Identification and functional analysis of 'hypothetical' genes expressed in Haemophilus influenzae

Abstract

The progress in genome sequencing has led to a rapid accumulation in GenBank submissions of uncharacterized`hypothetical' genes. These genes, which have not been experimentally characterized and whose functions cannot be deduced from simple sequence comparisons alone, now comprise a signi®cant fraction of the public databases. Expression analyses of Haemophilus in¯uenzae cells using a combination of transcriptomic and proteomic approaches resulted in con®dent identi®ca-tion of 54`hypothetical' genes that were expressed in cells under normal growth conditions. In an attempt to understand the functions of these proteins, we used a variety of publicly available analysis tools. Close homologs in other species were detected for each of the 54`hypothetical' genes. For 16 of them, exact functional assignments could be found in one or more public databases. Additionally, we were able to suggest general functional characterization for 27 more genes (comprising~80% total). Findings from this analysis include the identi®cation of a pyruvate-formate lyase-like operon, likely to be expressed not only in H.in¯uenzae but also in several other bacteria. Further, we also observed three genes that are likely to participate in the transport and/or metabolism of sialic acid, an important component of the H.in¯uenzae lipo-oligosaccharide. Accurate functional annotation of uncharacterized genes calls for an integrative approach, combining expression studies with extensive computational analysis and curation, followed by eventual experimental veri®cation of the computational predictions.

Global profiling of Shewanella oneidensis MR-1: Expression of hypothetical genes and improved functional annotations

“…Although a recent pilot study of 54 hypothetical proteins expressed in Haemophilus influenzae obtained similar results (11), in the study presented here there was a substantial (10-fold) increase in the number of expressed hypothetical proteins. In this study, we were able to find homologs to 520 proteins (97% of 538), which would facilitate further annotation and functional characterization of these proteins.…”

“…The same S. oneidensis arrays, consisting of Ϸ95% of the predicted genes (17), were used for all gene expression analyses. The expression values and corresponding standard errors were estimated by using the maximum likelihood analysis (18), and the resulting set included confidently expressed genes (11). Analysis of differential expression was not part of this study.…”

“…11 and 19-24; (ii) LC coupled to Fourier transform ion cyclotron resonance MS (25,26); (iii) LC coupled to quadruple-TOF MS (27); and (iv) 2D gel electrophoresis followed by LC-MS͞MS (16,23). Common approaches used in different proteome analyses included: LC-MS͞MS implemented by using the LCQ platform (Thermo Electron, San Jose, CA), standard sample processing (Supporting Materials), standard top-down data-dependent ion selection (11,(19)(20)(21)(22)(23)(24), use of the TURBO-SEQUEST (Thermo Electron) search engine, and use of the recently developed standard mixtures for proteome studies (24). These approaches, in turn, allowed implementation of the same stringent criteria (28) for all peptide and protein identifications included in this study (Supporting Materials).…”

“…Several criteria were used for gene selection, namely to identify expressed hypothetical genes to be further analyzed (11). Among all S. oneidensis genes that showed statistically significant expression levels in microarray experiments, we identified those whose products were confidently detected by at least two independent protein analysis approaches.…”

“…Although this result represents an improvement, it also serves to point out one of the emerging challenges of modern biology: namely, the rapid accumulation of uncharacterized hypothetical genes (7)(8)(9)(10)(11). This assignment is given to genes that have not been experimentally characterized and whose functions cannot be deduced from simple sequence comparisons.…”