Construction and characterization of centric circular and acentric linear chromosomes in fission yeast

Summary Gene disruption and gap repair of chromosomal DNA have been frequently employed techniques in yeast genetics. To extend the possibility of using these gene manipulations for larger genomic regions, we have examined the maximal sizes of chromosomal DNA disrupted or repaired in vivo. Here we report a simple, potentially general, method for selectively deleting a 150 kb region, or gap-filling a 100 kb region, in the fission yeast genome. This enables the generation of acentric linear chromosomes by deletion, or the cloning of large functional centromeric DNAs into circular minichromosomes by gap-filling. The fidelity of the resulting gap-filling is high, judging from partial-digestion mapping of gap-repaired DNAs. By analysing a series of such circular minichromosomes, we conclude that only a part of the repetitive centromeric region, including the central domain, is essential for mitotic and meiotic chromosome segregation. Acentric linear chromosomes, although unstable, could be maintained, indicating that it may be possible to construct an acentric vector for large DNA fragments in this organism.