RESUMEN
Identification of essential genes is critical to understanding the physiology of a species, proposing novel drug targets and uncovering minimal gene sets required for life. Although essential gene sets of several organisms have been determined using large-scale mutagenesis techniques, systematic studies addressing their conservation, genomic context and functions remain scant. Here we integrate 17 essential gene sets from genome-wide in vitro screenings and three gene collections required for growth in vivo, encompassing 15 Bacteria and one Archaea. We refine and generalize important theories proposed using Escherichia coli. Essential genes are typically monogenic and more conserved than nonessential genes. Genes required in vivo are less conserved than those essential in vitro, suggesting that more divergent strategies are deployed when the organism is stressed by the host immune system and unstable nutrient availability. We identified essential analogous pathways that would probably be missed by orthology-based essentiality prediction strategies. For example, Streptococcus sanguinis carries horizontally transferred isoprenoid biosynthesis genes that are widespread in Archaea. Genes specifically essential in Mycobacterium tuberculosis and Burkholderia pseudomallei are reported as potential drug targets. Moreover, essential genes are not only preferentially located in operons, but also occupy the first position therein, supporting the influence of their regulatory regions in driving transcription of whole operons. Finally, these important genomic features are shared between Bacteria and at least one Archaea, suggesting that high order properties of gene essentiality and genome architecture were probably present in the last universal common ancestor or evolved independently in the prokaryotic domains.
Asunto(s)
Regulación de la Expresión Génica Arqueal , Regulación Bacteriana de la Expresión Génica , Genes Esenciales , Genoma Arqueal , Genoma Bacteriano , Archaea/genética , Evolución Biológica , Burkholderia pseudomallei/genética , Escherichia coli/genética , Redes Reguladoras de Genes , Anotación de Secuencia Molecular , Mycobacterium tuberculosis/genética , Streptococcus/genéticaRESUMEN
The determination of genomic DNA sequence flanking a known region is often problematic. Existing technologies depend on multiple, efficient enzyme-catalysed preparative processing steps and/or rely on relatively inefficient one-sided' PCR mechanisms. I demonstrate the application of a simple two-sided' PCR-based approach, lariat-dependent nested PCR for rapid amplification of genomic DNA ends (LaNe RAGE), applied to the mouse GAPDH and PGK1 gene flanking sequences. This demonstration offers great promise in applications such as genome walking, transposon mutagenesis mapping and DNA fingerprinting.