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Specific rearrangement of internal chromosomal regions occurs during development of the somatic macronucleus in Tetrahymena thermophila and results in elimination of germ-line (micronuclear) DNA sequences. The timing and mechanism of genome rearrangement within one particular 9.3-kilobase region, which contains three distinct eliminated sequences, were investigated. Portions of this cloned region were used as probes in Southern hybridization experiments to analyze DNA from developing macronuclei (anlagen). All three deletions were found to occur predominantly within a 2-hr time period in which the nuclear DNA contents increased from 4C to 8C (1C represents the amount of DNA present in a haploid genome). The three deletion events can occur independently because intermediate forms, having sustained one or two deletions, were detected. One of the deletions occurs in two alternative ways, resulting in two equally abundant products of different size. Because reciprocal products expected from unequal sister chromatid exchange were not detected, an intramolecular DNA splicing mechanism is suggested.

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...

Cryomicrodissection makes possible the measurement of the entire in vivo protein content of the amphibian oocyte nucleus and provides a heretofore missing baseline for estimating protein loss during nuclear isolation by other methods. When oocyte nuclei are isolated into an aqueous medium, they lose 95% of their protein with a half-time of 250 s. This result implies an even more rapid loss of protein from aqueously isolated nuclei of ordinary-size cells.Cell nuclei are isolated in aqueous media in many laboratories, and their analyses are used to characterize structures and functions of the in vivo nucleus. Because the nuclear envelope contains pores permeable to macromolecules (1-4), it is understood that some proteins must be lost (5). However, the magnitude of the loss is unknown, because the in vivo (preisolation) protein content of nuclei has not been determined and compared to the protein remaining in isolated nuclei. We present here a two-step approach to this problem. First, we determined the in vivo protein content of the large nucleus (400-500-~m diameter) of the amphibian oocyte isolated by cryomicrodissection. Second, with this in vivo content as a baseline, we measured the kinetics of protein loss from oocyte nuclei isolated directly into an aqueous medium. MATERIALS AND METHODSCryomicrodissection (6, 7) is a method in which individual oocytes are frozen in liquid nitrogen and subsequently maintained at less than -45"C while the nucleus is microsurgically isolated with fine-tipped stainless steel microtools (Fig. I). The low temperature prevents diffusive relocations of nuclear and cytoplasmic solutes from their in vivo locations. Clean, intact nuclei cyomicrodissected from Xenopus oocytes (stages V and VI) (8) varied somewhat in wet weight from animal to animal, but their size distribution was narrow for cells from the same animal (standard error of the mean <5%). Nuclear water contents, determined from wet and dry weights of cryomicrodissected nuclei, were relatively constant (even between animals) at 87.2 +_ 0.3%, with the dry mass consisting almost entirely of protein. The nuclei of the oocytes from the two animals used in the present study had protein contents of 3.8 _ 0.4 and 5.5 _+ 0.7 ug (Fig. 2, upper and lower curves, respectively.)To isolate nuclei into aqueous solution, we punctured and compressed individual oocytes with forceps (9, 10) until the nucleus was extruded. The medium was Ca2÷-free and formulated (legend, Fig. 2) to mimic the oocyte's intracellular free monovalent cation concentrations (7). After extrusion, each nucleus was gently pipetted through the medium to remove traces of adherent cytoplasm, incubated without agitation in fresh medium for time t~, and assayed for protein content. Nonspherical (damaged) nuclei were discarded. RESULTS AND DISCUSSIONThe aqueous isolation procedure we used is gentle compared to the mass cell shearing or homogenization employed in most studies. Nevertheless, even under these conditions, 1240 loss of nuclear protein was 90% by 1 h, an...

Regulated DNA deletions are known to occur to thousands of specific DNA segments in Tetrahymena during macronuclear development. In this study we determined the precision of this event by examining the junction sequences produced by three different deletions in many independent caryonidal lines. 0.9 kb deletions in region M produce at least 3 types of junction sequences, of which two have been determined and found to be different by 4 bp. The alternative 0.6 kb deletions in this region are much less variable. 1.1 kb deletions in region R, known from a previous study to be slightly variable, produce two types of junction sequences which are different from each other by 3 bp. Thus, developmentally regulated deletions in Tetrahymena can produce sequence microheterogeneity at their junctions. This process contributes significantly to the diversification of Tetrahymena's somatic genome.