Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila.

Abstract

DNA deletion by site-specffic chromosome breakage and rejoining occurs extensively during macronuclear development in the ciliate Tetrahymena thermophila. We have sequenced both the micronuclear (germ line) and rearranged macronuclear (somatic) forms of one region from which 1.1 kilobases of micronuclear DNA are reproducibly deleted during macronuclear development. The deletion junctions lie within a pair of 6-base-pair direct repeats. The termini of the deleted sequence are not inverted repeats. The precision of deletion at the nucleotide level was also characterized by hybridization with a synthetic oligonucleotide matching the determined macronuclear (rejoined) junction sequence. This deletion occurs in a remarkably sequence-specific manner. However, a very minor degree of variability in the macronuclear junction sequences was detected and was shown to be inherent in the mechanism of deletion itself. These results suggest that DNA deletion during macronuclear development in T. thermophila may constitute a novel type of DNA recombination and that it can create sequence heterogeneity on the order of a few base pairs at rejoining junctions.Developmentally regulated DNA rearrangement via chromosome breakage and rejoining is known to occur with important phenotypic consequences in several diverse systems, from the cyanobacterium Anabaena sp. (21) to the vertebrate immune system (reviewed in references 26 and 45). Among the genomes most extensively rearranged during development are the macronuclear genomes of several ciliates; within the same nucleus, chromosome breakage is followed by telomere addition in some cases and chromosome rejoining in others (reviewed in references 6, 8, and 32). Some of these rearrangement events result in the elimination of significant amounts of micronuclear (germ line) DNA sequence from the macronuclear (somatic) genome. Studies of DNA renaturation kinetics have suggested that 10 to 20% of the micronuclear genome of the holotrich Tetrahymena thermophila (52) and over 95% of the micronuclear genomes of the hypotrichs Stylonychia mytilus (4) and Oxytricha sp. (34) are eliminated from the macronuclear genomes of these ciliates.Interstitial deletion (i.e., deletion-rejoining) is known to occur in T. thermophila (11,51,54) during a specific stage of macronuclear development (5, 9). Such rearrangements appear to be the primary mechanism of sequence elimination (28, 51). Molecular cloning and Southern hybridization experiments suggest that deletion occurs on average once every 30 to 40 kilobases (kb) and that some 5,000 such sites exist in the genome (51). Characterized deletion events appear to be highly regulated, occurring in over 90% of the cells in a conjugating population within a 2-h period (5). The recognition sequences and the mechanism(s) responsible for such extensive, developmentally regulated genome rearrangement, as well as the functions of these deletions, are unknown.In this paper, we present the sequences involved in a previously characterized 1.1-kb deletion in T (5, 51). T...

A distant 10-bp sequence specifies the boundaries of a programmed DNA deletion in Tetrahymena.

“…Our recent analysis of the neighboring R-element renders further support to this view. The R-element shares no sequence identity with the M-element {Austerberry and Yao 1987Yao , 1988), yet its deletion requires its immediately flanking sequences but not sequences at its junctions (A. La Terza, A. Wilson, and M.-C. Yao, unpubl.).…”

“…Tetrahymena DNA was isolated by phenol extraction and polyethylene glycol precipitation (Austerberry and Yao 1987). Restriction enzyme digestion, agarose gel electrophoresis, Southem blotting, and hybridization were carried out following standard protocols as described earlier .…”

“…However, the underlying mechanisms may be rather different. Sequences of two deletion elements in Tetrahymena characterized so far share very few common features {Austerberry and Yao 1987. They also contain no prominent structures often found with mobile genetic elements, such as long open reading frames, long terminal repeats, or inverted terminal repeats.…”

“…for some of the elements studied (Austerberry and Yao 1987;Austerberry et al 19891. Similar processes have been found in other ciliates, including Paramecium [Forney and Blackburn 1988}, Oxytricha, and Euplotes (for review, see Klobutcher and Prescott 1986;lahn 1991).…”

Precise excision of telomere-bearing transposons during Oxytricha fallax macronuclear development.

“…This, however, does not necessarily mean that all TBEI-1 and TBE1-2 excisions are precise. Some IESs are known not to be excised exactly the same way in each event (Austerberry and Yao 1987;Austerberry et al 1989); microheterogeneity results, even within a single developing macronucleus (rearrangements occur generally only after a few rounds of amplification). We have not analyzed the immediate products of macronuclear development but, instead, have analyzed macronuclear DNAs from cells derived from exconjugants by many generations of vegetative growth.…”