Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

Abstract

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C. F. Austerberry, C. D. Alis, and M.-C. Yao, Proc. Nati. Acad. Sci. USA 81: [7383][7384][7385][7386][7387]. In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.Elimination of germ line-specific sequences from developing somatic nuclei, which was first observed (as "chromatin diminution") a century ago (10), has now been shown to occur in several species of organisms (reviewed in references 2, 3, 8, 9, 11, 22, 29, and 30). In the holotrichous ciliate Tetrahymena thermophila, 10 to 20% of the DNA sequences in the micronucleus (germ line nucleus) are selectively eliminated (35) from the somatic macronucleus (16). Most of this elimination is the result of internal deletion (12,34,36), which occurs in more than 5,000 specific DNA segments during a 2-h period of differentiation (4, 34).We have previously cloned and analyzed the DNAs containing two neighboring deletion sites in T. thermophila which we have named region R and region M (4, 5, 34). Deletion in region R consistently eliminates 1.1 kilobases (kb) of DNA; the sequences in this region are the only such sequences in T. thermophila reported to date (5). A surprising result from these studies was the absence of obvious sequence structures at or near the deletion junctions that might appear to be sufficient to account for the high efficiency and site specificity of this deletion (4, 5).To further understand DNA deletions we have characterized the sequence structure of the deletion in region M. Unlike the deletion in region R, this deletion occurs in two alternative ways, eliminating either 0.6 or 0.9 kb of DNA (4). Alternative DNA deletions have also been suggested for several other T. thermophila DNAs (19,31). Analysis of the region M sequence should provide not only a second example of a sequence involved in deletion in T. thermophila but also the molecular basis for alternative DNA deletions.To rule out the possibility that heterozygous alleles in the germ line are responsible for generating the two alternatively sized deletions in region M, we established cell lines with homozygous genomes from individual progeny (caryonides) of the second round of genomic exclusion matings (CU427 or CU428 x A*III) (1). Southern blots (28) of HindlIl-digested DNAs of these caryonidal lines were hybridi...

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

“…cis-acting signals have been identified for only one of the five developmentally regulated rearrangements studied to date in T. thermophila. Ten-base-pair A5G5 polypurine tracts near the junctions of the M rearrangement have been shown to be both necessary and sufficient cis-acting sequences (5,23,24). Since the other four rearrangements in T. thennophila analyzed to date all lack A5G5 sequences, the polypurine tracts cannot be the universal signal for DNA rearrangement in T. thermophila.…”

“…The distance between the rearrangement junctions of Tlrl is greater than for developmentally regulated DNA rearrangements previously described for T. thermophila. The four deletion-ligation rearrangements analyzed to date result in the elimination of 600 bp to 2.9 kb of DNA from the macronucleus (2,4,5,26,37). The Tlrl rearrangement joins sequences which are separated by 13 kb or more in the micronuclear genome.…”

“…DNA rearrangement occurs after the genome has undergone one or two rounds of DNA replication (2,11), when there are four to eight copies of the genome present in the macronuclear anlagen. Thus, for rearrangements having alternate junctions, a single macronucleus may produce more than one rearrangement product (5).…”

“…Although the four deletion-ligation rearrangements studied thus far have different sequences at the rearrangement junctions, it is not expected that each of the 5,000 rearrangements in the T. thermophila genome has unique cis-acting sequences. It has been suggested that the rearrangements belong to several classes, each specified by different flanking sequences (5).…”

Programmed Genome Rearrangements in Tetrahymena

“…Impressively, this is not too far from the actual number and sizes, which it is now possible to measure using micronuclear genomic sequence data (see below). In the following two decades, a number of deletion elements (or IESs for internal eliminated sequence) were cloned and characterized, which revealed several common features (27,28,29,30,31,32,33,34). First, they were all simple deletions not accompanied by any additional sequence change at the junction.…”