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. . I N wild-type Paramecium tetraurelia stock 51, the A surface protein is coded by the A gene located close to the telomere in macronuclear chromosomes (reviewed by PREER 1986). In each wild type cell, the polyploid macronuclear chromosomal DNA contains about 2000 copies of the A gene. Macronuclear chromosomes range in size from about 100-600 kb. The micronucleus (containing about 2 100 kb per average chromosome; PREER 1986) is diploid. T h e A gene is present in the micronucleus of a mutant line called d48; however the macronucleus of this mutant contains few, if any copies of the intact A gene (RUDMAN et al . 199 1). As a result, the A protein is not detectably expressed by d48 (EPSTEIN and FORNEY 1984). At autogamy (a self-fertilization process that occurs periodically in P. tetraurelia) and conjugation, a new macronucleus and new micronuclei are formed from DNA processing of the old micronuclei. T h e old macronucleus degenerates as the new macronucleus develops. Although the molecular details of autogamy and conjugation are unknown in Paramecium, the DNA processing steps involved in generating polyploid macronuclear chromosomes from diploid micronuclei include DNA cleavage, telomere addition and DNA amplification. During formation of the macronucleus in the d48 mutant, processing of the A Genetics 129 727-734 (November, 1991) surface antigen gene is aberrant. A large chromosomal deletion which begins near the 5’ end of the gene (EPSTEIN and FORNEY 1984; FORNEY and BLACKBURN 1988) eliminates virtually the whole A gene from