1887

Abstract

The minimal length of integrated homologous donor DNA tracks in transformation and factors influencing the location and length of tracks were determined. Donor DNA contained the gene region (kanamycin resistance, Km). This region carried nine approximately evenly spaced silent nucleotide sequence tags and was embedded in heterologous DNA. Recipient cells carried the normal gene with a central 10 bp deletion (kanamycin-sensitive). The Km transformants obtained had donor DNA tracks integrated covering on average only 4.6 (2–7) of the nine tags, corresponding to about 60 % of the 959 nt homologous donor DNA segment. The track positions were biased towards the 3′ end of . While the replication direction of recipient DNA did not affect track positions, inhibited transcription (by rifampicin) shifted the beginning of tracks towards the promoter. Absence of the RecJ DNase decreased the length of tracks. Absence of SbcCD DNase increased the integration frequency of the 5′ part of , which can form hairpin structures of 43–75 nt, suggesting that SbcCD DNase interferes with hairpins in transforming DNA. In homology-facilitated illegitimate recombination events during transformation (in which a homologous DNA segment serves as a recombinational anchor to facilitate illegitimate recombination in neighbouring heterologous DNA), on average only about half of the approximately 800 nt long tagged anchor sequences were integrated. From donor DNA with an approximately 5000 nt long homologous segment having the gene in the middle, most transformants (74 %) had only a part of the donor integrated, showing that short track integration occurs frequently also from large homologous DNA. It is discussed how short track integration steps can also accomplish incorporation of large DNA molecules.

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2008-12-01
2020-04-05
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