Construction of a mini-chromosome by deletion and its mitotic and meiotic behaviour in fission yeast

Summary A highly stable, partial aneuploid, HM248, of the fission yeast Schizosaccharomyces pombe was obtained from the unstable aneuploid disomic for chromosome III by -irradiation. It contained a 500 kb mini-chromosome (designated Ch¹⁶) that was separated as a single band by pulsed field gradient electrophoresis. Genetic analysis showed that Ch¹⁶ was deleted for most of chromosome III except for the pericentric region; three centromere-linked markers encompassing the centromere region remained. This was further substantiated by integrating the cloned fragments of Ch¹⁶ DNA extracted from the agarose gel; integrations took place in the pericentric region. A 400 kb derivative (Ch^16D1) was constructed which appeared to lack a part of Ch¹⁶. A single haploid cell of S. pombe could stably maintain Ch¹⁶ and Ch^16D1 in addition to the three regular chromosomes. Ch¹⁶ was visualized as a minute chromosomal body in the nucleus of a -tubulin mutant under restrictive conditions. A single copy of Ch¹⁶ was highly stable and behaved like a natural chromosome in mitosis and meiosis. The frequency of chromosomal loss was 10^-4. In meiosis, it segregated independently of the regular chromosome III. Segregation of two Ch¹⁶s per cell could be monitored by specifically marked Ch¹⁶s containing the Saccharomyces cerevisiae LEU2 gene (designated Ch^16LE) of the fluorouracil resistance marker (Ch^16FR). Two copies of Ch¹⁶ were mitotically unstable (chromosomal loss, 10^-3) and frequently failed in meiotic segregation. The frequency of meiotic recombination between the two Ch¹⁶s was greatly reduced.