Figure 5. Insertional mutants of the CrTERT gene have shorter telomeres.
(A) Mutants tel-m1 and tel-m2 have shorter telomeres in TRF analyses (three independent subclones are shown). Control: mutant LMJ.RY0402.239308 from the CliP library, which has an insertion in a gene unrelated to CrTERT. Paromomycin resistance phenotype is indicated (“[ParoS/R]”; “S”: sensitive, and “R”: resistant). (B) Prolonged liquid cultures of telomerase mutants lead to rearranged TRF patterns. Cells were cultured in liquid medium for 2 mo before TRF analysis. Additional bands and slow-migrating DNA molecules (red dots and dotted vertical line, respectively) are indicated for tel-m1 and tel-m2 and are not present in the CC4533− reference strain TRF pattern. (C) Tetrad analysis of the cross between tel-m1 and T222+ shows a 2:2 cosegregation of paromomycin resistance and shortened telomeres after 21 and 42 d after the cross (see also Fig S5B). Mating types “mt+” and “mt−” are indicated. (D) Tetrad analysis of the cross between tel-m2 and T222+ shows a 2:2 cosegregation of paromomycin resistance and shortened telomeres after ∼80 d after the cross (see also Fig S5C). Mating types “mt+” and “mt−” are indicated.