Polarity of recombination in transformation of Streptococcus pneumoniae

In transformation of Streptococcus pneumoniae DNA enters the cell as single -strand fragments and integrates into the chromosome by homologous recombin ation, Deletions and insertions of a few hundred base pairs frequently stop the recombination process of a donor strand. In this work we took advantag e of such interruptions of recombination to compare the transformation effi ciencies of the segments 5'- and 3'-ward from a deletion. The deletion was created in the center of a fragment of the ami locus, and sites around the deletion were labeled by a frameshift generating a restriction site. Hetero duplexes were constructed containing two restriction sites on one strand an d two different ones on the complementary strand. ami(+) bacteria were tran sformed with such heteroduplexes. ami(-) transformants were isolated and in dividually underwent amplification of the transformed ami region. We have o btained two kinds of amplification products: short when the deletion was in tegrated, long when recombination stops at the deletion. Each long fragment was tested by the four restriction enzymes to detect which strand and whic h side of the deletion had recombined, We found that 80% of the cuts were l ocated 5' to the deletion, showing that, in vivo, the 5' side is strongly f avored by recombination, Further results suggest that exchanges occurring f rom 5' to 3' relative to the donor strand are more efficient than in the op posite direction, thus accounting for the 5' preference.