Genomic organization and developmental fate of adjacent repeated sequences in a foldback DNA clone of Tetrahymena thermophila.

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.

Methylation site within a facultatively persistent sequence in the macronucleus of Tetrahymena thermophila.

DNA methylation occurs at the adenines in the somatic macronucleus of Tetrahymena thermophila. We report on a methylation site within a DNA segment showing facultative persistence in the macronucleus. When the site is present, methylation occurs on both strands, although only 50% of the DNA molecules are methylated.

The 5S ribosomal RNA gene clusters in Tetrahymena thermophila: strain differences, chromosomal localization, and loss during micronuclear ageing.

The organization of the 5S genes in the genome of Tetrahymena thermophila was examined in various strains, with germinal ageing, and the 5S gene clusters were mapped to the MIC chromosomes. When MIC or MAC DNA is cut with the restriction enzyme EcoRI, electrophoresed, blotted, and probed with a 5S rDNA probe, the banding patterns represent the clusters of the 5S rRNA genes as well as flanking regions. The use of long gels and 60 h of electrophoresis at 10 mA permitted resolution of some 30-35 5S gene clusters on fragments ranging in size from 30-2 kb (bottom of gel). The majority of the 5S gene clusters were found in both MIC and MAC genomes, a few being MIC limited and a few MAC limited. The relative copy number of 5S genes in each cluster was determined by integrating densitometric tracings made from autoradiograms. The total number of copies in the MAC was found to be 33% greater than in the MIC. When different inbred strains were examined, the majority of the 5S gene clusters were found to be conserved, with a few strain-specific clusters observed. Nine nullisomic strains missing both copies of one or more MIC chromosomes were used to map the 5S gene clusters. The clusters were distributed non-randomly to four of the five MIC chromosomes, with 17 of them localized to chromosome 1. A deletion map of chromosome 1 was constructed using various deletion strains. Some of these deletion strains included B strain clones which had been in continuous culture for 15 years. Losses of 5S gene clusters in these ageing MIC could be attributed to deletions of particular chromosomes. The chromosomal distribution of the 5S gene clusters in Tetrahymena is unlike that found for the well-studied eukaryotes, Drosophila and Xenopus.