RESUMEN
A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I- Sce I, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I- Sce I site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.
Asunto(s)
ADN Bacteriano/genética , Escherichia coli/genética , Técnicas de Transferencia de Gen , Deleción Cromosómica , Cromosomas Bacterianos , ADN/metabolismo , ADN Bacteriano/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/biosíntesis , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Electroporación , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Mutación , Recombinación Genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
The risk of thromboembolism during oral contraceptive (OC) use is increased among factor V Leiden (FVL) carriers compared to women with wild-type genotype of the gene for coagulation factor V (FV). The carrier frequency in the general population is too high for FVL alone to be responsible for the reported association. Additional risk factors may be required to explain the increased risk of thromboembolism of carriers during OC use. We conducted a case-control study to compare the titer of anti-beta2-glycoprotein I immunoglobulin G (IgG) and the frequency of elevated titer of IgG type anti-beta2-glycoprotein I antibody between FVL carriers and individuals with FV wild-type genotype with and without pill use. An asymptomatic population of 313 unrelated nonpregnant women were screened for FVL and for the presence of anti-beta2-glycoprotein I IgG antibody. Sixty-six women were FVL carriers and 247 had normal genotype. One-hundred and thirty-five women used OC at the time of screening and 178 did not. Among FVL carriers, OC pill users had a higher mean anti-beta2-glycoprotein I IgG titer than nonusers (9.2 SGU/mL vs. 4.7 SGU/mL, p = 0.0485). Among women with FV wild-type genotype, there was no significant difference in anti-beta2-glycoprotein I IgG titers between users and nonusers of OCs (6.4 SGU/mL and 6.0 SGU/mL, respectively; p = 0.7010). The odds of an elevated anti-beta2-glycoprotein I IgG titer during OC use in FVL heterozygous women was 2.41 (95% confidence interval: 0.79-7.39) relative to users with-type genotype. FVL may contribute to the development of elevated titer of IgG type anti-beta2-glycoprotein I antibody during OC use. The elevated titer of IgG type anti-beta2-glycoprotein I antibody may select women among FVL carriers during OC use with an increased risk of thromboembolism.