Filtering "genic" open reading frames from genomic DNA samples for advanced annotation

BMC Genomics

Research

Sara D'Angelo¹  Nileena Velappan¹  Andrew RM Bradbury¹  Csaba Kiss¹  Claudio Santoro²  Daniele Sblattero²  Flavio Mignone³ 

[1] Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA;Department of Medical Sciences and IRCAD, University of Eastern Piedmont, Novara, Italy;Department of Structural Chemistry and Inorganic Stereochemistry, School of Pharmacy, University of Milan, Milan, Italy;

关键词: Signal Recognition Particle;    Genic ORFs;    Multiple Displacement Amplification;    Nitrocefin;    Ampicillin Concentration;   

DOI  :  10.1186/1471-2164-12-S1-S5

来源: Springer

【 摘 要 】

BackgroundIn order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA) for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the “domainome”.ResultsIn this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP), normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the “domainone” that is accessible.ConclusionsThe availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of functional genic ORFs. ORF libraries represent, moreover, a useful tool to proceed towards high-throughput functional annotation of newly sequenced genomes.

【 授权许可】

Unknown   
© D'Angelo et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.