XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Posters
22.
SITE SPECIFIC INTEGraTION OF BACTERIOPHAGE µ1/6 INTO THE
STrePTOMYCES aureoFACIenS CHROMOSOME
Jarmila Farkašovská and Andrej Godány
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava
Phage integrases are enzymes that mediate unidirectional site-specific recombination
between two recognition sequences, the phage attachment site, attP, and the bacterial
attachment site, attB. Site-specific recombinases can be classified into two major families
based on amino acid sequence homology and catalytic residues, either tyrosine or
serine. The properties of site-specificity and efficiency make phage integrases excellent
tools for the manipulation of DNA.
The phage µ1/6 integrates site-specifically into the chromosome of tetracycline producing
Streptomyces aureofaciens strains. The region of µ1/6 genome involved in this process
have been identified. The deduced 416 amino acid protein encoded by orf5 displays
significant similarity with site-specific recombinases of the tyrosine family (sometimes
referred to as integrase family). The phage attachment site (attP) was localized downstream
of the putative integrase gene. The junctions (attL and attR sites) of the prophage
with the host genome have been determined, allowing identification of the host attachment
site (attB), which has a common 45-nucleotide core region with attP. In attB
this core sequence consists of the 3 ’ end of the putative tRNA (Thr) (ACA) gene. To prove
the functionality of the putative integrase gene (orf5), an integrative vector pCTint was
constructed. This integration plasmid, consisting of the µ1/6 putative integrase gene
and the phage µ1/6 attP, and a selectable marker (thiostrepton resistance gene for
streptomycete) inserted stably into the chromosome of S. aureofaciens and S. lividans.
The µ1/6 integrase with a deduced molecular mass of 47,5 kDa was overproduced in
Escherichia coli and further analyzed.
XXII. Biochemistry Congress, Martin
139