7578 Nucleic Acids Research, 2007, Vol. 35, No. 22 MultiFun, Gene Ontology and other standard controlled vocabularies? We put forth EcoCyc as a model for the annotation of other genomes. Its ﬁrst dimension of genome annotation includes the highest fraction of experimentally validated annotations of any free-living organism, and experimental annotations are identiﬁed as such. EcoCyc provides textual summaries for every gene for which published literature has been identiﬁed by our curation team, along with extensive literature citations. Controlled vocabularies are used extensively within EcoCyc, and the database schema behind EcoCyc provides an exceptional level of computability. EcoCyc describes the metabolic, transport and transcriptional regulatory networks of E. coli. We argue that all genome annotations for important experimental organisms should be performed in the context of a larger model-organism DB project that couples ongoing computational reannotation of the genome with a literature-based curation eﬀort for the genome, and for the cellular networks of the organism, so as to integrate the best computational and experimental information available within a single resource. We further argue that another key to producing a rich and compre- hensive genome annotation is the underlying software and database schema: without a suitable vessel to hold it (i.e. a database schema that can accurately capture each dimension of the annotation), a genome annotation can attain only limited dimensionality. The Pathway Tools software (5) that is used to construct EcoCyc is being used by groups outside SRI to create EcoCyc-like DBs for more than 100 other organisms. Furthermore, SRI has created 370 EcoCyc-like DBs within our BioCyc DB collection (see BioCyc.org). We make these DBs available for adoption (see http://biocyc.org/intro.shtml#adopt) by scientists who will reﬁne them through ongoing curation. MATERIALS AND METHODS The results in this article pertain to version 11.0 of the EcoCyc DB released in March 2007. Analyses were performed using DB queries written in the Common Lisp language. Dead-end metabolites were identiﬁed by a Common Lisp program that considers a compound C to be a dead end if and only if the following conditions are true. (i) C or a parent compound class of C is a substrate in only one reaction of the set of small-molecule reactions occurring in EcoCyc. (ii) C is not transported into E. coli by transporters deﬁned in EcoCyc, nor are parent classes of C. (iii) C is a small molecule. A parent class of C is a database object describing a class of chemical compounds. For example, the function of a protein that transports all known amino acids is modeled in EcoCyc by a transport reaction among whose substrates is the class Amino Acids. That class is a parent of every individual amino acid in EcoCyc. This approach allows EcoCyc to compactly describe enzymes and transporters of broad substrate speciﬁcity. An alternative way to construct condition (i) would be for C to be a substrate of reactions that only produce C, or that only consume C. However, because the reaction direction information in EcoCyc is incomplete, this approach does not yield acceptable results. The deﬁnition that we used, based on condition (i), will be incomplete in the sense that it will fail to detect those true dead-end metabolites that are only produced, or only consumed, by multiple reactions. Nonetheless, the deﬁni- tion that we used produced useful results. RESULTS Genes and gene products EcoCyc contains 4465 gene objects, of which 4460 are assigned locations within the E. coli MG1655 complete genome sequence. Five genes are not mapped to the sequence because they are known only on the basis of phenotypes reported in the literature, not their location in the genome. The set of genes with assigned chromosomal locations is very close to being the same as the recently updated gene set deﬁned by the E. coli genome annotation consortium (6), although EcoCyc has introduced several new genes that have since been reported in the literature. The nucleotide sequence, and the nucleotide start and stop positions (i.e. their coding region) for each gene shared with (6) are the same as those in (6) for the vast majority of genes, although in a few cases we have adjusted gene start and stop positions based on published experimental information. Each gene is associated in EcoCyc with at least one gene product. In the case of gene products that are chemically modiﬁed, such as by phosphorylation, the DB contains a distinct object for each diﬀerent chemical state of the protein. EcoCyc also contains a distinct object for each isoform coded for by a gene (for example, the two proteins generated from dnaX are separate DB objects). In contrast, post-translational processing of a protein, such as removal of signal sequences or starting methio- nines, is represented as features on the protein sequence using the EcoCyc protein feature ontology (see http:// biocyc.org/ECOLI/NEW-IMAGE?object=Protein- Features). Each gene product within EcoCyc is of a given type, which is deﬁned by its parent DB class. Table 1 shows the distribution of E. coli gene products among these types. EcoCyc does not include objects for mRNA species, although it does include objects for other RNAs, as reﬂected in Table 1. EcoCyc contains queryable descriptions of the multi- meric structure of E. coli gene products as described in the experimental literature. A total of 774 multimeric protein complexes are deﬁned in EcoCyc, ranging in complexity from simple homodimers, to the NADH dehydrogenase I complex of 13 gene products, to the ribosome. Table 1. Statistics on EcoCyc gene products Because of programmed frame shifting and other causes discussed in the text, there are more total gene products than genes. Proteins tRNAs rRNAs Misc RNAs 4316 89 22 64