Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector.
A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T. thermophila by microinjection. Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously. However, in one transformant, the circular plasmid initially outreplicated the endogenous rDNA and was the major rDNA form for up to 65 generations. Stable circular replicons have not been reported previously in Tetrahymena. A single point mutation (+G) was identified in the repeated promoter of the plasmid maintained in this transformant. After recovery from the Tetrahymena transformant and recloning in Escherichia coli, the mutated circular plasmid again transformed Tetrahymena with stable maintenance of the circular rDNA plasmid. Transformants containing circular replicons were also obtained by using a similar plasmid from which the repeated promoter, but not the repeated replication origin, had been removed by BAL-31 deletion. We therefore propose that repeated rRNA promoters are deleterious in vivo in Tetrahymena, which normally lacks them. Transformants were obtained in 2-5 days compared with the 7-14 days required for transformation with unmutated rDNA plasmids by recombination. Similar results were obtained when a 550-base-pair segment containing the telomerase RNA gene of T. thermophila was inserted in the polylinker of the plasmid. We suggest that this plasmid is a useful vector system for transformation of Tetrahymena.
Yu
G L
GL
Department of Molecular Biology, University of California, Berkeley 94720.
Blackburn
E H
EH
D000818:Animals; D001483:Base Sequence; D004270:DNA, Circular / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013768:Tetrahymena / Q000235:genetics*
D004270:DNA, Circular; D004275:DNA, Ribosomal