Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

Abstract

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5’-TA-3’ dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60 000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.

Introduction

A common characteristic shared by all unicellular eukaryotes belonging to the monophyletic group of ciliates is the presence, within the same cytoplasm, of two types of nuclei, which play distinct roles throughout the cell life cycle (figure 1; see 〚1〛). The diploid micronucleus divides mitotically but remains transcriptionally silent during vegetative growth. It can be viewed as the germline nucleus since it undergoes meiosis during sexual reproduction, and provides the gametic nuclei which contribute to the formation of the zygotic nucleus (figure 1, stages I–III). The macronucleus is highly polyploid, although various ploidy levels have been reported in different ciliates (45n in Tetrahymena thermophila, around 1000n in Paramecium aurelia or Euplotes crassus). It divides amitotically and is actively transcribed during vegetative growth, but is destroyed at each sexual cycle. The macronucleus can therefore be considered as the ciliate somatic nucleus, since it governs the cell phenotype but does not transmit its genome to sexual progeny.

The precise number of vegetative macro- and micronuclei is variable among ciliates: P. aurelia carries one macronucleus and two micronuclei, while T. thermophila and E. crassus harbour one macronucleus and a single micronucleus. However, the general outline of the sexual processes is largely similar in all ciliates and each new sexual generation is faced with the problem of deriving a new macronucleus from a mitotic product of the zygotic nucleus.

Two modes of sexual reproduction have been identified in P. aurelia and can easily be induced experimentally. Mixing reactive cells of complementary mating types leads to conjugation, during which karyogamy takes place after a reciprocal exchange of gametic nuclei between two sexual partners. During the self-fertilisation process called autogamy, which can be obtained following extensive starvation of cells belonging to a single mating type, the two gametic nuclei from a single cell fuse to give the zygotic nucleus (see figure 1, stage III for details). In each case, the diploid zygotic nucleus undergoes two successive mitotic divisions (figure 1, stage IV): depending on their cellular localisation, two of the resulting nuclei become the new micronuclei while the other two differentiate into new macronuclei (figure 1, stage Va, and 〚2〛). The whole process of macronuclear development is accompanied by intense DNA synthesis to reach a final ploidy level of 800-1000n and extends over two cell cycles following the formation of the zygotic nucleus: at the first cell division, also called karyonidal division, one developing macronucleus, or anlage, is distributed to each daughter cell (figure 1, stage Vb), and mature macronuclei are obtained at the end of the second cycle (figure 1, stage Vc).

It should be emphasised that progressive degradation of the parental macronucleus starts shortly after meiosis of the germline nuclei. The parental macronucleus becomes fragmented and DNA replication rapidly stops within the resulting fragments, which persist within the cytoplasm and contribute to about 80% of total RNA synthesis throughout the whole period of formation of the new macronucleus 〚3〛. Macronuclear fragments are eventually diluted out during the subsequent vegetative cell divisions, and can be more rapidly degraded when cells are maintained under severe starvation conditions 〚4〛.

Not only do both types of ciliate nuclei differ in their cellular functions, their genomes also exhibit striking differences. A comparison of their respective DNA content has revealed that extensive and developmentally programmed DNA rearrangements participate in the formation of the macronuclear genome, in a highly reproducible manner from one sexual generation to the next 〚1〛, 〚5〛, 〚6〛, 〚7〛.

In the P. aurelia group of species, the germline genome is composed of 30 to 63 chromosome pairs, depending on the species or strain, and its haploid DNA content has been estimated to be around 100–200 Mbp, which would give an average chromosome size of 1–7 Mbp 〚1〛, 〚8〛. In contrast, the acentromeric macronuclear ‘chromosomes’ are shorter molecules of 300–800 kb in length 〚9〛. Thus, chromosomal fragmentation within reproducible regions, followed by de novo addition of telomeric repeats, is involved in the formation of the somatic genome (figure 2). Alternative fragmentation regions separated by 2–20 kbp can be used, and for each of those, the exact point of telomere addition varies within a 0.2–2 kbp range. Chromosomal fragmentation is associated with the imprecise loss of germline repetitive sequences, but, in contrast to the situation observed in T. thermophila or E. crassus, it does not appear to be determined by any specific consensus nucleotide sequence 〚10〛. Therefore, the molecular mechanism of chromosome fragmentation in P. primaurelia remains largely unknown.

The second type of DNA rearrangements involved in macronuclear development is the precise deletion of interstitial DNA segments specifically found in the germline genome (figure 2). In P. aurelia, these internal eliminated sequences (IESs) can be found in non-coding regions, including introns, but most of them interrupt open reading frames: therefore, IES elimination must be efficient and precise at the nucleotide level to allow the reconstitution of an active somatic genome. An extrapolation of the available data has led to an estimated number of 50 000–60 000 IESs per haploid genome (i.e., one IES every 1–2 kbp), each element being present as a single copy 〚11〛. Thus, IES elimination in P. aurelia is not restricted to a few specific loci, but is a genome-wide phenomenon.

Kinetic analyses have allowed the determination of the relative chronology of both types of DNA rearrangements in several ciliates, and have pointed to the diversity of the developmental programs involved in different organisms. In T. thermophila, all DNA rearrangements take place within the same time window 〚12〛, while precise IES elimination is completed prior to chromosome fragmentation in E. crassus 〚13〛. This type of study has long been delayed in Paramecium, because of experimental limitations in obtaining large amounts of synchronous cells undergoing macronuclear development, but a link between IES deletion and chromosome fragmentation has been suggested in P. primaurelia 〚14〛. Comparison of the timing of both reactions during macronuclear development should provide a better understanding of the relationships that may exist between the molecular mechanisms involved in the two types of DNA rearrangements.