7580 Nucleic Acids Research, 2007, Vol. 35, No. 22 Thereafter, our standard procedure for curation of genes in EcoCyc includes updating of both MultiFun and GO term assignments, which are displayed near the top of EcoCyc gene and protein pages. Rather than storing GO terms with gene objects in EcoCyc, they are stored in gene products and in complexes, allowing EcoCyc to annotate both monomeric gene products, and multimers, with GO terms in a uniform fashion. EcoCyc contains a total of 8155 MultiFun term assignments for 3361 of its genes (2.4 terms assigned per gene on average). It contains 5939 GO term assignments for 2866 of its genes (2.1 terms assigned per gene). Computational update to the E. coli genome annotation As new sequence-analysis methods are developed, new genome sequences are published, and the functions of genes in organisms other than E. coli are elucidated, it is probable that it will become possible to predict the functions of some E. coli genes of unknown function. To keep the annotation of E. coli genes in EcoCyc as up-to-date as possible, we will perform a regular annual computational re-annotation of the E. coli genome using the robust genome auto-annotation pipeline (10) at the J. Craig Venter Institute (JCVI). This pipeline runs a gene prediction algorithm, Glimmer 3, followed by various homology searches, including a BLAST-based search against a nonredundant protein database, HMM searches against TIGRFAM and PFAM protein family models, PROSITE motif searches, and a BLAST search against the NCBI COG database; and analyses of protein properties, including prediction of signal peptides, other sorting signals and transmembrane helices. We ran an automated re-annotation of E. coli K-12 in December 2006. A comparison of the automated annota- tion with the manually curated E. coli annotation in EcoCyc showed that 3124 genes were identical or essentially identical in both annotations. Two hundred and seventeen genes in the EcoCyc annotation were missed by the auto annotation, as they were not called as open reading frames by the Glimmer 3 gene ﬁnder. Manual examination of this set of genes indicated that these were mostly very small genes and included 62 pseudogenes, 11 leader peptides, 43 phage-related genes or IS elements and 67 small hypothetical genes. Seven hundred and ninety-six genes diﬀered between the two annotations, with the auto annotation giving more general or generic gene calls than the existing E. coli K-12 annotation. For example, kdsD, encoding D-arabinose 5-phosphate isomerase, was called as a putative isomerase by the automated annotation. Manual inspection of these diﬀerences indicated that in all these instances, the original annotation was correct and frequently supported by experimental evidence. One hundred and ninety-one genes diﬀered, with the auto annotation either providing a functional call for a gene annotated as ‘hypothetical’ or ‘conserved hypothe- tical’ in the original annotation, or by giving a more precise function to a gene annotated with a generic function. The underlying evidence for the auto annotation of each of these 191 genes was manually examined by a curator. For 94 out of the 191, there was suﬃcient evidence to modify the existing annotation in EcoCyc with information from the automated annotation. In most cases, this involved updating annotation as ‘conserved hypothetical’ or ‘hypothetical’ to a generic prediction such as ‘putative lipoprotein’, ‘dehydrogenase’, ‘kinase’, ‘lipase’, ‘carboxylase’, ‘protease’, and so on. In some cases, it involved providing a more detailed prediction such as updating ‘putative transporter’ to ‘putative amino acid transporter’. While these updates do not provide exact functions for these genes, we felt they did contain better information that might be helpful for experimen- talists trying to ﬁnd functions of novel E. coli genes. We used our controlled evidence codes to label these functional assignments as speculative and based on predictions, to distinguish them from experimentally determined functions. For the remaining 97 out of 191 genes where no changes were made, 24 had experimental data that supported the original EcoCyc annotation, and for the other 73 there was insuﬃcient evidence to support the automated annotation over the original assignments. Comparison to experimentally elucidated gene fractions of other model organisms How does the ﬁgure of 66% of E. coli genes with experimentally determined functions compare to other model organisms? The answer is found in Table 3 by comparing lines two (‘EcoCyc deﬁnition’) and four (‘Def 2’): E. coli has the highest absolute number of genes with experimental functions of all the model Table 3. Genes of experimentally deﬁned function in the major model organisms Total genes EcoCyc deﬁnition Def 1. Strict evidence, leaf terms only Def 2. Loose evidence, leaf terms only Def 3. Strict evidence, all terms Def 4. Loose evidence, all terms Escherichia coli Saccharomyces cerevisiae Drosophila melanogaster Mus musculus Arabidopsis thaliana EcoCyc 2941 4460 66% 953 2319 1666 3627 SGD 6203 15% 37% 27% 58% 369 614 737 1302 FlyBase 14 816 2% 4% 5% 9% MGI 35 887 947 1456 2053 4080 3% 4% 6% 11% 630 1816 1058 3021 TAIR 37 186 2% 5% 3% 8% For comprehensiveness, we compare the EcoCyc deﬁnition of experimentally deﬁned function with four alternative deﬁnitions based on the Gene Ontology, although we consider Deﬁnition 2 to be the closest to the EcoCyc deﬁnition.