RESUMEN
The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.
Asunto(s)
Bacterias/genética , Microbiología Ambiental , Genes Bacterianos , Integrasas/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , Integrasas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Moldes GenéticosRESUMEN
A phospholipase A(2) inhibitor has been identified in the serum of the common tiger snake (Notechis scutatus). The inhibitor is composed of two chains, an alpha-chain and a beta-chain, that form a non-covalently associated complex capable of inhibiting the enzymatic activity of all phospholipase A(2) enzymes it was tested against. The alpha and beta-chains have been purified to homogeneity, digested and sequenced. From the peptide sequence generated, degenerate PCR primers were designed and used to elucidate the complete cDNA sequence of the chains using 5' and 3' RACE PCR. A total of three alpha-chain isoforms were identified, only one isoform of the beta-chain was detected. The two-dimensional structure of the three alpha-chains and one beta-chain were predicted using five prediction programs (discrimination of secondary structure class; nearest neighbour secondary structure, profile network from Heidelberg; self-optimised prediction method from multiple alignment, SSPAL). For each protein chain a consensus prediction was generated. Results are discussed in relation to the function of the protein, and how they may influence the three-dimensional structure of the inhibitor. Additionally, the sequences of several snake phospholipase A(2) inhibitors were used as the input for a motif prediction algorithm (MEME). The results are discussed in relation to the activity of these proteins.
Asunto(s)
Proteínas Sanguíneas/química , Elapidae , Fosfolipasas A/antagonistas & inhibidores , Alquilación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Sanguíneas/genética , Clonación Molecular , Disulfuros/metabolismo , Elapidae/sangre , Elapidae/genética , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.