Publisher Summary In addition to sequence homology, proteins have other recognizable features that are useful for functional annotation. These include signal sequences, which serve to facilitate localization or retention of proteins in specific cellular compartments. This information may help in pinpointing the candidate gene for a missing function. This chapter examines two approaches that require no other information than the genome sequences with correctly recognized open-reading frames. One of these methods is based on the analysis of conservation of gene order and gene clusters across multiple species, and the other is based on the analysis of the presence and absence of homologous genes across multiple genomes. The chapter starts with gene clustering and gene-order conservation. Clustering of genes on the chromosome may occur in three different forms. The most extreme one is translational fusion: when two proteins are combined into one multidomain protein. Another level of gene clustering is transcriptional fusion, in which one RNA encodes several open-reading frames, which can direct the synthesis of several distinct proteins, using either bacterial strategy of internal initiation of translation or various virus-like mechanisms of translating many proteins from one transcript. The third type of gene clustering is simply a tendency of certain genes to be positioned close to each other on a chromosome.