EXPERIMENTAL FIGURE 8-41 Yeast transformation experiments were used to identify the functional chromosomal elements necessary for normal chromosome replication and segregation. In these experiments, plasmids containing the LEU gene from normal yeast cells are constructed and introduced into leu⁻ cells by transfection. If the plasmid is maintained in the leu⁻ cells, they are transformed to LEU⁺ cells by the LEU gene on the plasmid and can form colonies on medium lacking leucine. (a) Sequences that allow autonomous replication (ARS) of a plasmid were identified because their insertion into a plasmid vector containing a cloned LEU gene resulted in a high frequency of transformation to LEU⁺. However, even plasmids with ARS exhibit poor segregation during mitosis and therefore do not appear in each of the daughter cells. (b) When randomly broken pieces of yeast DNA are inserted into plasmids containing ARS and LEU, some of the subsequently transfected cells produce large colonies, indicating that a high rate of mitotic segregation among their plasmids is facilitating the continuous growth of daughter cells. The DNA recovered from plasmids in these large colonies contains yeast centromere (CEN) sequences. (c) When leu⁻ yeast cells are transfected with linearized plasmids containing LEU, ARS, and CEN, no colonies grow. Addition of telomere (TEL) sequences to the ends of the linear DNA gives the linearized plasmids the ability to replicate as new chromosomes that behave very much like a normal chromosome in both mitosis and meiosis. See A. W. Murray and J. W. Szostak, 1983, Nature 305:89, and L. Clarke and J. Carbon, 1985, Ann. Rev. Genet. 19:29.