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The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained aHl cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T.

The tandemly repeated hexanucleotide C-C-C-C-A-A has previously been found near the termini of extrachromosomal gene coding for ribosomal RNA as well as in many other locations of the genome of Tetrahymena. Moreover, the organization of these clusters of repeats in the somatic macronucleus is different from that in the germinal micronucleus. In this study we used the exonuclease Bal 31 to show that the repeats are located near free ends ofDNA in the macronucleus. When whole cell DNA or macronuclear DNA was digested with Bal 31 to remove approximately 600 base pairs from free ends, 80% of the C4A2 repeats were removed, as judged by hybridization. Because no particular cluster was resistant to exonuclease digestion, we believe that essentially all the CA2 repeats are located near free ends of DNA. The C4A2 repeats in the micronucleus, on the other hand, were not digested by Bal 31. The ciliated protozoan Tetrahymena thermophila normally contains a macronucleus and a micronucleus in each cell. During conjugation the macronucleus degenerates and the micronucleus goes through a series of events to produce the new macronucleus and micronucleus for the following asexual generation. Thus, the two nuclei share the same genetic origin, but it is the micronucleus that maintains the genetic continuity of the organism (1). Recent studies have shown that the genome of this organism is significantly altered during the formation of the macronucleus (2). The gene coding for ribosomal RNA (rDNA) is selectively amplified several hundredfold (3), and about 15% of the genome is eliminated (4,5).Another observation related to genome alteration has been made recently. In the macronucleus the amplified rDNA exists as extrachromosomal palindromic molecules (6, 7). A tandemly repeated hexanucleotide, (C-C-C-C-A-A)20-70, was found at or near the free ends of this linear molecule (8). This repeated sequence was later found in other locations of the genome (3, 9).It exists in many clusters in both the macronucleus and micronucleus. After restriction enzyme digestion the DNA fragments containing this repeated sequence are found to be of different sizes in these two nuclei. These results suggest that the genome is somehow altered in regions associated with the C4A2 repeats.In this report we present evidence which reveals the nature of this alteration. Using the exonuclease Bal 31 we were able to show that the C4A2 repeats are located near free DNA ends in the macronucleus. These free ends are apparently generated through chromosome fragmentation during development. Thus in Tetrahynena, as in two other ciliates (10, 11), the simple repeated sequences seem to play a significant role in the fragmentation of chromosomes. MATERIALS AND METHODSCells and Culture Conditions. T. thermophila inbreeding strain B, obtained from P. Bruns (Cornell University), was used throughout this study. The cells were cultured in axenic media as described (12). Macronuclei and micronuclei were isolated from cells in late logarithmic phase of growth by using t...