to Swiss-Prot (9) and the Entrez Nucleotide and Gene data- bases. Arabidopsis genes are linked to TAIR (The Arabidopsis Information Resource). Enzymes are often linked to protein structure databases such as PDB (10) when applicable. 5 In addition, whenever possible, literature references are linked to PubMed. ENHANCEMENTS TO THE PATHWAY TOOLS SOFTWARE The Pathway Tools software provides query and visualization 10 services to users and editing functions to curators (2,11). Recent enhancements to the software that are relevant to MetaCyc users include the following: Improved displays: The pathway display algorithms have been modified to produce more compact pathway diagrams 15 that are more likely to fit within a single page. Enzyme/gene naming: Protein and gene names within a path- way display are now labeled with the initials of an organism’s genus and species name (e.g. an Escherichia coli enzyme and gene are written as ‘acetylornithine decarboxylase (Ec)’ and 20 ‘Ec-argD’, respectively). This notation aids in identifying individual proteins and genes from multiple organisms that are assigned to the same pathway. Chemical drawing tools: The Pathway Tools software now includes interfaces for both the Marvin (http:// 25 www.chemaxon.com/marvin/) and JME (http://www. molinspiration.com/jme/) chemical drawing editors, permit- ting the user to enter or modify chemical structures within MetaCyc or other PGDBs. A new suite of comparative genomics tools is available in 30 Pathway Tools in conjunction with the preceding expansion of the BioCyc database collection (see below). These tools include comparisons of the full pathway, reaction and meta- bolite sets present in a specified group of organisms; compar- isons of genes associated with a single pathway or a single 35 reaction across a specified group of organisms, including the operon distributions of those genes; and a comparative gen- ome browser for visualizing chromosomal regions around a specified set of orthologous genes. 40 Expansion of BioCyc A major application of MetaCyc is its use for predicting the metabolic pathways of an organism from its sequenced gen- ome (12–14), using the PathoLogic program (15). We recently automated PathoLogic, allowing it to be applied to large num- 45 bers of genomes. Jointly with the European Bioinformatics Institute, we used this feature to expand our BioCyc collection of PGDBs to >200 organism-speciﬁc databases, each of which contains the predicted metabolic pathways of the organism, based on its annotated genome (16). These PGDBs are avail- 50 able for adoption and ongoing curation by the scientiﬁc community. Each PGDB contains the genome of the organism, which is accessible using the new Pathway Tools genome browser (http://biocyc.org/ANTHRA/NEW-IMAGE?type¼FULL- 55 MAP&object¼DNAA), predicted operons (only for bacteria) (17) and predicted metabolic pathways (15). In addition, since predicted pathways often contain pathway holes (reactions in a Nucleic Acids Research, 2006, Vol. 34, Database issue D515 predicted metabolic pathway for which no enzymes have been identiﬁed in the sequenced genome), we applied our pathway- hole ﬁller algorithm to all t6h0e BioCyc PGDBs. This algorithm searches the sequenced genome and identiﬁes candidate genes for these missing enzymes (18). All the PGDBs in the BioCyc collection can be used to analyze gene and protein expression data using the Omics Viewer, a Pathway Tools f65eature that allows expression data and metabolomics data to be painted onto the full metabolic network of an organism. DATABASE AND SOFTWARE AVAILABILITY MetaCyc is freely available via the Web at http://Metacyc.org/ (updated four times a year)70. It is also available for download, free of charge to non-proﬁt organizations or for a fee to com- mercial institutions, as a stand-alone application program for Linux, Windows and Solaris workstations (updated two times a year). A set of ﬂat data ﬁles that is updated four times a year is also available online at 7h5ttp://BioCyc.org/download.shtml. ACKNOWLEDGEMENTS We thank Teresa Steininger and Thomas Kilduff for their help with the catecholamine biosynthesis pathway, Eurie L. Hong and Rama Balakrishnan for the Saccharomyces pathways they submitted to MetaCyc, and80 Tanya Berardini, Leonore Reiser and Wolf Frommer for reviewing the cellular component ontol- ogy. We thank Sunita Reddy for contributions to the chemical compound data, and Aleksey Kleytman and Thomas Yan for curation assistance. This work was supported by grant R01- RR07861-01 from the NI85H National Institute of General Medical Sciences, and by grant R01-HG02729-01 from the NIH National Human Genome Research Institute. P.Z. and S.Y.R. were supported in part by NSF grant DBI-0417062 and C.T. was supported, in part, by a gift from Pioneer Hi-Bred. Funding to pay th9e0 Open Access publication charges for this article was provided by grant GM70065 from the NIH National Institute of General Medical Sciences. Conflict of interest statement. None declared. REFERENCES 1. Krieger,C.J., Zhang,P., Muelle95r,L.A., Wang,A., Paley,S., Arnaud,M., Pick,J., Rhee,S.Y. and Karp,P.D. 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