RESUMEN
BACKGROUND: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. RESULTS: To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a "landing pad" (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. CONCLUSIONS: Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.
Asunto(s)
Cromosomas Bacterianos , Ingeniería Genética/métodos , Integrasas/genética , Rhizobium etli/enzimología , Rhizobium etli/genética , Conjugación Genética , ADN , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Replicación del ADN , Escherichia coli/genética , Citometría de Flujo , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Operón Lac , Plásmidos/genética , Regiones Promotoras Genéticas , Recombinación GenéticaRESUMEN
DNA polymerase activity has been measured in placentas of normal and protein-restricted rats and correlated with the mean percent daily increase in DNA. During normal placental growth, increases in DNA fell rapidly from 13 to 19 days and polymerase activity using denatured DNA template showed a similar pattern falling from values of 10,000 mumu mols dAMP incorporated per mg DNA at 12 days of gestation to 3,100 at 19 days. Protein restriction during gestation reduced placental DNA content after 14 days; by 19 days the DNA content was 81% of normal. The increase in DNA between 13 and 19 days in placentas of malnourished animals paralleled the normal but was significantly lower. Malnutrition markedly reduced enzyme activity at 12, 14, and 16 days; at 19 days, when DNA synthesis has normally ceased, values of DNA polymerase were not different in control and malnourished placentas. Thus DNA polymerase activity using denatured DNA as template, as measured in vitro, was an index of proliferative cell growth in both normal and malnourished placentas. Furthermore, the decrease in enzyme activity in malnourished samples preceded by at least two days any measurable decrease in total placenta DNA content. It is suggested that future clinical application of this technique may provide an index of nutritional status in "at risk" pregnancies.