RESUMO
In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.
Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos/genética , RNA Bacteriano/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA Bacteriano/metabolismo , DNA Bacteriano/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Dados de Sequência Molecular , Ligação Proteica , RNA Bacteriano/metabolismo , RNA Bacteriano/farmacologia , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência/farmacologia , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
In this study we report on a novel pair of cis-regulatory motifs in promoter sequences of the nematode Caenorhabditis elegans. The motif pair exhibits extraordinary genomic traits: The order and the orientation of the two motifs are highly specific, and the distance between them is almost always one of two frequent distances. In contrast, the sequence between the motifs is variable across occurrences. Thus, the motif pair constitutes a nearly combinatorial sequence configuration. We further show that this module is conserved among, and unique to, the entire Caenorhabditis genus. By analyzing several gene expression data sets, our data suggest that this motif pair may function in germline development, oogenesis, and early embryogenesis. Finally, we verify that the motifs are indeed functional cis-regulatory elements using reporter constructs in transgenic C. elegans.
