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. 1994 Dec;60(12):4279–4283. doi: 10.1128/aem.60.12.4279-4283.1994

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

M T Alegre 1, B Cournoyer 1, J M Mesas 1, M Guérineau 1, P Normand 1, J L Pernodet 1
PMCID: PMC201981  PMID: 7811067

Abstract

pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.

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Selected References

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