Reverse transcriptase-polymerase chain reaction validation of 25 "orphan" genes from Escherichia coli K-12 MG1655

Despite the accumulation of sequence information sampling from a broad spec trum of phyla, newly sequenced genomes continue to reveal a high proportion (50%-30%) of "uncharacterized" genes, including a significant number of st rictly "orphan" genes, i.e., putative open reading frames (ORFs] without an y resemblance to previously determined protein-coding sequences. Most genes found in databases have only been predicted by computer methods and have n ever been experimentally validated. Although theoretical evolutionary argum ents support the reality of genes when homologs are found in a variety of d istant species, this is not the case for orphan genes. Here, we report the direct reverse transcriptase-polymerase chain reaction assay of 25 strictly orphan ORFs of Escherichia coli. Two growth conditions, exponential and st ationary phases, were tested. Transcripts were identified for a total of 19 orphan genes, with 2 genes found to be expressed in only one of the two gr owth conditions. Our results suggest that a vast majority of E. coli ORFs p resently annotated as "hypothetical" correspond to bona fide genes. By exte nsion, this implies that randomly occurring "junk" ORFs have been actively counter selected during the evolution of the dense E. coli genome.