Micronuclear Dna Sequences Which Are Normally Eliminated Can Persist In Macronuclei Of Tetrahymena Thermophila (rearrangement, Methylation).

During conjugation in Tetrahymena thermophila, a somatic macronucleus (MAC) develops from the germinal micronucleus (MIC). Ten to twenty per cent of the haploid MIC genome is eliminated during this process. The remainder is replicated approximately 45 - 64 fold. A MIC DNA clone was isolated which contains a representative from each of three families of eliminated sequences. One, present in 200 - 300 copies in the MIC, is almost completely eliminated from the MAC. A second is present in approximately fifty copies in the MIC; approximately one tenth of the members of this family persist in the MAC. The third family has 3 to 4 members, all of which are eliminated from the MAC. These three families are clustered in several locations throughout the genome. The persistent sequences from two of these repetitive families were analysed in the MAC of cell lines from a variety of sources. The results suggest that sequences which remain in the MAC have a genetic predisposition to persist. However, only some of the persistent sequences will actually be observed in a particular MAC. Furthermore, the sequences which persist as a MAC develops may not be present in all 45 haploid units of the MAC. During vegetative growth, this heterogeneity may be resolved by the process of phenotypic assortment. A MAC DNA clone was isolated which contains a fragment with one of these persistent sequences as well as some flanking sequences. This cloned fragment was used to construct restriction maps of this region in cell lines which do or do not contain the persistent sequence. The restriction maps show a large amount of variation flanking this region. These different DNA arrangements are epigenetically determined during the formation of the MAC. A MAC can be formed which contains more than one type of DNA arrangement in its 45 haploid units. This epigenetically induced heterogeneity is then resolved by the process of phenotypic assortment. In Tetrahymena, methylation occurs at 0.6 - 0.8% of the adenines of the MAC. A specific site of double stranded methylation occurs within the persistent sequence which was cloned. However, only 50% of the DNA molecules are methylated at this site. A similar amount of methylation is present at another methylation site 5 kb from the persistent region. Three other methylation sites appear to be associated with persistent sequences which are normally eliminated from the MAC.Ph.D.Biological SciencesMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127732/2/8512535.pd