Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.
The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.
Jessop-Murray
H
H
Department of Biology, Indiana University, Bloomington 47405.
Martin
L D
LD
Gilley
D
D
Preer
J R
JR
Polisky
B
B
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D004252:DNA Mutational Analysis; D016054:DNA, Protozoan / Q000235:genetics*; D008845:Microinjections; D009154:Mutation; D010247:Paramecium / Q000235:genetics*; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic
D000954:Antigens, Surface; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan