Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila.

Abstract

We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines.A long-standing question in eucaryote development is the extent and nature of DNA rearrangement during development. Irreversible differential gene alterations have been found in a number of widely separated systems. Developmentally programmed examples of these include the chromosomal diminution found in nematodes (reviewed in reference 36), some insects and crustaceans (reviewed in references 5 and 35), and ciliates (reviewed in references 15, 24, and 25) and DNA rearrangements which occur in antibody-producing lymohocytes (reviewed in reference 34).The ciliated protozoan Tetrahymena thermophila affords an excellent model system to determine the extent, nature, and developmental timing of rearrangements during development of the somatic nucleus. T. thermophila is a unicellular organism containing two nuclei, a transcriptionally inactive germline nucleus (micronucleus) and a somatic, amitotically dividing macronucleus which is the site of gene expression. In the sexual process, conjugation, haploid micronuclei generated by meiosis are exchanged between the paired cells and subsequently fused. A division product of the newly diploid micronucleus in each cell differentiates into a new macronucleus, replacing the old macronucleus, which is destroyed (for reviews, see references 12, 14, and 15). Since these cells subsequenly divide vegetatively by binary fission in the absence of conjugation (which requires specific culture conditions as well as interaction between two cells of different mating types), large clonal populations can be obtained at given stages of development, and pure preparations can be made of macronuclei and their parent micronuclei.

A family of DNA sequences is reproducibly rearranged in the somatic nucleus ofTetrahymena

“…The three macronuclear fragments could have been the result of variable rearrangements of one of the micronuclear sequences In Individual cells of the population as have been previously reported (10,12). In Tltrahymena, genes which are alelic In the macronucleus undergo a process known as phenotypic assortment (33) hybridization pattern of mac DNA from these cells was the same as the parental cells (Fig.…”

“…That Is, a mic sequence may be rearranged In more than one way. Such variability has been reported In studies of rearranged sequences In caryonides (the four cells from a mating pair) (12,13).…”

“…This Is not the case for all of the rearranged sequences studied In I. thermoJhila. Austerberry et al (13) and Howard Blackburn (12) have shown that some rearranged sequences display alternate processing In different caryonides. One case has been observed where two different rearrangements are present In a clonal cell line, and only one or the other Is maintained after a number of vegetative fissions as Is expected for a sequence undergoing phenotypic assortment.…”

“…However, following vegetative cell division, clonal cell lines contain only one form. It has been suggested that this process Is the physical basis of the genetic phenomenon of phenotypic assortment (12 protoose peptone (Dlfco), 0.1% yeast extract, and 0.003% sequestrine (CibaGeigy)) prepared as descrlbod by Gorovsky et al (19). Stocks were maintained in 1% PPYS as described by Karror (20).…”

Formation of stable chromatin structures on the histone H4 gene during differentiation in Tetrahymena thermophila.

“…Like most or all genes in T. thermophila, it appears to be transcriptionally silent in the germinal micronucleus and active in the macronucleus. Frequent DNA sequence rearrangements (chromosome fragmentation, interstitial deletions) accompany gene activation during macronuclear development in T. thermophila (1,2,6,10,16,22,29,30). Although the role of these rearrangements, if any, in the expression of macronuclear genes has yet to be demonstrated (3,22), they nonetheless offer the possibility that ciliates have unique mechanisms of gene activation.…”