Biochimie 83 (2001) 1009−1022
© 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
S0300908401013499/REV
Developmentally programmed excision of internal DNA
sequences in Paramecium aurelia
Ariane Gratias, Mireille Bétermier*
Laboratoire de Génétique Moléculaire, CNRS UMR 8541, École Normale Supérieure, 46, rue d’Ulm, 75005 Paris, France
(Received 1 October 2001; accepted 6 November 2001)
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. © 2001 Société française de biochimie et biologie
moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
site-specific recombination / DNA deletion / circularisation / transposition / ciliates
1. Introduction
1.1. Nuclear dimorphism in ciliates: separation
of germline and somatic functions in a single-cell
organism
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
*Correspondence and reprints.
E-mail addresses: [email protected] (M. Bétermier),
[email protected] (A. Gratias).
Abbreviations: IES, internal eliminated sequence; bp, basepair;
Mbp, megabase pairs; kbp, kilobase pairs; nt, nucleotides; Pddp,
programmed DNA degradation protein.
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.
1.2. Macronuclear development in P. aurelia
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
1010
Figure 1. The sexual cycle in P. aurelia. In the vegetative cell,
the germline micronuclei are drawn as white circles and the
somatic macronucleus is shown in black. The different steps of
the sexual cycle are depicted as follows: Stage I: sexual
processes are initiated by micronuclear meiosis, which generates
eight haploid nuclei; Stage II: seven haploid nuclei quickly
degenerate (grey circles), while a single one migrates to a
specialised cell compartment and undergoes one mitotic division
to produce two identical haploid gametic nuclei (white circles).
In the meantime, the parental macronucleus becomes fragmented
into approximately 30 fragments (shown in black); Stage III: two
alternative fertilisation pathways, the occurrence of which depends on the presence or absence of a sexual partner, lead to the
formation of the zygotic nucleus (white circle). During conjugation, reciprocal fertilisation results from the migration of one
gametic nucleus from each mating cell into its sexual partner
(not represented on the figure): fusion of the incoming and
resident nuclei in each exconjugant produces genetically identical zygotic nuclei in both cells. During autogamy, a fully
homozygous zygotic nucleus is formed by the fusion of the two
identical gametic nuclei of a single cell; Stage IV: the diploid
zygotic nucleus divides twice mitotically to produce four identical nuclei harbouring the same germline genome (white
circles); Stage V: macronuclear development extends over two
cell cycles. Va: two mitotic products of the zygotic nucleus
become the new micronuclei (white circles) and the other two
differentiate into the new macronuclear anlagen (shown in grey),
in the presence of the fragments of the parental macronucleus
(black dots). During this period, intense DNA replication and
massive genome rearrangements take place within the developing anlagen. Vb: at the first cell division (or karyonidal division),
the two developing macronuclei become separated into each
daughter cell. Active DNA synthesis during the second cell cycle
accounts for the final ploidy level reached in the mature
macronucleus. For simplicity, only one daughter cell issued from
each cell division is represented. Vc: macronuclear development
is completed at the end of the second cell cycle and vegetative
growth resumes.
Gratias et Bétermier
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].
1.3. Developmental DNA rearrangements in P. aurelia:
a genome-wide affair
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, 5–7].
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.
IES excision in Paramecium
1011
2. General features of IESs in P. aurelia
2.1. Sequence analysis
Figure 2. DNA rearrangements during the formation of the
somatic genome in P. aurelia. The germline regions imprecisely
eliminated in association with chromosomal fragmentation are
shown as a grey box on the representation of the micronuclear
genome (top), and precisely excised IESs are drawn as black
boxes between two TA dinucleotides. The white arrow represents
an open reading frame. Only a few corresponding somatic
chromosomes are shown at the bottom of the figure. Telomeric
repeats are drawn as grey boxes at macronuclear chromosome
ends, and the 0.2–2 kbp heterogeneous regions that define their
point of addition are hatched. The precise macronuclear junctions formed after IES excision are represented by TA dinucleotides.
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.
The nucleotide sequence of 78 IESs of P. primaurelia
and P. tetraurelia was determined by different laboratories
([11, 14–26] and S. Duharcourt, O. Garnier, A. Le Mouël
and K.Y. Ling, personal communications). A striking
feature of P. aurelia IESs is their extremely high A/T
content (80% compared to 70% in their flanking
macronuclear-destined DNA regions), similar to that of
non coding sequences. All IESs are flanked by an absolutely conserved 5’-TA-3’ dinucleotide present as a direct
repeat on each side, a single copy of which is retained on
the chromosome after their precise deletion (figure 3A). P.
aurelia IESs are short, ranging from 26 to 882 bp, but are
not randomly distributed within this size range (figure 3B):
76% are shorter than 100 bp, and nearly one third are
26–30 bp long, which, interestingly, is within the size
range of Paramecium introns (18–35 nt: see [27, 28]).
These features relate P. aurelia IESs to the family of short
‘TA’ IESs also found in E. crassus [29].
A statistical analysis of the nucleotide sequence of 20
IESs of P. aurelia has allowed to propose a degenerate
consensus sequence present as an inverted repeat at both
ends [18]. This loosely conserved 8-bp sequence (5’
TAYAGYNR 3’) includes the flanking TA and is very
similar to the ends of Tc1-related transposons (5’
TACAGTKS 3’), which duplicate a target TA dinucleotide
upon insertion (figure 3A and [30]). Extension of this
analysis to the updated database of 78 IESs confirms the
general consensus and reveals an intriguing bias in the
nature of the pyrimidine present at the third position: most
IESs of 26-30 bp carry a T residue, while a C residue is
more statistically significant for longer IESs (figure 3C
and legend). Although the biological significance of this
sequence bias is unclear, it could reflect differences in the
evolution of the two classes of IESs or in the molecular
mechanism of their elimination. The similarity between
IES ends and the terminal inverted repeats of transposable
elements was extended to the ‘TA’ IESs of E. crassus, in
which the Tec elements, transposon-like elements belonging to the Tc1/mariner family, are massively eliminated
from the developing macronuclear genome [6]. This led to
the hypothesis that ‘TA’ IESs in ciliates may have evolved
from ancestral transposons by losing their coding capacity
while being kept under selective pressure for sequence
features allowing their precise elimination during macronuclear development.
2.2. Sequence determinants required for deletion
Genetic evidence points to a functional role of Paramecium IES ends in the elimination process. Mutations of
the TA dinucleotide or other positions in the consensus
1012
Gratias et Bétermier
Figure 3. Sequence analysis of P. aurelia IESs. A. IES
structure and general consensus for the ends. The IES is drawn
as a black box flanked by two TA repeats and white triangles
indicate the additional six nucleotides that define the consensus
for the terminal inverted repeats. The consensus for P. aurelia
IES ends is compared to the terminal inverted repeats of
Tc1/mariner elements and to the consensus for E. crassus short
IESs and Tec transposon-like elements. Y = C or T, R = A or G,
K = G or T, S = C or G (adapted from [29]). B. Size repartition
of IESs in P. aurelia. The histogram is based on the available
sequences of 78 IESs from the micronuclear versions of the
genes encoding the surface antigens A51, B51, α-51D, ε-51D
and G51, and of the ICL1d, PAK1, PAK11 and pwB genes of P.
tetraurelia strain 51, the A29 and sm19 genes of P. tetraurelia
strain d4.2, the 156G and 156φG loci and one chromosome
fragmentation region of P. primaurelia strain 156 (see section
2.1 for references). When alternative ends were reported for a
given IES, the size of every alternative element was recorded.
For the two 370-bp IESs of the A51 surface antigen gene
reported to carry internal shorter IESs, the deleted forms of 341
(for IES 51A6649) and 342 bp (for 51A2591) that result from
internal IES excision were considered as alternative IESs. One
882-bp IES was omitted in this representation. The size of IESs
under epigenetic control (77, 222, 229 bp and two IESs of 370
bp) is indicated by asterisks. C. Variation of the consensus
according to IES size. For the two groups of IESs (26–30 bp
and > 30 bp), the frequency of each nucleotide for the first five
positions is displayed (starting from the conserved TA). The
two variant consensus sequences were determined using an
adaptation of the ConsTrans computer program (S. Graziani,
personal communication), by normalising these values relatively to the expected frequencies calculated from the overall
nucleotide composition of the 78 IESs of our database (41% for
A, 7% for C, 12% for G and 40% for T: these frequencies do
not vary significantly if the calculation is restricted to the 25
IESs shorter than 30 bp).
result in the constitutive maintenance of the mutant IES in
the macronuclear genome [17, 20, 21, 23]. Moreover, a
comparison of excision patterns of two allelic versions of
the same IESs has indicated that, when one TA boundary
is mutated, a closely located TA dinucleotide can be
recruited for IES elimination. This suggests that the
boundaries of a deleted DNA segment can also result from
an adaptative convergent evolution of their sequence,
leading to a better match to the consensus for IES ends
[15]. This is further supported by the observation that, in
rare cases, alternative excision boundaries are used in the
same macronuclear anlage for a given IES [15, 17].
IES excision in Paramecium
1013
Although the 8-bp consensus sequence is involved in
specifying the ends of an IES, it is probably not the sole
determinant for elimination since it can be found in many
non-IES sequences of the Paramecium genome [18]. The
overall A/T-richness of IESs might favour the formation of
internal secondary DNA structures involved in elimination. In addition, as was demonstrated for T. thermophila
IESs (see [7]), important information may be contained
within the flanking DNA of P. aurelia IESs: indeed, it has
been reported that the removal of DNA sequences flanking
a particular 28-bp IES inhibits its developmental elimination [31].
2.3. Homology-dependent epigenetic control of IES
elimination
The existence of an epigenetic regulation of IES
elimination was first suggested by the study of the mtFE
mutation, inherited in a classical Mendelian manner,
which was shown to affect the elimination of a 222-bp IES
during macronuclear development in P. tetraurelia [22].
Surprisingly, when the wild-type mtF+ allele is reintroduced by conjugation into the micronucleus of the mutant
cell line, elimination of the IES from the macronuclear
genome is not restored in its sexual progeny. Instead, a
new cell line is obtained, which is homozygous for the
mtF+ allele but still maintains the 222-bp IES in its
macronucleus, even after multiple successive rounds of
autogamy. In this cell line, the alternative macronuclear
DNA rearrangement pattern, initially observed in the mtFE
mutant, is transmitted from one sexual generation to the
next, even though the macronuclear genome of each new
sexual generation is derived from a fully wild-type germline genome.
The demonstration that this epigenetic control of IES
elimination is exerted by the parental macronucleus was
obtained by transforming the vegetative macronucleus of
a wild-type cell with a plasmid carrying the cloned IES
sequence (figure 4, see [32]). These experiments clearly
showed that the presence of the 222-bp IES in the parental
macronucleus inhibits the elimination of the same IES
from the genome of the developing macronucleus of the
next sexual generation. Inhibition depends on the copy
number of the plasmid injected in the parental macronucleus, and also on sequence homology, since no other
IES was found to be affected. In addition, efficiency of
inhibition increases with the length of the flanking sequences present on the injected construct. Thus, in each
cell line, although the genome of the parental macronucleus is not transmitted to the sexual progeny, it can
influence the way the new macronuclear genome is
formed from the inherited germline genome. In Paramecium, by analogy to sexual reproduction in other eukaryotic systems, this type of ‘macronuclear’ heredity is also
referred to as ‘maternal’ heredity (reviewed in [10]). An
extension of this study revealed that five out of the 13
Figure 4. Homology-dependent epigenetic control of IES excision by the parental macronucleus (adapted from [10]). A.
Wild-type cell line. B. Cell line injected with a plasmid carrying
a cloned copy of an IES under epigenetic control. After injection
into the macronucleus, the plasmid DNA is linearised by the cell
and forms multimers to the ends of which terminal telomeric
repeats are added. The injected plasmid is maintained, throughout vegetative growth, as an autonomous macronuclear minichromosome. Like the rest of the macronuclear genome, it is
lost at each sexual cycle. A simplified view of the germline (mic)
and somatic (MAC) genomes is displayed. The chromosome
ends are hatched, and the telomeres added to the ends of the
linearised injected plasmid are drawn as open boxes. IESs are
represented by black boxes.
IESs tested are submitted to the same kind of maternal
effect [11]. No obvious size difference distinguishes the
regulated IESs from those which are not (see figure 3B).
Interestingly, however, two short 28- and 29-bp IESs do
escape the effect, even though they are inserted within
larger IESs showing inhibition of their excision. The only
noticeable feature of four out of the five IESs under
epigenetic control is that the nucleotide sequence of their
ends (5’ TATT 3’) differs at the fourth position from the
general consensus (5’ TAYA… 3’).
Homology-dependent maternal inhibition of IES elimination was also described in T. thermophila [33], which
suggests that a general epigenetic regulatory mechanism
1014
of DNA rearrangements may exist in ciliates [10]. Two
models have been proposed to explain this phenomenon.
In the first one, a cytoplasmic factor could be titrated out
by the IES copies present in the parental macronucleus.
However, to account for the sequence specificity, one has
to imagine that one specific factor exists for each IES
showing inhibition, which, given the probable high number of these sequences in the germline genome, makes it
unlikely to be a protein. In the second model, sequences
originating from the parental macronucleus could pair
with the homologous germline sequence in the developing
nucleus. This pairing would interfere with the elimination
process either by directly blocking access to a putative
enzymatic machinery (steric inhibition) or by inducing an
epigenetic modification of the rearranged sequence, such
as DNA methylation for instance, that would prevent it
from being recognised for elimination. It has been proposed that an IES-specific RNA, transcribed from the
parental macronucleus and imported into the anlage,
might be mediating this trans-nuclear sequence specificity. However, the presence of specific transcription promoters, either within the regulated IESs or in their
flanking sequences, has not been directly assayed, and the
existence and transport of this putative RNA molecule still
have to be demonstrated.
3. Precise elimination of IESs : a site-specific excision
reaction
3.1. Models for IES elimination in P. aurelia
Formally, the elimination of ‘TA’ IESs can be viewed as
the precise deletion of a DNA sequence located between
two short direct TA repeats. Three models can be proposed
for the molecular mechanism of this particular type of
DNA rearrangements. The first one relies on DNA polymerase slippage during replication [34]: for Paramecium
IESs, this would involve polymerase pausing at the first
TA, or immediately downstream of it, followed by reannealing of the nascent DNA strand to the second TA
repeat. After several rounds of replication, the IES would
be under-amplified or completely absent from the new
somatic chromosomes. In a second model, an internal
double strand break would be introduced within the IES,
perhaps at the level of an internal secondary structure.
Double strand break repair through a pathway related to
single strand annealing (see [35] for a review) would
produce 3’-overhangs able to anneal at the level of the TA
repeats. Trimming of the unpaired 3’ extensions followed
by gap-filling and ligation would produce the macronuclear chromosome sequence. The last type of mechanism is related to the recombination reactions which
participate in DNA site-specific excision/integration or
transposition [36]. In this case, endonucleolytic cleavages
would be introduced at IES ends, in the region of the TA
Gratias et Bétermier
repeats. This would release the excised IES while the
flanking macronuclear DNA ends would be precisely
rejoined by direct strand transfer or DNA repair.
3.2. Extrachromosomal forms of excised IESs
Of the three models presented above, only the third one
would allow the liberation of extrachromosomal forms of
the excised sequence. The analysis of IES excision products in P. tetraurelia has provided strong experimental
support for this model [37]. Southern blots of total
genomic DNA from large-scale cultures of autogamous
cells revealed the accumulation, during macronuclear
development, of extrachromosomal forms for all IESs
tested larger than 200 bp (figure 5A). Further analysis of
the electrophoretic behaviour of the excised molecules
obtained for two IESs of 222 bp and 370 bp, and their
treatment by restriction enzymes and exonuclease III,
indicated that the major excision product in each case is a
covalently closed, double stranded DNA circle. Interestingly, the exact number of extrachromosomal bands observed on the electrophoresis gel increases with IES size
and their relative abundance varies during macronuclear
development (figure 5A). These bands have tentatively
been assigned to topoisomers of the excised IES circles,
namely nicked or relaxed forms for the slowest migrating
species, and differentially supercoiled circles for the faster
migrating ones. However, the exact nature of these forms
and the biological significance of their different timing of
appearance still have to be elucidated.
Sequence analysis of the circles obtained for three
different IESs showed that their ends are precisely joined
on these molecules and separated by one copy of the
flanking TA repeat ([37] and Gratias, unpublished). Thus,
the same precision is observed in the formation of the
circular and chromosomal junctions following IES excision (figure 5B). This is in favour of a model where
excision involves DNA cleavage near the TA dinucleotide
at each IES end, and results in the release of an excised
form which, at least for IESs larger than 200 bp, can be
mainly detected as a circular molecule. The homogeneity
of the macronuclear junction and the accurate formation
of both chromosomal and circular junctions characterise
IES excision in P. aurelia. In E. crassus, excision of ‘TA’
IESs also results in the formation of a precise TA
macronuclear junction and produces abundant circular
molecules [38]. However, the circular junction is composed of two copies of the TA repeat, separated by a 10-bp
partial heteroduplex originating from the flanking DNA
(figure 5B, see [39, 40]). Moreover, although sequence
similarities have been observed between the ends of ‘TA’
IESs and Tc1/mariner transposons, the products of IES
developmental excision differ significantly from those
produced during the ‘cut-and-paste’ transposition of Tc1
elements (figure 5B): the latter are excised as linear
molecules and, when the donor sequence is repaired by
IES excision in Paramecium
1015
Figure 5. Detection of extrachromosomal products of IES
excision in P. tetraurelia. A. Southern blot analysis of genomic
DNA from vegetative (V) and autogamous cells at different
stages of macronuclear development, till the karyonidal division. The blot was successively hybridised with four radioactively labelled probes corresponding to IESs 51A4578,
51A2591, 51A6649 and 51G4404, as described in [37]. For
each IES, an arrowhead indicates the minor product migrating
at the size expected for a putative linear excised form (respectively 882 bp, 370 bp, 341 bp and 222 bp). B. Schematic
representation of the excision products obtained for E. crassus
and P. aurelia IESs, and comparison with Tc1/mariner transposons. The excised sequences are drawn as a double continuous line and dotted lines represent the flanking chromosomal
DNA. The structure of the linear excised forms of Paramecium
IESs is unknown, as indicated by the ? at their ends. The
double-strand cleavages at Tc1 ends are shown: although
circular versions of excised Tc1 have been detected, these are
most likely by-products of transposition and are therefore not
represented.
cellular enzymes, the two flanking TA dinucleotides are
generally retained at the chromosomal junction, separated
by a ∼ 2-bp footprint originating from the transposon
sequence [30].
More than 70% of P. aurelia IESs are shorter than 80
bp (figure 3B), which is about the minimal length compatible with the formation of double stranded DNA circles
in vivo [41]. All attempts to detect circles for 77-bp and
even 150-bp IESs have been unsuccessful so far (Gratias,
unpublished): thus, circular molecules may not be produced at all for the shortest IESs and may be only
secondary products of excision for the longest ones. In
support of this hypothesis, a minor product exhibiting the
electrophoretic behaviour expected for a linear molecule
has been detected at early time points of macronuclear
development for the four IESs examined so far (figure 5A).
This suggests that, as proposed for T. thermophila IESs
[42], which do not belong to the ‘TA’ IES family, IES
excision in P. aurelia may result in the early release of
linear molecules. These would then be rapidly degraded,
or, although this remains to be assayed directly, could be
converted into circles, as demonstrated for the signal
joints produced by V(D)J recombination during the assembly of immunoglobulin genes in mammals (reviewed
in [43]).
4. IES excision is regulated during macronuclear
development
4.1. Timing of IES excision
Various aspects of macronuclear development have
been studied using similar techniques for the synchroni
1016
sation of small-scale cultures of well-fed exconjugants of
P. aurelia. Microscopic analysis of radioactively pulselabelled cells led to the determination of DNA synthesis
rates within the developing macronucleus and to the
detection of anlage-specific transcription as early as 3 to 4
hours after exconjugant separation [3], which takes place
between the first and second divisions of the zygotic
nucleus [44]. Semi-quantitative PCR using convergent
pairs of primers was used to specifically amplify the
chromosomal (unexcised) copies of several IESs [37].
Shortly after exconjugant separation, IESs start to be
amplified in the developing macronuclear genome, in
association with their flanking chromosomal sequences
(figure 6A). Halfway through the first cell cycle, which
extends over a 10- to 12-hour period, a rapid decrease in
the chromosomal signal for the three IESs tested reflects
their massive elimination, which is essentially completed
when karyonidal division takes place. Massive IES excision is reproducibly detected within a time window
corresponding to the middle of the first cell cycle,
independently of the P. tetraurelia strain used or of
variations in the extent of this cycle (Gratias, unpublished). However, excision could be initiated earlier:
indeed, for two particular IESs of 28 bp and 29 bp, it has
been possible to directly monitor the transient appearance
of the chromosomal junctions, which are not present in
the parental macronucleus since these sequences are
located within two larger IESs of 370 bp (51A2591 and
51A6649, respectively). Excision of these short IESs is
detected approximately 2 h before the decrease in the
chromosomal signal observed for the larger ones (figure
6A). Analysis of the excision products of the larger IESs
revealed striking differences in the amounts of full-sized
circles compared to those from which the internal IESs
have been deleted: the circles observed for IES 51A6649
are mostly 349-bp molecules deleted from the internal
element, while most of IES 51A2591 circles are 370 bp
long and retain the short IES (figure 5A). This indicates
that some excision events are initiated early, at least for
short IESs, with an efficiency that may vary from one
element to the other.
Massive IES excision takes place during a period of
intense DNA synthesis within the macronuclear anlage
(figures 6A, B). Such a correlation in the timing of IES
elimination and DNA replication has also been reported in
other ciliates [45–47], but no general scheme has emerged
from these studies for a participation of DNA replication
in excision. In T. thermophila, inhibition of DNA polymerase α by aphidicolin treatment just after the beginning
of macronuclear development does not prevent or delay
excision [48]. In contrast, addition of hydroxyurea decreases the efficiency of ‘TA’ IES excision in E. crassus
[46]. Similar studies have not been carried out in P.
aurelia.
Gratias et Bétermier
4.2. Detection and fate of circular products
In P. aurelia, massive elimination of IESs larger than
200 bp is associated with the formation of abundant
circular products that can be amplified by PCR with
divergent primers [37]. These molecules transiently accumulate during macronuclear development till the end of
the first cell cycle, at which time they quickly disappear
(figure 6C). This suggests that circles are degraded by an
active mechanism, rather than simply being diluted out
during successive cell divisions. Similarly, although IES
circles are hardly detected in T. thermophila, time-course
studies have indicated that they do persist in this ciliate
until karyonidal division [49].
During the second cell cycle following meiosis in P.
aurelia, a second round of production of DNA junctions
characteristic of circularised IESs has been detected, in
two independent experiments and for two different IESs,
while no previous increase in the chromosomal IES signal
can be observed (figure 6C, and Gratias, unpublished).
This may result from the excision of the few remaining
chromosomal IES copies that, for some reason, would
have escaped the massive round of elimination during the
first cell cycle and would be deleted concomitantly with
their replication. In support of this hypothesis is the
observation that, in some cases, cloned IESs injected into
the developing macronucleus after karyonidal division can
still be excised, which indicates that the excision machinery is still present and active at this stage [31].
5. Towards the molecular mechanism of IES excision
in P. aurelia
5.1. DNA transactions leading to excision
The available data suggest that excision of P. aurelia
IESs involves DNA cleavage at their ends, near the
flanking TA repeats, but no information has been obtained
on the precise number of initial cleavage events or on the
existence of a concerted cleavage at both ends. Several
mechanisms have been proposed for the developmental
deletion of germline sequences in other ciliates (figure
7A). In E. crassus, the unusual structure of the circular
DNA junctions formed by ‘TA’ IESs has led to an excision
model involving the simultaneous introduction of staggered, double-stranded DNA breaks at each end: one
strand would be cleaved adjacent to the TA repeat and the
other one within the flanking DNA, to generate 5’overhangs. The macronuclear junction would be formed
by the pairing of the TA dinucleotides carried by the
flanking DNA ends, followed by gap filling and ligation,
while the circle heteroduplex junctions would result from
the alignment of the non-complementary overhangs generated at IES ends [29]. Direct experimental evidence was
obtained in T. thermophila for the existence of double
IES excision in Paramecium
1017
Figure 6. Time-course analysis of IES excision in well-fed
exconjugants of P. tetraurelia. Semi-quantitative PCR reactions with convergent or divergent primers were performed on
total lysates from cells issued from 5 synchronised mating pairs
(as described in [37]), to amplify chromosomal and circular
IES forms, respectively. A. Amplification of IES chromosomal
forms as a function of time, following exconjugant separation
until the end of the second cell cycle. Elimination of IESs
51A–712 (77 bp) and 51G4404 (222 bp) is displayed relative to
the left axis, and the right axis refers to 51A6649 (370 bp). The
arrow indicates the first excision events detected for the 29-bp
IES within 51A6649. B. Peaks of DNA synthesis during the
first cell cycle, as measured by radioactive pulse-labelling of
exconjugants (from [3]). Since the extent of the first cell cycle
was longer in this experiment than in A, the scale of the
horizontal axis was adapted to superimpose exconjugant separation and karyonidal division in both experiments. C. Semiquantitative PCR detection of the chromosomal and circular
forms of IES 51G4404 during macronuclear development,
from exconjugant separation till the end of the second cell
cycle. The black box on the horizontal axis represents karyonidal division.
strand breaks generating 5’-overhangs of 4 nucleotides
[50, 51]. However, recent results have suggested that, in
this ciliate, the initiating break occurs at a single end, and
cleavage of the other end is mediated by a nucleophilic
attack by the 3’OH group from the flanking macronuclear
DNA (figure 7A and [42]). This first attack would give rise
to a branched intermediate, from which a linear form of
the excised IES could be released after a second cleavage
step. A similar transesterification mechanism was proposed for the precise excision of TBE-1 elements in
Oxytricha fallax (figure 7A and [52]). In this model,
however, the 3’OH groups liberated on both strands by the
initial double strand break on one end would attack the
phosphodiester backbone of the two DNA strands at the
1018
Gratias et Bétermier
Figure 7. Molecular models for DNA transactions involved in IES excision. A. Models proposed for E. crassus, T. thermophila and
O. trifallax. IESs are drawn as thick grey lines and the excision process is described in three steps: 1 , the initial DNA cut; 2 , the
nucleophilic attack involved in cleavage of the other end for T. thermophila and O. trifallax elements (such an intermediate step has
not been proposed for E. crassus); and 3 the obtention of the final products. Detailed experimental data were obtained for IES
excision in T. thermophila [42, 51]. The initial 4-bp staggered double-strand break is introduced at a single end, 3’ to an adenine residue
in the consensus sequence 5’-ANNNNT-3’: either end can be cleaved first, and alternative cleavage sites have been demonstrated for
a given end. Nucleophilic attack of the same strand at the other end by the free 3’OH group also takes place at alternative positions,
which explains the heterogeneity of the macronuclear junctions in this ciliate. The excised IES is released as a linear molecule by an
additional cleavage at variable positions on the other strand: the linear IES exhibits a typical structure, with a 5’-overhang at one end,
corresponding to the initial cleavage, and a 3’ overhang at the other, corresponding to the transesterification step. The macronuclear
junction is repaired by degradation of the unpaired overhangs and gap filling (dotted line in step 3 ). The reactive 3’OH groups
involved in transesterification reactions are drawn as full circles. B. Proposed models for IES excision in Paramecium, that would
generate circular (left) or linear molecules (right) as primary excision products (see text for discussion). DNA cleavage events are
symbolised by scissors, and DNA strand-transfers by arrows, or by a cross within the nucleoprotein complex involving both ends (left
part of B).
other end: this would directly result in the circularisation
of the excised element and the formation of the chromosomal junction.
Although the exact positions of the initial cleavages
need to be determined for P. aurelia IESs, two excision
models can tentatively be proposed (figure 7B), which
mainly differ by the nature of the excised molecule
produced by the reaction. Circular molecules could directly be generated through pathways involving DNA
strand transfers between both ends, such as those proposed
for TBE-1 or various other transposition and site-specific
recombination systems [36]: the linear molecules would
correspond to excision by-products or degradation intermediates of the circles. This type of model implies that
IES ends are able to be physically brought together, which
can be facilitated by DNA structure, such as intrinsic
bending, or the binding of an architectural protein, but
seems hard to reconcile with the short size of most P.
aurelia IESs and the early detection of linear excised
molecules. Alternatively, DNA double strand breaks intro
IES excision in Paramecium
duced simultaneously at both ends, or a single initiating
break followed by a transesterification step analogous to
the one proposed for T. thermophila IESs, would release a
linear form of the excised sequence. For IESs larger than
200 bp, this linear molecule could subsequently be circularised. With respect to this hypothesis, the biological
significance of the circles should be addressed, since these
are the major excision products observed for large IESs
and accumulate in significant amounts prior to their
degradation. IES excision could potentially generate one
double strand break every 1–2 kb in the developing
macronuclear genome. Consequently, P. aurelia may have
evolved a highly efficient DNA repair system, and the
great precision observed for the circular junctions could
reflect the fidelity of this double strand break repair
pathway. It has also been proposed that circle formation
could prevent the ‘reactive’ ends of an excised IES from
reintegrating into macronuclear chromosomes [52].
5.2. Protein factors involved in DNA cleavage
The identity of the enzymatic machinery responsible
for the initial cleavages of IES ends remains an open
question in ciliates. Given the lack of coding capacity of
‘TA’ IESs of P. aurelia, it can be postulated that the
reaction is carried out by functions encoded elsewhere in
the genome. One mutation, mtFE, has been reported to
inhibit IES excision in P. tetraurelia, in addition to
causing pleiotropic effects during development [22]. However, since only a few IESs appear to be affected in this
mutant, the mtFE gene probably corresponds to an accessory factor.
An interesting hypothesis for a family of putative
excision factors has emerged from the discovery of a
‘transposon link’ for ciliate IESs [6]. Indeed, Tc1-related
transposons encode a transposase carrying a characteristic
DDE catalytic domain [30] and the Tec transposon-like
elements of E. crassus harbour several open reading
frames, one of which encodes a putative DDE transposase
[53]. Their precise excision produces the same type of
circular junctions as those reported for E. crassus short
‘TA’ IESs [54]: this led to the suggestion that IESs and
Tecs could be excised by the Tec transposase. However,
the very low levels of Tec transcripts detected during
macronuclear development do not support the sole participation of the transposase to the massive developmental
excision of the Tecs and of the ‘TA’ IESs [55]. Instead, it
has been proposed that these transcripts may be sufficient
for low-level Tec transposition within the germline genome, although such transposition events have not been
described.
Transposon-like elements belonging to the Tc1/mariner
family have also been discovered in the germline genome
of P. aurelia [10], but further work is needed to evaluate
the contribution of their putative proteins to IES excision.
In other systems, in vitro assays have revealed that
1019
transposition-related DNA excision is always initiated by
the liberation of a reactive 3’OH group through a single
strand cleavage reaction catalysed by the DDE domain of
the recombinase [56]. The various strategies used for the
second strand cleavage leading to excision of the internal
sequence are characterised by the formation of specific
molecular intermediates [57]. Second strand cleavage can
be performed by another protein, not necessarily carrying
a DDE catalytic site, as illustrated for the bacterial Tn7
transposon [58]. Alternatively, nucleophilic attack of the
facing strand by the free 3’OH group generates a hairpin
structure, as demonstrated for some cut-and-paste transposons and for the coding ends generated by V(D)J
recombination. In these first two situations, the internal
sequence is primarily excised as a linear molecule. For
some bacterial insertion sequences, such as IS911, however, a 3’OH group is liberated at one end only and
nucleophile attack of the other end gives a typical ‘figureof-eight’ molecule, which is resolved as a double stranded
circle by a host-encoded machinery: additional cleavage
of the circular junction by the transposase generates a
reactive linear transposition intermediate [59].
No clear experimental evidence has been obtained so
far that would link P. aurelia IES excision to transpositional recombination. The initial DNA breaks could be
produced by topoisomerases or site-specific recombinases,
which generate transient covalent intermediates between
the protein and the cleaved DNA [36, 60], or by any other
type of endonucleases. Molecular analysis of intermediate
products and extensive search for the putative developmental recombinase should contribute to a better understanding of the reaction.
5.3. Targeting the cleavage machinery to the excised
sequences
The TA repeats flanking Paramecium IESs, and their
adjacent nucleotides corresponding to the consensus for
IES ends, are most likely part of the site cleaved by the
recombinase during the initiating step of excision. These
sequences, however, are probably not sufficient to precisely target the recombinase and several speculations can
be made about the pathways that could allow the specification a germline sequence to be excised.
Although the macro- and micronucleus of ciliates are
derived from identical mitotic products of the zygotic
nucleus, significant differences exist in their chromatin
structure, as shown in E. crassus and T. thermophila (see
[61–65] and references therein). Macronuclear development should, therefore, be accompanied by profound
chromatin changes within the anlage, such as the incorporation of histone variants or other chromatin-associated
proteins, and the post-translational modification of histones which could account for transcriptional activation of
the developing macronucleus [66]. Chromatin remodelling could also epigenetically label IES sequences for
1020
excision, by discriminating them from their macronucleardestined flanking sequences, or for subsequent degradation of their excised forms. This has been suggested on the
basis of the unusually compact chromatin structures
formed by the Tec elements of E. crassus during macronuclear development, which have been attributed to the
specific incorporation of a developmental variant of core
histone H3 [67]. In T. thermophila, three proteins, named
Pdd1p, Pdd2p and Pdd3p, have been reported to specifically associate with eliminated germline DNA in electrondense structures, during the formation of the macronucleus [48, 68–70]. Expression of Pdd1p and Pdd2p is
required for IES excision and, strikingly, Pdd1p and
Pdd3p contain characteristic conserved protein motifs
called chromodomains, originally found in the Drosophila
heterochromatin associated protein HP1 and in the Polycomb protein [71]. This suggests a possible involvement
of the Pddp proteins in heterochromatin formation, via
protein-protein interactions with specific modified forms
of histones [72–74] or protein-RNA interactions [75].
In P. aurelia, activation of transcription within the
macronuclear anlage takes place during a time interval
that could correspond to the first detectable IES excision
events, a few hours prior to their massive excision (figure
6). Interestingly, in T. thermophila, specific transcription
of the IESs, initiated from their flanking DNA sequences,
was also detected prior to their elimination and suggested
to be required for excision [76]. Read-through transcription could result in a local change in the germline
chromatin structure at IES ends, as a consequence of
nucleosome displacement or histone acetylation [77, 78].
As proposed for other systems such as V(D)J [79, 80] or
the class switch recombination of immunoglobulin genes
(reviewed in [81]), this could specifically make the ends
accessible to a recombinase. Alternatively, a direct role
can be proposed for IES specific transcripts: through
pairing to their homologous DNA, they could specifically
target the cutting machinery to IES ends. The existence
and mode of action of such ‘guide’ RNA molecules need
to be investigated, but they would provide an alternative
explanation for the homology-dependent maternal inhibition of IES excision (section 2.3). During macronuclear
development, putative aberrant transcripts produced from
IES copies retained in the parental macronucleus could
pair to the guide RNAs and induce their degradation
through a pathway related to RNA interference [82]. The
existence of a post-transcriptional, homology-dependent
gene silencing system in P. aurelia has been demonstrated
in vegetative cells ([83] and A. Galvani and L. Sperling,
personal communication) and provides support to this
hypothesis.
6. Conclusion
Significant progress has been made, in the past few
years, in the characterisation of the cis requirements, the
Gratias et Bétermier
description of intermediate products and the determination
of the timing of IES excision during macronuclear development. This information should be of great help in the
understanding of the molecular mechanisms that participate in the recognition and excision of P. aurelia eliminated sequences, and in the regulation of these processes.
This area of research will greatly benefit from the use of
new powerful tools, such as homology-dependent gene
silencing [83] and the development of a genomic program
for the discovery of genes in P. aurelia [28], and should
provide important contributions to the general knowledge
of site-specific recombination and homology-dependent
epigenetic effects.
Acknowledgments
We wish to thank Sandra Duharcourt, Angélique Galvani,
Olivier Garnier, Anne Le Mouël, Kit-Yi Ling and Linda Sperling
for the communication of unpublished results and Stéphane
Graziani for his help in adapting his ConsTrans computer
program to the statistical analysis of IES ends. We are grateful to
all former and present members of Eric Meyer’s lab for
extremely rich and stimulating discussions, and to E. Meyer for
critical reading of the manuscript. The work in the Ciliate
Molecular Biology group was supported by the Association pour
la Recherche sur le Cancer (grant no. 5733), the Centre National
de la Recherche Scientifique (Programme Génome), the Ministère de l’Education Nationale, de la Recherche et de la
Technologie (Programme de Recherche fondamentale en Microbiologie et Maladies infectieuses et parasitaires), and the Comité
de Paris de la Ligue Nationale contre le Cancer (grant no.
75/01-RS/73). A. Gratias is the recipient of a doctoral fellowship
from the French Ministère de la Recherche.
References
[1] Prescott D.M., The DNA of ciliated protozoa, Microbiol. Rev. 58
(1994) 233–267.
[2] Grandchamp S., Beisson J., Positional control of nuclear differentiation in Paramecium, Dev. Biol. 81 (1981) 336–341.
[3] Berger J.D., Nuclear differentiation and nucleic acid synthesis in
well-fed exconjugants of Paramecium aurelia, Chromosoma 42
(1973) 247–268.
[4] Berger J.D., Selective autolysis of nuclei as a source of DNA
precursors in Paramecium aurelia exconjugants, J. Protozool. 21
(1974) 145–152.
[5] Coyne R.S., Chalker D.L., Yao M.C., Genome downsizing during
ciliate development: nuclear division of labor through chromosome restructuring, Annu. Rev. Genet. 30 (1996) 557–578.
[6] Klobutcher L.A., Herrick G., Developmental genome reorganization in ciliated protozoa: the transposon link, Prog. Nucleic Acid
Res. Mol. Biol. 56 (1997) 1–62.
[7] Yao M.C., Duharcourt S., Chalker D.L., Genome-wide rearrangements of DNA in ciliates, in: Craig N., Craigie R., Gellert M.,
Lambowitz A. (Eds.), Mobile DNA II, American Society for
Microbiology, Washington D.C., 2001 in press.
[8] Sonneborn T.M., Paramecium aurelia, in: King R.C. (Ed.), Handbook of genetics : Plants, plant viruses and protists, Plenum Press,
New York, 1974, pp. 469–594.
IES excision in Paramecium
[9] Caron F., Meyer E., Molecular basis of surface antigen variation in
paramecia, Annu. Rev. Microbiol. 43 (1989) 23–42.
[10] Meyer E., Garnier O., Non-Mendelian inheritance and homologydependent effects in ciliates, Adv. Genet. (2001) in press.
[11] Duharcourt S., Keller A.M., Meyer E., Homology-dependent
maternal inhibition of developmental excision of internal eliminated sequences in Paramecium tetraurelia, Mol. Cell. Biol. 18
(1998) 7075–7085.
[12] Yao M.C., Site-specific chromosome breakage and DNA deletion
in ciliates, in: Berg D.E., Howe M.M. (Eds.), Mobile DNA,
American Society for Microbiology, Washington, D.C., 1989,
pp. 715–734.
[13] Tausta S.L., Klobutcher L.A., Internal eliminated sequences are
removed prior to chromosome fragmentation during development
in Euplotes crassus, Nucleic Acids Res. 18 (1990) 845–853.
[14] Amar L., Chromosome end formation and internal sequence
elimination as alternative genomic rearrangements in the ciliate
Paramecium, J. Mol. Biol. 236 (1994) 421–426.
[15] Dubrana K., Le Mouël A., Amar L., Deletion endpoint allelespecificity in the developmentally regulated elimination of an
internal sequence (IES) in Paramecium, Nucleic Acids Res. 25
(1997) 2448–2454.
[16] Dubrana K., Amar L., Programmed DNA under-amplification in
Paramecium primaurelia, Chromosoma 109 (2000) 460–466.
[17] Haynes W.J., Ling K.Y., Preston R.R., Saimi Y., Kung C., The
cloning and molecular analysis of pawn-B in Paramecium tetraurelia, Genetics 155 (2000) 1105–1117.
[18] Klobutcher L.A., Herrick G., Consensus inverted terminal repeat
sequence of Paramecium IESs: resemblance to termini of Tc1related and Euplotes Tec transposons, Nucleic Acids Res. 23
(1995) 2006–2013.
[19] Ling K.Y., Vaillant B., Haynes W.J., Saimi Y., Kung C., A
comparison of internal eliminated sequences in the genes that
encode two K+-channel isoforms in Paramecium tetraurelia,
J. Euk. Microbiol. 45 (1998) 459–465.
[20] Mayer K.M., Mikami K., Forney J.D., A mutation in Paramecium
tetraurelia reveals function and structural features of developmentally excised DNA elements, Genetics 148 (1998) 139–149.
[21] Mayer K.M., Forney J.D., A mutation in the flanking 5’-TA-3’
dinucleotide prevents excision of an internal eliminated sequence
from the Paramecium tetraurelia genome, Genetics 151 (1999)
597–604.
[22] Meyer E., Keller A.M., A mendelian mutation affecting matingtype determination also affects developmental genomic rearrangements in Paramecium tetraurelia, Genetics 143 (1996) 191–202.
[23] Ruiz F., Krzywicka A., Klotz C., Keller A., Cohen J., Koll F.,
Balavoine G., Beisson J., The SM19 gene, required for duplication
of basal bodies in Paramecium, encodes a novel tubulin, etatubulin, Curr. Biol. 10 (2000) 1451–1454.
[24] Scott J., Leeck C., Forney J., Analysis of the micronuclear B type
surface protein gene in Paramecium tetraurelia, Nucleic Acids
Res. 22 (1994) 5079–5084.
[25] Steele C.J., Barkocy-Gallagher G.A., Preer L.B., Preer J.R.,
Developmentally excised sequences in micronuclear DNA of
Paramecium, Proc. Natl. Acad. Sci. USA 91 (1994) 2255–2259.
[26] Vayssié L., Sperling L., Madeddu L., Characterization of multigene families in the micronuclear genome of Paramecium tetraurelia reveals a germline specific sequence in an intron of a centrin
gene, Nucleic Acids Res. 25 (1997) 1036–1041.
[27] Dupuis P., The beta-tubulin genes of Paramecium are interrupted
by two 27 bp introns, EMBO J. 11 (1992) 3713–3719.
[28] Dessen P., Zagulski M., Gromadka R., Plattner H., Kissmehl R.,
Meyer E., Bétermier M., Schultz J.E., Linder J.U., Pearlman R.E.,
Kung C., Forney J., Satir B., van Houten J.L., Keller A.M.,
Froissard M., Sperling L., Cohen J., Paramecium genome survey:
a pilot project, Trends Genet. 17 (2001) 306–308.
[29] Jacobs M.E., Klobutcher L.A., The long and the short of developmental DNA deletion in Euplotes crassus, J. Euk. Microbiol. 43
(1996) 442–452.
1021
[30] Plasterk R.H., Izsvak Z., Ivics Z., Resident aliens: the Tc1/mariner
superfamily of transposable elements, Trends Genet. 15 (1999)
326–332.
[31] Ku M., Mayer K., Forney J.D., Developmentally regulated excision of a 28-base-pair sequence from the Paramecium genome
requires flanking DNA, Mol. Cell. Biol. 20 (2000) 8390–8396.
[32] Duharcourt S., Butler A., Meyer E., Epigenetic self-regulation of
developmental excision of an internal eliminated sequence in
Paramecium tetraurelia, Genes Dev. 9 (1995) 2065–2077.
[33] Chalker D.L., Yao M.C., Non-Mendelian, heritable blocks to DNA
rearrangement are induced by loading the somatic nucleus of
Tetrahymena thermophila with germ line-limited DNA, Mol. Cell.
Biol. 16 (1996) 3658–3667.
[34] Viguera E., Canceill D., Ehrlich S.D., Replication slippage involves DNA polymerase pausing and dissociation, EMBO J. 20
(2001) 2587–2595.
[35] Karran P., DNA double strand break repair in mammalian cells,
Curr. Opin. Genet. Dev. 10 (2000) 144–150.
[36] Hallet B., Sherratt D.J., Transposition and site-specific recombination: adapting DNA cut-and-paste mechanisms to a variety of
genetic rearrangements, FEMS Microbiol. Rev. 21 (1997)
157–178.
[37] Bétermier M., Duharcourt S., Seitz H., Meyer E., Timing of
developmentally programmed excision and circularization of
Paramecium internal eliminated sequences, Mol. Cell. Biol. 20
(2000) 1553–1561.
[38] Tausta S.L., Klobutcher L.A., Detection of circular forms of
eliminated DNA during macronuclear development in E. crassus,
Cell 59 (1989) 1019–1026.
[39] Tausta S.L., Turner L.R., Buckley L.K., Klobutcher L.A., High
fidelity developmental excision of Tec1 transposons and internal
eliminated sequences in Euplotes crassus, Nucleic Acids Res. 19
(1991) 3229–3236.
[40] Klobutcher L.A., Turner L.R., LaPlante J., Circular forms of
developmentally excised DNA in Euplotes crassus have a heteroduplex junction, Genes Dev. 7 (1993) 84–94.
[41] Ringrose L., Chabanis S., Angrand P.O., Woodroofe C., Stewart A.F., Quantitative comparison of DNA looping in vitro and in
vivo: chromatin increases effective DNA flexibility at short distances, EMBO J. 18 (1999) 6630–6641.
[42] Saveliev S.V., Cox M.M., Product analysis illuminates the final
steps of IES deletion in Tetrahymena thermophila, EMBO J. 20
(2001) 3251–3261.
[43] Grawunder U., Harfst E., How to make ends meet in V(D)J
recombination, Curr. Opin. Immunol. 13 (2001) 186–194.
[44] Diller W.F., Nuclear reorganization processes in Paramecium
aurelia, with description of autogamy and hemixis, J. Morphol. 59
(1936) 11–67.
[45] Austerberry C.F., Allis C.D., Yao M.C., Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena, Proc.
Natl. Acad. Sci. USA 81 (1984) 7383–7387.
[46] Frels J.S., Jahn C.L., DNA rearrangements in Euplotes crassus
coincide with discrete periods of DNA replication during the
polytene chromosome stage of macronuclear development, Mol.
Cell. Biol. 15 (1995) 6488–6495.
[47] Frels J.S., Tebeau C.M., Doktor S.Z., Jahn C.L., Differential
replication and DNA elimination in the polytene chromosomes of
Euplotes crassus, Mol. Biol. Cell 7 (1996) 755–768.
[48] Nikiforov M.A., Smothers J.F., Gorovsky M.A., Allis C.D.,
Excision of micronuclear-specific DNA requires parental expression of Pdd2p and occurs independently from DNA replication in
Tetrahymena thermophila, Genes Dev. 13 (1999) 2852–2862.
[49] Saveliev S.V., Cox M.M., The fate of deleted DNA produced
during programmed genomic deletion events in Tetrahymena
thermophila, Nucleic Acids Res. 22 (1994) 5695–5701.
[50] Saveliev S.V., Cox M.M., Transient DNA breaks associated with
programmed genomic deletion events in conjugating cells of
Tetrahymena thermophila, Genes Dev. 9 (1995) 248–255.
1022
[51] Saveliev S.V., Cox M.M., Developmentally programmed DNA
deletion in Tetrahymena thermophila by a transposition-like reaction pathway, EMBO J. 15 (1996) 2858–2869.
[52] Williams K., Doak T.G., Herrick G., Developmental precise
excision of Oxytricha trifallax telomere-bearing elements and
formation of circles closed by a copy of the flanking target
duplication, EMBO J. 12 (1993) 4593–4601.
[53] Doak T.G., Doerder F.P., Jahn C.L., Herrick G., A proposed
superfamily of transposase genes: transposon-like elements in
ciliated protozoa and a common ’D35E’ motif, Proc. Natl. Acad.
Sci. USA 91 (1994) 942–946.
[54] Jaraczewski J.W., Jahn C.L., Elimination of Tec elements involves
a novel excision process, Genes Dev. 7 (1993) 95–105.
[55] Jaraczewski J.W., Frels J.S., Jahn C.L., Developmentally regulated, low abundance Tec element transcripts in Euplotes crassusimplications for DNA elimination and transposition, Nucleic Acids
Res. 22 (1994) 4535–4542.
[56] van Gent D.C., Mizuuchi K., Gellert M., Similarities between
initiation of V(D)J recombination and retroviral integration, Science 271 (1996) 1592–1594.
[57] Turlan C., Chandler M., Playing second fiddle: second-strand
processing and liberation of transposable elements from donor
DNA, Trends Microbiol. 8 (2000) 268–274.
[58] Hickman A.B., Li Y., Mathew S.V., May E.W., Craig N.L.,
Dyda F., Unexpected structural diversity in DNA recombination:
the restriction endonuclease connection, Mol. Cell 5 (2000)
1025–1034.
[59] Rousseau P., Normand C., Loot C., Turlan C., Alazard R.,
Duval-Valentin G., Chandler M., Transposition of IS911, in:
Craig N., Craigie R., Gellert M., Lambowitz A. (Eds.), Mobile
DNA II, American Society for Microbiology, Washington D.C.,
2001 in press.
[60] Champoux J.J., DNA Topoisomerases: Structure, function, and
mechanism, Annu. Rev. Biochem. 70 (2001) 369–413.
[61] Huang H., Wiley E.A., Lending C.R., Allis C.D., An HP1-like
protein is missing from transcriptionally silent micronuclei of
Tetrahymena, Proc. Natl. Acad. Sci. USA 95 (1998) 13624–13629.
[62] Jahn C.L., Ling Z., Tebeau C.M., Klobutcher L.A., An unusual
histone H3 specific for early macronuclear development in Euplotes crassus, Proc. Natl. Acad. Sci. USA 94 (1997) 1332–1337.
[63] Stargell L.A., Bowen J., Dadd C.A., Dedon P.C., Davis M.,
Cook R.G., Allis C.D., Gorovsky M.A., Temporal and spatial
association of histone H2A variant hv1 with transcriptionally
competent chromatin during nuclear development in Tetrahymena
thermophila, Genes Dev. 7 (1993) 2641–2651.
[64] Strahl B.D., Ohba R., Cook R.G., Allis C.D., Methylation of
histone H3 at lysine 4 is highly conserved and correlates with
transcriptionally active nuclei in Tetrahymena, Proc. Natl. Acad.
Sci. USA 96 (1999) 14967–14972.
[65] Sweet M.T., Jones K., Allis C.D., Phosphorylation of linker
histone is associated with transcriptional activation in a normally
silent nucleus, J. Cell. Biol. 135 (1996) 1219–1228.
[66] Jenuwein T., Allis C.D., Translating the histone code, Science 293
(2001) 1074–1080.
Gratias et Bétermier
[67] Jahn C.L., Differentiation of chromatin during DNA elimination in
Euplotes crassus, Mol. Biol. Cell 10 (1999) 4217–4230.
[68] Coyne R.S., Nikiforov M.A., Smothers J.F., Allis C.D., Yao M.C.,
Parental expression of the chromodomain protein Pdd1p is required for completion of programmed DNA elimination and
nuclear differentiation, Mol. Cell. 4 (1999) 865–872.
[69] Madireddi M.T., Coyne R.S., Smothers J.F., Mickey K.M.,
Yao M.C., Allis C.D., Pdd1p, a novel chromodomain-containing
protein, links heterochromatin assembly and DNA elimination in
Tetrahymena, Cell 87 (1996) 75–84.
[70] Nikiforov M.A., Gorovsky M.A., Allis C.D., A novel chromodomain protein, Pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila, Mol. Cell. Biol. 20 (2000) 4128–4134.
[71] Cavalli G., Paro R., Chromo-domain proteins: linking chromatin
structure to epigenetic regulation, Curr. Opin. Cell Biol. 10 (1998)
354–360.
[72] Bannister A.J., Zegerman P., Partridge J.F., Miska E.A., Thomas J.O., Allshire R.C., Kouzarides T., Selective recognition of
methylated lysine 9 on histone H3 by the HP1 chromo domain,
Nature 410 (2001) 120–124.
[73] Lachner M., O’Carroll D., Rea S., Mechtler K., Jenuwein T.,
Methylation of histone H3 lysine 9 creates a binding site for HP1
proteins, Nature 410 (2001) 116–120.
[74] Nielsen A.L., Oulad-Abdelghani M., Ortiz J.A., Remboutsika E.,
Chambon P., Losson R., Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins, Mol.
Cell 7 (2001) 729–739.
[75] Akhtar A., Zink D., Becker P.B., Chromodomains are protein-RNA
interaction modules, Nature 407 (2000) 405–409.
[76] Chalker D.L., Yao M.C., Nongenic, bidirectional transcription
precedes and may promote developmental DNA deletion in
Tetrahymena thermophila, Genes Dev. 15 (2001) 1287–1298.
[77] Flaus A., Owen-Hughes T., Mechanisms for ATP-dependent chromatin remodelling, Curr. Opin. Genet. Dev. 11 (2001) 148–154.
[78] Marmorstein R., Roth S.Y., Histone acetyltransferases: function,
structure, and catalysis, Curr. Opin. Genet. Dev. 11 (2001)
155–161.
[79] McBlane F., Boyes J., Stimulation of V(D)J recombination by
histone acetylation, Curr. Biol. 10 (2000) 483–486.
[80] McMurry M.T., Krangel M.S., A role for histone acetylation in the
developmental regulation of V(D)J recombination, Science 287
(2000) 495–498.
[81] Kinoshita K., Honjo T., Linking class-switch recombination with
somatic hypermutation, Nat. Rev. Mol. Cell. Biol. 2 (2001)
493–503.
[82] Matzke M., Matzke A.J., Kooter J.M., RNA: guiding gene
silencing, Science 293 (2001) 1080–1083.
[83] Ruiz F., Vayssie L., Klotz C., Sperling L., Madeddu L., Homologydependent gene silencing in Paramecium, Mol. Biol. Cell 9 (1998)
931–943.