction. This program improves the efficiency of identification of genes that may be useful for particular phylogenetic studies and identifies taxa and taxonomic branches that are under-represented in sequence databases.

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We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.

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Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.

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The taxonomically broad organelle genome database (GOBASE) organizes and integrates diverse data related to organelles (mitochondria and chloroplasts). The current version of GOBASE focuses on the mitochondrial subset of data and contains molecular sequences, RNA secondary structures and genetic maps, as well as taxonomic information for all eukaryotic species represented. The database has been designed so that complex biological queries, especially ones posed in a comparative genomics context, are supported. GOBASE has been implemented as a relational database with a web-based user interface (http://megasun.bch.umontreal.ca/gobase/gobas e.html ). Custom software tools have been written in house to assist in the population of the database, data validation, nomenclature standardization and front-end design. The database is fully operational and publicly accessible via the World Wide Web, allowing interactive browsing, sophisticated searching and easy downloading of data.

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The rules that govern the dynamics of protein characterisation by peptide-mass fingerprinting (PMF) were investigated through multiple interrogations of a nonredundant protein database. This was achieved by analysing the efficiency of identifying each entry in the entire database via perfect in silico digestion with a series of 20 pseudo-endoproteinases cutting at the carboxy terminal of each amino acid residue, and the multiple cutters: trypsin, chymotrypsin and Glu-C. The distribution of peptide fragment masses generated by endoproteinase digestion was examined with a view to designing better approaches to protein characterisation by PMF. On average, and for both common and rare cutters, the combination of approximately two fragments was sufficient to identify most database entries. However, the rare cutters left more entries unidentified in the database. Total coverage of the entire database could not be achieved with one enzymatic cutter alone, nor when all 23 cutters were used together. Peptide fragments of > 5000 Da had little effect on the outcome of PMF to correctly characterise database entries, while those with low mass (near to 350 Da in the case of trypsin) were found to be of most utility. The most frequently occurring fragments were also found in this lower mass region. The maximum size of uncut database entries (those not containing a specific amino acid residue) ranged from 52,908 Da to 258,314 Da, while the failure rate for a single cutter in identifying database entries varied from 10,865 (8.4%) to 23,290 (18.1%). PMF is likely to be a mainstay of any high-throughput protein screening strategy for large-scale proteome analysis. A better understanding of the merits and limitations of this technique will allow researchers to optimise their protein characterisation procedures.

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The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

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The staphylococcal beta-lactamase (Bla) transposon Tn4002 has previously been reported to have a high level of insertional specificity for a 1.8-kb region on the plasmid pSK1. Nucleotide sequences of this region and of a related region on plasmid pI9789 were determined. Sequence analysis revealed that these two plasmids contain sin, a gene whose deduced product shows similarity to a family of DNA recombinases. Southern hybridisation analysis indicated that sin is located on alpha-, beta- and gamma-families of Bla plasmids and on pSK1 family plasmids. A region of dyad symmetry located upstream from sin on pSK1 and pI9789 was identified as the site of insertions of Tn552 and Tn4002 in separate isolates.

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The occurrence of resistance to antiseptics and disinfectants in clinical isolates of coagulase-negative staphylococci (CNS) was examined. Of 164 clinical strains of CNS isolated in the early 1980s, 65 were resistant to cationic antimicrobial compounds such as cetyltrimethylammonium bromide. Further characterisation of 40 resistant isolates by DNA-DNA hybridisation analysis and phenotypic resistance studies revealed that this resistance was mediated by the multidrug export genes qacA and qacC, characterised previously in Staphylococcus aureus. Of the resistant CNS isolates, 50% contained only qacA, 10% contained only qacC, and the remaining 40% contained both qacA and qacC. Both qacA and qacC genes resided on plasmids in all cases, with qacA located on plasmids of > 10 kb, whereas qacC was located primarily on plasmids of 2-3 kb. Representative qacA and qacC plasmids were characterised by restriction endonuclease mapping, and were found to be similar in some cases, but different in others, to those plasmids on which these genes are found in S. aureus.

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Entrez is a molecular sequence and reference database. We present a tool called CLEVER which permits flexible access to the Entrez database by other applications and interactively by users. In this way, CLEVER is both a search engine and a command-line browser of the Entrez database.

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Nucleotide sequence analysis of ORF1 from the integron on the broad-host-range plasmid R751 revealed that the first 94 of 110 codons of ORF1 from R751 are identical to ORF4, an open reading frame from the 3' conserved segment of other integrons found in gram-negative bacteria, after which point they diverged completely. The predicted products of both ORF1 and ORF4 share homology with the multidrug exporter QacC. Phenotypic analysis revealed that ORF1 specifies a resistance profile to antiseptics and disinfectants almost identical to that of qacC, whereas ORF4 specifies much lower levels of resistance to these compounds. ORF4, whose product lacks the C-terminal 16 amino acids of the ORF1 protein, may have evolved by the interruption of ORF1 from the insertion of a DNA segment carrying a sulI sulfonamide resistance determinant. Hence, ORF1 was designated qacE, and its partially functional deletion derivative, ORF4, was designated qacE delta 1. Fluorimetric experiments indicated that the mechanism of resistance mediated by QacE, the protein specified by qacE, is active export energized by proton motive force. Amino acid sequence comparisons revealed that QacE is related to a family of small multidrug export proteins with four transmembrane segments.

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The amdR gene of Aspergillus nidulans regulates a number of structural genes in response to omega amino acid inducers. The site of action of the amdR product on expression of the amdS gene was investigated by studying the effects of changes in the 5' region of amdS, generated in vitro, on the induction, and on responses of an amdS-lacZ fusion gene to an amdRc allele. A sequence was identified that is sufficient for amdR regulation and that shows identity with sequences involved in amdR regulation of the gatA and lam genes. This sequence includes a CCAAT sequence and it was shown that this sequence is an important element in setting the basal level of amdS expression.

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Clinical isolates of Staphylococcus aureus carry various antiseptic and disinfectant resistance determinants (qac genes) on a variety of plasmids. The biochemistry and specificity of these resistance genes in S. aureus is the subject of this report. The qac genes were separated into two families on the basis of resistance profiles and DNA homology. Isotopic and fluorimetric assays demonstrated that the qac genes encode efflux systems that rely on proton motive force.

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Resistance to antiseptics and disinfectants in Staphylococcus aureus, encoded by the qacC/qacD gene family, is associated with genetically dissimilar small, nontransmissible (pSK89) and large conjugative (pSK41) plasmids. The qacC and qacD genes were analysed in detail through deletion mapping and nucleotide sequence analysis, and shown to encode the same polypeptide, predicted to be 107 aa in size. Direct repeat elements flank the qacD gene, elements which also flank the qacC gene in truncated forms. These elements contain palA sequences, regions of DNA required for replication of some plasmids in S. aureus. The qacC gene is predicted to have evolved from the qacD gene, and in the process to have become reliant on new promoter sequences for its expression. The entire sequence of the 2.4-kb plasmid pSK89 (which contains qacC) was determined, and is compared with other plasmids from Gram + bacteria.

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Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium-type antiseptic compounds, mediated by the Staphylococcus aureus plasmid pSK1 is specified by an energy-dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36 bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins responsible for export-mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N- and C-terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram-negative, but not those from Gram-positive, bacteria.

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The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.

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