Sequence microheterogeneity is generated at junctions of programmed DNA deletions in Tetrahymena thermophila.

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.

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

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. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). 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.

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

DNA deletion by site-specific 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.

Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena.

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 elimination in Tetrahymena: a developmental process involving extensive breakage and rejoining of DNA at defined sites.

Elimination of specific DNA sequences occurs during macronuclear development in the ciliate Tetrahymena thermophila. Recombinant DNA clones containing a segment of micronuclear (germinal) DNA involved in elimination and the corresponding segment of macronuclear (somatic) DNA produced after elimination were isolated. Detailed comparisons of the cloned DNAs, as well as the genomic DNAs, by hybridization indicated that DNA elimination is accompanied by specific DNA rearrangements. In this 9.5 kb region three defined DNA segments are deleted and the remaining sequences are linked together as one contiguous piece in the macronucleus. Specific DNA rearrangement of this kind occurs widely in the genome. Analysis of 20 randomly selected DNA clones suggests that there are more than 5000 such rearrangement sites in the genome. Thus specific breakage and rejoining of DNA occurs extensively during development, and might play an essential role in nuclear differentiation.

Protein loss during nuclear isolation.

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.

In vivo distribution of proteins within a single cell.

Using the oocyte of Xenopus laevis, we present an experimental system, involving two-dimensional gel electrophoresis, for measuring unambiguously the nucleocytoplasmic distribution of proteins within a living cell.

Isolation and partial purification of phthalate ester hydrolyzing enzyme(s) from the brine shrimp, Artemia.

Isolation and 30-fold purification of a di-n-butyl phthalate (DNBP) hydrolyzing enzyme from synchronously hatched larvae of the brine shrimp, Artemia, are reported. The activity of the enzyme increases during larval development simultaneously with the acute toxic action of DNBP on the larvae. The enzyme is not stimulated by 10 mM sodium cholate, is stable for days to weeks in solution at 4 degrees C, indefinitely stable at -70 degrees C and is distinct from the previously reported trypsin- and chymotrypsin-like proteases [1].