Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

Abstract

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

“…Plasmid DNAs were extracted and analyzed to differentiate the pTLU-type (telomere-associated linear) from the pRLU1-type (circular), based on restriction analysis and Southern blotting with a telomere probe. The telomere-association at the plasmid ends was observed in rad52 mutant of S. cerevisiae (16, 17), but not in telomerase-subunit mutants of TLC1 encoding RNA template (21) and EST2 encoding reverse-transcriptase (22), as shown in Table 1. Only circularized plasmids were found in these mutants while the frequency of the plasmid relocation into the nucleus was not affected, supporting the view that the event was due to the action of host telomerase rather than RAD52 -dependent recombination with chromosomal telomeric repeats.…”

“…The telomere addition occurred in rad52 mutant (16, 17), but not in tlc1 and est2 mutants, strongly suggesting the involvement of host telomerase rather than recombination with chromosome telomeric repeats. While the TG 1-3 organization varied among the individual pTLU plasmids, it was symmetrically identical at the left and the right ends of any one pTLU.…”

“…Clonal analysis showed that the slow growing trait of small colonies resulted from low expression of the URA3 gene due to instability of the endogeneous pTLU siblings whereas large colonies on SC-Ura constantly contained cells with relatively stable pTLU. A possibility that the low expression of the URA3 gene may be due to a position effect of the nearby telomeric sequence (28, 29) was ruled out by the fact that Ura - clones growing on SC with 5-fluoroorotic acid (30) were all devoid of pTLU plasmids (17). …”

“…The linear form, termed pTLU-type, lacks TP but carries the host telomeric repeats (TG) 1-6 TG 2-3 (TG 1-3 in short) of about 300-350 bp long at the plasmid ends. This event was further studied using a monitor linear plasmid pCLU1 (8.4 kb) carrying the cytoplasmic (UCS-Sc LEU2 ) and the nuclear (Sc URA3 ) markers on pGKL1 (15-17). Like pGKL1, pCLU1 normally replicates in the cytoplasm with the helper pGKL2.…”