The streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system.

نویسندگان

  • Patricia Combes
  • Rob Till
  • Sally Bee
  • Margaret C M Smith
چکیده

The integrase from the Streptomyces phage (phi)C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, (phi)C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ((Delta)attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and (Delta)attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family.

The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous h...

متن کامل

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Strepto...

متن کامل

Identification of a Specific Pseudo attP Site for Phage PhiC31 Integrase in Bovine Genome

Background: PhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene. Objectives: Identification of a novel pseudo attP site. Materials and Methods...

متن کامل

Site-specific integration with bacteriophage ΦC31 integrase.

Few nonviral techniques exist for efficient and stable eukaryotic gene transfer and fewer still are broadly useful in both cell culture and whole-organism applications. C31 integrase, a site-specific bacteriophage recombinase, is able to catalyze chromosomal transgene insertion under a diverse range of experimental and therapeutic conditions. The enzyme recognizes and catalyzes unidirectional r...

متن کامل

Sequences in attB that affect the ability of ϕC31 integrase to synapse and to activate DNA cleavage

Phage integrases are required for recombination of the phage genome with the host chromosome either to establish or exit from the lysogenic state. C31 integrase is a member of the serine recombinase family of site-specific recombinases. In the absence of any accessory factors integrase is unidirectional, catalysing the integration reaction between the phage and host attachment sites, attP x att...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Journal of bacteriology

دوره 184 20  شماره 

صفحات  -

تاریخ انتشار 2002