Download Control of DNA excision efficiency in Paramecium

4654–4662 Nucleic Acids Research, 2001, Vol. 29, No. 22
© 2001 Oxford University Press
Control of DNA excision efficiency in Paramecium
Karine Dubrana1 and Laurence Amar1,2,*
1Laboratoire
de Génétique Moléculaire, Ecole Normale Supérieure, 46 Rue d’Ulm, 75230 Paris Cedex 05, France
and 2UPRES-A 8080, IBAIC, Bat 444, 91405 Orsay Cedex, France
Received July 17, 2001; Revised and Accepted September 26, 2001
ABSTRACT
Programmed excision of internal eliminated
sequences (IESs) occurs at thousands of sites in
ciliate genomes. How this is controlled is largely
unknown. Here, we report the characterization of
the non-efficiently excised 156ψG-11 IES from
Paramecium primaurelia strain 156 and that of the
efficiently excised 168ψG-11 IES, an allelic variant
from strain 168. Then, we report a genetic and molecular analysis of IES excision efficiency in F1 progeny
derived from interstrain crosses and in F2
homozygous progeny derived from F1 autogamy. IES
168ψG-11 excision efficiency was ∼100% in all cases.
IES 156ψG-11 excision efficiency was 19 ± 13% in F1
progeny and 0.6 ± 1.1% in F2 progeny. No transexcision event between IESs 156ψG-11 and 168ψG11 was detected within the F1 progeny. These data
demonstrate that the excision efficiency of this IES is
variable and controlled by a cis-acting element. This
should encompass positions 8 and/or 9 of the right
IES end, which display allele differences. Finally, the
30-fold stimulation of IES 156ψG-11 excision efficiency
within F1 progeny relative to F2 progeny demonstrates that Paramecium IES excision efficiency is
sensitive either to a conjugation-specific trans-acting
factor provided by the zygotic genome, or to homologous chromosome cross-talk.
INTRODUCTION
Programmed DNA rearrangements occur in a wide range of
genomes. However, in no genome do they occur as frequently
as in ciliate genomes. These are rearranged every 100–1000 bp
on average, thereby offering great opportunities to study some
of the regulatory systems and mechanisms affecting eukaryotic
genome plasticity.
Ciliates are unicellular eukaryotes that harbor two types of
nuclei, macronuclei and micronuclei. These carry out different
functions within the cell. Macronuclei are transcriptionally
active and carry out vegetative functions. Micronuclei are
DDBJ/EMBL/GenBank accession nos AF384101, AF384102
transcriptionally silent and provide genetic continuity between
sexual generations. In the course of sexual reproduction the
micronuclei undergo meiosis, whereas the macronuclei
degenerate. The fusion of two gametic nuclei produces a
zygotic nucleus. This nucleus divides twice and the daughter
nuclei then differentiate into a micronucleus or a macronucleus. In the second case, the whole genome is processed
through chromosome fragmentation, telomere addition to the
broken ends and excision of internal eliminated sequences
(IESs) that are eventually eliminated. Massive DNA amplification also occurs; this accounts for the large size, and hence the
name, of the macronuclei (1,2).
In Paramecium, IESs are excised from the micronuclear
genome every 1000 bp on average. Since their first identification
in 1994, some 80 Paramecium IESs have been characterized.
IESs are usually short (26–882 bp), AT-rich (80%) DNA
segments. They are invariably surrounded by 5′-TA-3′ repeats,
one copy of which is retained at the excision junction. Two
IESs have been shown to form extrachromosomal circles in the
course of macronucleus development, the IES ends being
joined by one 5′-TA-3′ copy (3).
How Paramecium IESs are excised and how Paramecium
IES excision is controlled are still largely unknown. IES excision can be negatively regulated by a trans nuclear epigenetic
mechanism. Injection of one IES into the parental macronucleus results in inhibition of excision of the endogenous IES
within the developing macronucleus (4). However, in nine
other cases injection of IESs in similar amounts results in
either partial inhibition of excision of the endogenous IES or
does not result in any excision inhibition (5).
Functional analysis of natural and mutagenesis-derived IES
alleles has demonstrated that the 5′-TA-3′ dinucleotide invariably flanking IES ends is strictly required for IES excision. A
mutation in the 5′-TA-3′ dinucleotide is associated with either
defective excision of the mutated IES (6) or a shift of the IES
end towards the next two 5′-TA-3′ dinucleotides, located 1 and
5 bp downstream (7). Functional analysis has also shown that a
mutation of nucleotide position 5 from one IES (numbering of
IES nucleotide positions starts at the 5′-TA-3′ dinucleotide)
totally impairs its excision (8). Yet, a previous comparison of
IESs demonstrated nucleotide preferences for IES nucleotide
positions 3, 4, 5, 6 and 8 (9). These preferences point to a role
of these nucleotides in IES recognition and/or excision (9).
*To whom correspondence should be addressed at: UPRES-A 8080, IBAIC, Bat 444, 91405 Orsay Cedex, France. Tel: +33 1 69 15 66 38;
Fax: +33 1 69 15 68 03; Email: [email protected]
Present address:
Karine Dubrana, Département de Biologie Moléculaire, Université de Genève, 30 quai Ernest Ansermet, 1205 Genève, Switzerland
Nucleic Acids Research, 2001, Vol. 29, No. 22 4655
Figure 1. IESs from the G gene and ψG pseudogene of P.primaurelia. (A) Maps of the micronuclear version of the G gene and ψG pseudogene. IESs are shown
in red. Large insertion/deletion differences between the G gene and ψG pseudogene loci are indicated in gray. Probes BS (left) and XH (right) are drawn in blue.
(B–D) Sequences of the homologous 156ψG-11, 168ψG-11 and 156G-11 IESs. IESs are flanked by 5′-TA-3 ′ dinucleotides, one copy of which is retained at the
macronuclear excision junction. Nucleotides differing between IESs 168ψG-11 or 156G-11 and IES 156ψG-11 are underlined. Note that IES 156G-11 is 1 nt
longer than IESs 156ψG-11 and 168ψG-11. The extra nucleotide is shown as an excluded nucleotide to preserve IES sequence alignment.
Recently, a determinant(s) lying external to one 28 bp IES,
31–72 bp from its end, has been characterized as essential for
its excision (10).
In a previous study, we characterized the micronuclear version
of the G gene encoding the G surface antigen in Paramecium
primaurelia strain 156 and that of a ψG pseudogene (11). By
comparing the micronuclear version of the G gene to one of its
macronuclear copies, we identified six IESs (Fig. 1A). All of
them had paralogs in the ψG pseudogene, i.e. counterparts
inserted at homologous positions. However, a comparison of
the micronuclear version of the ψG pseudogene with one of its
macronuclear copies showed that one of the paralogous IESs,
IES 156ψG-11, failed to be excised. These data suggest that
IES 156ψG-11 is defective for excision.
Here we provide an en masse analysis of IES 156ψG-11
excision indicating that IES 156ψG-11 is excised at low
frequency. We show that non-efficient IES 156ψG-11 excision
is due to a defective cis-acting element(s). Our data strongly
suggest that this element(s) encompasses nucleotide positions
8 and/or 9 of the IES right end, thereby providing the first functional demonstration of the importance of these positions for
IES excision. In addition, our data demonstrate that a transacting factor encoded by the zygotic genome participates in
the control of IES 156ψG-11 excision efficiency. Together,
these data help us understand how IES excision is regulated
in Paramecium.
MATERIALS AND METHODS
Cell lines and culture
Paramecium primaurelia strains 156 and 168 are wellcharacterized strains which have been used extensively in
genetic studies of the surface antigen system. The karyonides,
the daughter cells from conjugant cells, harbor macronuclei
produced from single developmental events. All the F1 and F2
progeny cultures, as well as cultures 156/1–2, 156/16–17 and
168/1–6, were monokaryonidal cultures. Cultures 156/3–15
and 156/18 were polykaryonidal.
DNA extraction
Cultures of exponentially growing cells at 1000 cells/ml were
centrifuged. After washing in Dryl’s medium, the pellet was
resuspended in 1 vol of the same buffer and added to 4 vol of
lysis solution [0.44 M EDTA, pH 9.0, 1% SDS, 0.5% N-laurylsarcosine (Sigma) and 1 mg/ml proteinase K (Merck)] at 55°C.
The lysate was incubated at 55°C for at least 5 h, gently
extracted with phenol and dialyzed twice against TE (10 mM
Tris–HCl and 1 mM EDTA, pH 8.0).
DNA analysis and sequencing
DNA restriction and electrophoresis were carried out
according to standard methods. DNA was blotted onto Hybond
N+ membrane (Amersham) in 0.4 N NaOH after depurination
in 0.25 N HCl. Hybridization was carried out overnight in 7%
SDS, 0.5 M sodium phosphate and 1% bovine serum
albumin (BSA) at 60°C for probes BS and HX, at 35°C for
oligonucleotide A. Probes BS and HX were labeled using a
random priming kit (Boehringer-Mannheim, Mannheim,
Germany). Oligonucleotide A was labeled in a kinase reaction.
Membranes hybridized with probes BS and HX were washed
at 60°C in 0.1% SDS and 0.2× SSC (1× SSC is 0.15 M NaCl
and 0.015 M sodium citrate, pH 7.0). Membranes hybridized
with oligonucleotide A were washed at 50–58°C in 0.1% SDS
and 2× SSC. Hybridization signals were revealed and quantified
4656 Nucleic Acids Research, 2001, Vol. 29, No. 22
using a Fuji phosphorimager. PCRs were performed in
volumes of 25 µl comprising 1× PCR buffer supplied by the
manufacturer (Promega), 1.25 mM MgSO 4, 50 µM of each
dNTP, 1 µM oligonucleotides and 0.8 U Tfl thermostable DNA
polymerase. Amplification comprised 32 cycles of 92°C for
1 min, 63°C for 1 min and 72°C for 2 min, with a final extension at 72°C for 3 min. PCR was performed in a Perkin Elmer
Cetus thermocycler in capped 0.5 ml polypropylene microcentrifuge tubes (Sigma).
DNA cloning and library construction
To clone an insert encompassing IES 168ψG-11, PCR was
performed using an oligonucleotide that annealed within the
macronuclear-destined sequence located upstream from IES
168ψG-11 and an oligonucleotide that annealed within the
downstream IES 168ψG1416. Inserts encompassing the IES
168ψG-11 excision site were cloned from a partial DNA
library. This library was constructed from a DNA restricted
with BglII and size fractionated on a 0.8% agarose gel. The
DNA was eluted from the fractions using the GeneClean
procedure (Bio 101 Inc.) and screened by PCR for presence of
the ψG pseudogene. The >17 kb BglII-digested DNA fraction
was selected and cloned into the λ EMBL4 vector.
Sequence analysis
Sequences were analyzed with the Gap program from the
Wisconsin Package v.9.1 (Genetics Computer Group, Madison,
WI).
Nucleotide sequence accession numbers
The GenBank accession no. for IES 156ψG-11 and its flanking
sequence is AF384101; for IES 168ψG-11 and its flanking
sequence, AF384102.
RESULTS
IES 156ψG-11 is non-efficiently excised from
macronuclear genomes
In a previous study we identified IES 156ψG-11 from
P.primaurelia strain 156 as putatively defective for excision
(11). To accurately define the IES 156ψG-11 excision pattern,
we have analyzed macronuclear DNA molecules from 15 cell
cultures. PCR was performed using oligonucleotide primers A
and B that specifically anneal 60 bp downstream and 225 bp
upstream from IES 156ψG-11, respectively (Fig. 2A). Control
PCRs were performed on cloned inserts that harbored either
the micronuclear version of the ψG pseudogene or an
IES-deleted macronuclear copy (see below for a description of
this copy). PCR products were then run on an agarose gel and
hybridized with oligonucleotide A. PCR products of 285 and
329 bp were expected for IES 156ψG-11-deleted and IES
156ψG-11-harboring fragments, respectively. However, in
order to analyze the PCR data correctly, we first characterized
the ψG pseudogene macronuclear content of the 15 DNAs.
Usually, the PCR products generated from whole-cell DNA
only reflect the macronuclear DNA content of Paramecium
cells as a consequence of the low cell micronuclear to macronuclear DNA ratio (each cell harbors two diploid micronuclei
and a 800–1700n macronucleus). The ψG pseudogene has
been shown to be under-amplified within the polyploid
Figure 2. IES 156ψG-11 non-efficient excision. (A) Diagram of the PCR and
hybridization reactions designed to analyze the excision pattern of IESs
156ψG-11 and 168ψG-11. PCRs were performed using oligonucleotides A
and B. The PCR products were electrophoresed and then hybridized with
oligonucleotidic probe A. (B) PCR products generated from strain 156 and 168
cultures. (C) PCRs products generated from F2 progeny. The upper PCR products identified IES-harboring fragments; the lower PCR products identified
IES-deleted fragments. Control PCRs were performed on DNA inserts that
encompassed the ψG pseudogene micronuclear version from strain 156 (lane
Mic ψG) or a ψG pseudogene macronuclear copy from strain 168 (lane Mac
ψG). Note that no PCR products were generated from inserts harboring the
G gene micronuclear version (lane Mic G) or a G gene macronuclear copy
(lane Mac G).
macronuclear genome (11). Therefore, it was important to
check that the 329 bp PCR product always originated from
macronuclear DNA templates.
The ψG pseudogene macronuclear content was analyzed
relative to the G gene macronuclear content (this content was
constant amongst all DNAs). DNAs 156/1–15 were restricted
with BglII, electrophoresed and probed with fragment BS. This
fragment, derived from the DNA located >3.0 kb upstream
from the 5′-end of the ψG pseudogene, hybridized to DNA
from both the G and ψG loci (Fig. 1A). Signals of 2.8, 5.1–6.7,
8.0–9.7, 11.0–>12.2 and >>12.2 kb were identified (Fig. 3A).
No significant levels of alternative forms of DNA rearrangement could be detected over a >200 kb region that encompassed the G gene (12). Therefore, the 2.8 kb signal, which was
the only signal to be revealed in all DNAs, must identify a fragment from the G locus, and the four signals of 5.1–6.7, 8.0–9.7,
11.0–>12.2 and >>12.2 kb, which were present in only some
DNAs, must identify four fragment families from the ψG locus.
Families of fragments differing by 1–2 kb have been exclusively
associated with DNA fragmentation in Paramecium
(programmed DNA fragmentation occurs every 300 kb on
Nucleic Acids Research, 2001, Vol. 29, No. 22 4657
Figure 4. Macronuclear DNA molecules arising from the ψG locus. Hatched
boxes defined the nucleotide positions at which telomeric repeats are added.
From top to bottom, macronuclear DNA molecules end 1.0–2.6 kb upstream
from the ψG pseudogene start and 0.3–2.0, 3.3–4.3 and >15 kb downstream
from it. IES 156ψG-11, which is retained on most molecules, is shown in red.
Probes BS (left) and XH (right) are drawn in blue.
Figure 3. Analysis of the ψG pseudogene macronuclear content from (A) cultures of strain 156 and (B) F1 progeny arising from crosses between strains 156
and 168. Genomic DNA was restricted with BglII, separated by electrophoresis
on a 0.8% agarose gel and probed with probe BS derived from the 5′-side of the
ψG pseudogene (see Fig. 1). The 2.8 kb signal identified a fragment from the
G locus. The signals of 5.1–6.7, 8.0–9.7, 11.0–>12.2, >11.2 and >>12.2 kb
identified fragment families from the ψG locus. Probe BS faintly cross-hybridized
with two bands indicated with dots. F1 progeny 1–4 were raised from the
156/18 × 168/5 mating, F1 progeny 5–8 from the 156/18 × 168/6 mating,
F1 progeny 9–12 from the 156/18 × 168/7 mating and F1 progeny 13–16 from
the 156/18 × 168/8 mating. Macronuclear genomes from F1 progeny 1–2, 5–6,
9–10 and 13–14 developed within 168 parents, those from F1 progeny 3–4, 7–8,
11–12 and 15–16 developed within 156 parents.
average within the developing macronuclei). Heterogeneity of
1–2 kb reflects heterogeneity in the nucleotide position to
which telomeric repeats are added, and heterogeneity in the
length of the added telomeric repeats. Therefore, signals of
5.1–6.7, 8.0–9.7 and 11.0–>12.2 kb should identify families of
telomeric fragments ending 1.0–2.6 kb upstream of the ψG
pseudogene start and 0.3–2.0 and 3.3–4.3 kb downstream of it
(Fig. 4). The band >>12.2 kb should identify the full-size BglII
restriction fragment derived from DNA molecules ending
>12.0 kb from the ψG pseudogene start. PCRs performed
with a genomic oligonucleotide and an oligonucleotide
corresponding to Paramecium telomeric repeats confirmed that
macronuclear molecules were telomerized on both sides of the
ψG pseudogene start (data not shown). Only the 11.0–>12.2 and
>>12.2 kb signals identified fragments long enough to encompass IES 156ψG-11 with any certainty. The 5.1–6.7 kb signal
identified fragments ending far upstream of the ψG pseudogene. The 8.0–9.7 kb signal identified fragments ending close
to the ψG pseudogene start. However, size estimation from the
Southern blot analysis was not precise enough to ensure that
some of these fragments did not end immediately upstream of
the ψG pseudogene start, within IES 156ψG-11 itself. No
11.0–>12.2 and >>12.2 kb signals were detected within DNAs
156/8–14, indicating that these DNAs lack a significant level
of ψG pseudogene within their macronuclear genome. In
contrast, DNAs 156/1–7 and 156/15 displayed 11.0–>12.2 and
>>12.2 kb signals. In these DNAs, ratios of the 2.8 kb signal to
the 11.0–>12.2 and >>12.2 kb signals were >1:0.10 (Table 1, A).
Further analysis showed that a ratio of 1:0.10 defined a ψG
pseudogene macronuclear content >80n (data not shown).
Therefore, at least 8 of the 15 DNAs (DNAs 156/1–7 and 156/15)
harbored a ψG pseudogene macronuclear content large enough
to analyze IES 156ψG-11 excision.
No ∼300 bp PCR product was detected for DNAs 156/1–7,
156/9, 156/11–12 and 156/15 (Fig. 2B); instead, a PCR
product of ∼330 bp was detected. In the case of DNAs 156/9
and 156/11–12, the lack of IES-deleted fragments could be
accounted for by a lack of ψG pseudogene macronuclear
copies. However, this cannot be the case for DNAs 156/1–7
and 156/15, since these DNAs harbored a ψG pseudogene
macronuclear content >80n. In contrast to the PCRs performed
on DNAs 156/1–7, 156/9, 156/11–12 and 156/15, the PCRs
performed on DNAs 156/8, 156/10 and 156/13–14 generated
both ∼300 and ∼330 bp PCR products. These were quantified.
The ∼300 bp PCR product that identified IES 156ψG-11deleted fragments accounted for 2–15% of the total PCR products (Table 1, A). These fragments harbored a canonical
excision junction that corresponded to one of the IES flanking
5′-TA-3′ dinucleotides (Fig. 1B). Although the more sophisticated techniques available for quantitative PCR could have
provided a better estimation of the relative amount of
IES-deleted and IES-retaining fragments, the classical PCR
technique used here clearly established that IES 156ψG-11
was sporadically excised.
Together, these data demonstrate that IES 156ψG-11 is a
partially defective IES, characterized by a low excision
efficiency.
IES 156ψG-11 has an efficiently excised allelic counterpart
To identify the factor(s) responsible for non-efficient IES
156ψG-11 excision, we searched for an IES allelic variant
4658 Nucleic Acids Research, 2001, Vol. 29, No. 22
Table 1. IES 156ψ-11 excision efficiency from micronuclear genomes and
ψG pseudogene amplification level within macronuclear genomes of (A)
cultures from P.primaurelia strain 156, (B) F1 progeny arising from matings
between strains 156 and 168 and (C) F2 progeny arising from F1 autogamy
(only F2 progeny harboring the ψG pseudogene 156 sequence are shown)
A
B
C
Culture
ψG pseudogene content
in macronuclear genome
IES 156ψG-11
excision efficiency
156/3
0.27
0
156/4
0.40
0
156/5
0.43
0
156/6
0.20
0
156/7
0.31
0
156/8
0
0.02a
156/9
0
0
156/10
0
0.06a
156/11
0
0
156/12
0
0
156/13
0
0.06a
156/14
0
0.15a
156/15
0.10
0
156/1
0.25
0
156/2
0.24
0
156/18
0.05
0.05
156/19
0.11
0.13
F1 /3
0.22
0.10
F1 /4
0.30
0.22
F1 /7
0.13
0.23
F1 /8
0.18
0.09
F1 /11
0.12
0.33
F1 /12
0.10
0.36
F1 /15
0.18
0
F1 /16
0.11
0
Mean
0.17
0.17
Standard deviation
0.07
0.14
F1 /1
0.45
0.10
F1 /2
0.33
0.16
F1 /5
0.45
0.24
F1 /6
0.39
0.38
F1 /9
0.37
0.15
F1 /10
0.36
0.11
F1 /13
0.32
0.43
F1 /14
0.26
0.14
Mean
0.35
0.21
Standard deviation
0.06
0.13
F1 mean
0.19
F1 standard deviation
0.13
F2 /510
0.37
0.02
F2 /513
0.66
0
F2 /514
0.34
0
F2 /515
0.40
0
F2 /520
0.45
0
F2 /79
0.33
0
F2 /228
0.28
0.03
F2 /23
0.46
0
F2 /28
0.32
0.01
Mean
0.40
0.01
Standard deviation
0.12
0.01
displaying high excision efficiency. We cloned and sequenced
IES 168ψG-11 from P.primaurelia strain 168 (Fig. 1C) and
analyzed the DNA from four cell cultures. PCRs were
performed and analyzed as above using oligonucleotides A and
B, which are common to the 156 and 168 alleles (Fig. 2A). A
unique signal of ∼300 bp was revealed in all PCRs (Fig. 2B).
No ∼330 bp fragment, which would have been produced from
IES 168ψG-11-harboring macronuclear molecules, was identified. This demonstrates that IES 168ψG-11 is excised from
∼100% of the macronuclear DNA molecules.
IESs 156ψG-11 and 168ψG-11 display allele-specific
excision efficiencies
The excision efficiencies of IESs 156ψG-11 and 168ψG-11
were characterized in F 1 progeny arising from crosses between
strains 156 and 168. Conjugation in ciliates is a reciprocal
process which results in the formation of a genetically identical zygotic nucleus in each of the two parental cells. Each
parent then divides into two karyonidal cells, each of which
harbors an independently developed macronuclear genome. Of
the four progeny arising from an interstrain cross, two harbor
macronuclear genomes formed in the context of the 156 parent
and two harbor macronuclear genomes formed in the context
of the 168 parent. In the case where a trans-acting factor(s)
encoded by the 156 parental genome determines non-efficient
IES 156ψG-11 excision, IESs 156ψG-11 and 168ψG-11
should be non-efficiently excised in progeny developed within
parent 156 and efficiently excised in progeny developed within
parent 168. Where a trans-acting factor(s) encoded by the
zygotic genome determines non-efficient IES 156ψG-11 excision, the excision of both IESs 156ψG-11 and 168ψG-11
should be efficient (or both non-efficient, depending on which
factor-encoding allele is recessive), whatever the parent in
which the progeny genomes have developed. In contrast, in the
case where a cis-acting factor(s) determines non-efficient IES
156ψG-11 excision, this non-efficient excision should be allele
specific and occur in progeny from both parents.
We studied the DNA from 16 progeny obtained from four
interstrain matings. In a preliminary step, we determined the
ψG pseudogene to G gene macronuclear content as above.
Probing BglII-restricted DNAs from progeny F1/1–16 and
from parents 156 and 168 with fragment BS revealed the 2.8 kb
signal that is specific for the G locus and the 5.1–7.1, 7.1–10.2
and >11.2 kb signals that are specific for the ψG locus (Fig. 3B).
The >11.2 kb signal identified macronuclear molecules ending
>3.3 kb downstream of the ψG pseudogene start (see Fig. 4).
The ratio of the 2.8 to >11.2 kb signal ranged within the
interval 1:0.10–1:0.45 (Table 1B). This indicated that DNAs
from F1 progeny displayed a ψG pseudogene macronuclear
IES 156 ψ-11 excision efficiency is given in percent. It was established by
quantifying the IES 156 ψ-11-deleted PCR products relative to whole PCR
products. The ψG pseudogene macronuclear content is quantified relative to
the G gene macronuclear content. A ratio of 1:0.10 defines a ψG pseudogene
macronuclear content >80n.
aNote that in the case of DNAs 156/8, 156/10 and 156/13–14, quantification
of the IES 156 ψ-11-deleted PCR products did not allow calculation of IES
156 ψ-11 excision efficiency because the ratio of the micronuclear to macronuclear DNA was <<80n.
Nucleic Acids Research, 2001, Vol. 29, No. 22 4659
Figure 5. Analysis of the ψG pseudogene 156 and 168 sequences in F1 progeny.
Genomic DNAs were restricted with EcoRI and HindIII, separated by electrophoresis on a 0.8% agarose gel and hybridized with probe HX derived from the
5′-side of the ψG pseudogene (see Figs 1A and 4).
content >80n. In addition, we determined the levels of the 156
and 168 ψG pseudogene sequences in the F1 macronuclear
genomes. DNAs were restricted with HindIII and EcoRI,
electrophoresed and probed with fragment HX derived from
the IES 156ψG-11 nearby upstream region (see Fig. 1A). The
sequence of fragment HX was common to the 156 and 168
sequences with the exception of one nucleotide position.
Signals of 0.5 and 0.6 kb were revealed, which identified the
156 and 168 ψG pseudogene sequences, respectively (Fig. 5).
Both signals were quantified (data not shown). Although
sequence 168 displayed a short amplification advantage over
sequence 156, both were nearly equal within macronuclear
genomes (41 ± 7 versus 59 ± 9%). Finally, the analysis of F2
progeny showed that the 156 and 168 sequences were similarly
fragmented on both sides of the ψG pseudogene (see below).
These data indicate that DNA from all the F1 progeny harbored
a macronuclear content of the 156 and 168 ψG pseudogene
sequences large enough to allow analysis of IES 156ψG-11
and 168ψG-11 excision.
To determine the excision efficiency of IESs 156ψG-11 and
168ψG-11 in the F1 progeny, PCRs were done using oligonucleotides C and D, which are common to the 156 and 168
alleles. These oligonucleotides generated PCR products that
encompassed the HindIII and FokI restriction site polymorphisms of the 156 and 168 sequences. Oligonucleotide C
annealed 519 and 520 bp upstream of IESs 168ψG-11 and
156ψG-11, respectively; oligonucleotide D annealed 440 bp
downstream of them. Control PCRs were performed on the
same cloned inserts as above (the ψG pseudogene micronuclear version derived from strain 156 and the ψG pseudogene macronuclear copy derived from strain 168). The PCR
products were then restricted with HindIII and FokI, electrophoresed and hybridized with the internal oligonucleotide A.
PCR products of 698 and 742 bp were expected for the 156ψG11-deleted and 156ψG-11-harboring fragments, respectively,
and PCR products of 961 and 1005 bp for the 168ψG-11deleted and 168ψG-11-harboring fragments, respectively.
PCR products of ∼700 and ∼750 bp were detected in all F1
DNAs except for DNAs F1/15–16, which lacked PCR products
of ∼700 bp (Fig. 6). A PCR product of ∼1 kb that could identify
IES 168ψG-11-deleted or IES 168ψG-11-harboring DNA
fragments was detected in all DNAs. An analysis of the PCR
products generated in other PCRs indicated that the PCR products of ∼1 kb identified IES 168ψG-11-deleted fragments (data
not shown). No signal of ∼800 or ∼850 bp, which would have
attested to recombination between the FokI and HindIII restriction sites and thereby recombination between the flanking
sequences of IESs 156ψG-11 and 168ψG-11, was detected,
except perhaps in DNA F1/7.
The size difference (44 bp) between IES 156ψG-11-deleted
(962 bp) and IES 156ψG-11-retaining (1006 bp) templates is
<5%. In our hands, PCRs performed with two mixed cloned
inserts of ∼1 kb that have an ∼5% size difference generate PCR
products with similar efficiency (7). Therefore, quantification
of the restriction fragments of ∼700 and ∼750 bp was
performed and the relative proportion of IES 156ψG-11deleted templates was calculated for each DNA (Table 1B).
IES 156ψG-11 excision efficiency was similar between
macronuclear genomes developed within 156 (17 ± 14%) and
168 parents (21 ± 13%).
In summary, IESs 156ψG-11 and 168ψG-11 retained their
parental pattern of excision in F1 progeny, IES 156ψG-11 excision
efficiency remaining far lower (19 ± 13%) than IES 168ψG-11
excision efficiency (∼100%). Nevertheless, IES 156ψG-11 excision efficiency from the F1 progeny was significantly stimulated,
compared to that from cultures of the 156 parental strain (2 ± 4%).
Stimulation of IES 156ψG-11 excision efficiency did not exhibit
any parent dependence (17 ± 14 versus 21 ± 13%), thereby
excluding the possibility that it had been caused by a strainspecific trans-acting maternal factor(s). Altogether, these data
demonstrate that a cis-acting determinant(s) is responsible for
non-efficient IES 156ψG-11 excision and that a trans-acting
factor encoded by the zygotic genome participates in the control
of IES 156ψG-11 excision efficiency.
Figure 6. Excision efficiency from IESs 156ψG-11 and 168 ψG-11 in F 1 progeny. The PCR products were restricted with FokI and HindIII, which identified restriction site polymorphisms between the 156 and 168 sequences, separated on a 1.5% agarose gel and hybridized with oligonucleotide A. The upper PCR products
identified IES 168 ψG-11-deleted fragments. The intermediate PCR products identified IES 156ψG-11-harboring fragments. The lower PCR products identified
IES 156 ψG-11-deleted fragments. The nature of the minor PCR product indicated by the black dot is not known. Control PCRs were as in Figure 2.
4660 Nucleic Acids Research, 2001, Vol. 29, No. 22
IES 156ψG-11 displays different excision efficiencies
between F1 and F2 progeny
To characterize the zygotic trans-acting factor(s) that participates in the control of IES excision, we have investigated the
genetic linkage of the locus encoding this factor(s) to the
ψG-11 site. This was done by characterizing the excision efficiency from IESs 156ψG-11 and 168ψG-11 in F2 progeny
derived from F1 autogamy. The zygotic nuclear genome that
forms during autogamy results from fusion of two daughter
nuclei arising from mitosis of one haploid nucleus. As a consequence, the genome of the F2 autogamous progeny is entirely
homozygous.
We studied the DNA of 30 progeny arising from autogamy
of four F1 progeny. The DNAs were first typed for the ψG
pseudogene allele by looking for the HindIII restriction site,
which maps ∼350 bp upstream of IES 156ψG-11 (data not
shown). Nine progeny harbored the 156 allele and 21 progeny
the 168 allele. The DNAs were then analyzed for ψG pseudogene macronuclear content as above (data not shown). All
DNAs exhibited signals identifying G and ψG sequences, the
ratios of which were >1:0.10 (ratios from progeny harboring
the ψG pseudogene 156 allele are shown in Table 1C). Therefore, all DNAs harbored a ψG pseudogene macronuclear
content large enough to analyze IES 156ψG-11 and 168ψG-11
excision. The DNAs were then used to perform PCRs with
oligonucleotides A and B (Fig. 2C). An ∼300 bp PCR product
was generated from the DNA of all 21 progeny harboring the
168 allele. No ∼330 bp PCR product was identified. This indicated an ∼100% excision efficiency for IES 168ψG-11. In
contrast, almost no PCR product of ∼300 bp was produced
from the DNA of the nine progeny harboring the 156 allele. In
these DNAs a PCR product of ∼330 bp was mostly or exclusively generated. Both PCR products were quantified and an
IES 156ψG-11 excision efficiency of 0.6 ± 1.1% was established (Table 1, C).
These data confirm that a cis-acting determinant(s) is
responsible for non-efficient IES 156ψG-11 excision. Furthermore, they indicate that if another locus than the ψG locus
constantly participates in the control of IES 156ψG-11
excision, this locus is closely linked to the ψG locus (see
Discussion).
DISCUSSION
Non-efficient IES excision
Macronucleus differentiation in ciliates involves the excision
of IESs that are eventually eliminated. In a previous study we
characterized six IESs from the G gene of P.primaurelia strain
156 and their six paralogs from a ψG pseudogene. Comparison
of the micronuclear version of the G gene and ψG pseudogene
to one of their macronuclear copies showed that one of the
12 IESs, IES 156ψG-11, was defective for excision.
Here we have provided an en masse analysis of IES 156ψG-11
excision efficiency. By analyzing 15 independent cultures
from strain 156, we have shown that IES 156ψG-11 excision is
non-efficient rather than null. Having identified an efficiently
excised IES 156ψG-11 allelic variant, we have performed a
genetic analysis of the determinant(s) responsible for nonefficient IES 156ψG-11 excision. We have established that
non-efficient excision of IES 156ψG-11 does not result from
epigenetic control involving maternal factors. Indeed, efficient
IES 156ψG-11 excision never occurs within F1 progeny, even
for those that developed within 168 parents. Conversely, efficient IES 168ψG-11 excision occurs in all F1 progeny, even
those that developed within 156 parents. Furthermore, only
efficient IES 168ψG-11 excision and non-efficient IES
156ψG-11 excision occur within F2 progeny. Together, these
data demonstrate that a defective cis-acting determinant(s) is
responsible for non-efficient IES 156ψG-11 excision in strain
156.
Until now, Paramecium IESs have been assumed to display
an all-or-nothing excision pattern, although most of them were
characterized by comparison of a micronuclear DNA version
with a unique macronuclear copy. En masse analysis of
Paramecium IES excision has only shown sporadically
retained IESs (13). Further, the view of an all-or-nothing excision pattern was strengthened by characterization of mutant
IESs that were fully defective for excision rather than partially
defective. Our data are the first demonstration that IES
excision may be a non-efficient process and that IES excision
efficiency is controlled. Since IES insertion is frequent within
genes, the control of IES excision efficiency constitutes a way
of regulating the number of transcription templates in the
highly polyploid macronuclear genomes of ciliates.
The cis-determinant(s) of IES excision efficiency
A recent analysis of a series of molecules injected into
developing macronuclei of P.tetraurelia has shown that DNA
flanking the 28 bp 51A2591 IES is necessary for excision of
this IES (10). Sequences flanking allelic IESs 156ψG-11 and
168ψG-11 are >99% identical; the first nucleotide differences
outside these IESs map 165 bp upstream and 335 bp downstream of their left and right boundaries, respectively. In the
case where non-efficient excision of IES 156ψG-11 is caused
by an external cis-acting determinant(s), the above nucleotide
positions would be candidates for defining them. However,
given the distances that separate these nucleotide positions and
the IES ends, it seems more plausible that differences in IESs
156ψG-11 and 168ψG-11 excision efficiency result from
differences in the IES 156ψG-11 and 168ψG-11 sequences.
IESs 156ψG-11 and 168ψG-11 are identical except at right end
positions 8 and 9 (see Fig. 1B and C). A difference in right end
position 8 by itself could be responsible for the difference in
IES excision efficiency since IES position 8 displays a nucleotide
preference, an indication of its importance for IES excision (9).
Only left position 8 of IES 168ψG-11 fits the preferred
nucleotide. Alternatively, mutations at both left positions 8
and 9 could account for non-efficient IES 156ψG-11 excision.
This hypothesis would agree with the observation that the
paralogous IES 156G-11, the right end of which is identical to
that of IES 168ψG-11, is also efficiently excised (Fig. 1D). If
differences in IESs 156ψG-11 and 168ψG-11 excision efficiency result from differences in the IES 156ψG-11 and
168ψG-11 sequences, our data provide the first functional
demonstration of the importance of IES nucleotide positions 8
and 9 for IES excision.
The zygotic determinant(s) of IES excision
F1 and F2 progeny display a 30-fold difference in IES 156ψG-11
excision efficiency. This is true whether F1 progeny develop
from 156 or 168 parents. A cut at one end of IES 168ψG-11
Nucleic Acids Research, 2001, Vol. 29, No. 22 4661
could generate a 3′-OH terminus able to trans-attack one end
of IES 156ψG-11 and thereby trigger an enhancement of IES
156ψG-11 excision efficiency. However, the joining of IESs
156ψG-11 and 168ψG-11 flanking sequences should have produced 156 and 168 chimeric macronuclear DNA molecules.
No such chimeric molecules were detected, indicating that
enhancement of IES 156ψG-11 excision efficiency did not
result from IES 156ψG-11 and 168ψG-11 trans-excision. On
the other hand, difference in IES 156ψG-11 excision efficiency
between F1 and F2 progeny cannot be the consequence of a
difference in the amplification of IES 156ψG-11-deleted and
IES 156ψG-11-retaining molecules: IES 156ψG-11-retaining
and IES 168ψG-11-deleted allelic molecules were similarly
amplified within the macronuclear genome of the F2 progeny.
Rather, the difference in IES 156ψG-11 excision efficiency
between F1 and F2 progeny must be a consequence of a transacting factor differently provided by the zygotic genome in
autogamy and conjugation. A conjugation-specific factor(s)
could be responsible for the enhanced excision efficiency of
IES 156ψG-11 within F1 progeny (the F2 progeny arose from
autogamy). Alternatively, IES 156ψG-11 excision efficiency
enhancement in F1 progeny may result from the action of a
factor encoded by the 168 allele of a locus closely linked to the
ψG locus. Although this could occur by chance, it seems more
plausible that IES 156ψG-11 excision efficiency enhancement
in F1 progeny results from cross-talk between the 156 and
168 ψG pseudogene sequences. Cross-talk between alleles is
known to modulate many different mechanisms, including
gene expression in Drosophila, chromatin structure and meiotic
double-strand break frequency in Saccharomyces cerevisiae
(14,15) and transposon excision in plants (16). Homologous
chromosome cross-talk may involve indirect physical interactions via soluble factors or direct physical interactions.
Somatic pairing of the homologous chromosomes regularly
occurs in the developing macronuclei of some hypotrichous
ciliates. Although no such pairing has ever been characterized
in the holotrichous ciliates Paramecium and Tetrahymena, the
characterization of recombinant DNA molecules within the
developing macronuclear genome of Tetrahymena attests to
physical interactions between homologous chromosomes in
their genomes (17).
IES excision and chromosome fragmentation
In Paramecium, programmed chromosome fragmentation
usually occurs within DNA domains spaced by a few kilobases.
We have shown here that four fragmentation domains map
over the ψG locus: one domain maps 1.0–2.6 kb upstream of
the ψG pseudogene start, two domains within the ψG pseudogene and the last downstream of its 3′-end. A large difference
in the use of these four DNA domains between karyonides
must account for the previously reported large variation in the
ψG pseudogene macronuclear content (11). Determinants of
DNA fragmentation are unknown in Paramecium. Although
the G and ψG paralogous loci have arisen from duplication of
an ancestral genomic locus, their patterns of fragmentation are
highly distinct. None of the four fragmentation DNA domains
located around the ψG pseudogene has a paralog around the G
gene, except that lying at the ψG pseudogene 3′-end, the
paralog of which is non-efficiently used (13). Macronuclear
molecules that end within this paralog retain a 67 bp IES at
their ends, or close to them. This has led to the proposal that
chromosome fragmentation and IES excision could be alternative DNA rearrangements: the retained IESs would act as a
cis-acting determinant for fragmentation by either providing
accurate sites to cut or inducing cut sites in their flanking
sequences. In that hypothesis, the non-efficiently excised IES
156ψG-11 could act as a cis-acting determinant for DNA fragmentation. However, IES 156ψG-11 is retained not only on
macronuclear molecules ending close to it, but also on those
ending much further downstream. Furthermore, the 168
sequence displays the same DNA fragmentation pattern as the
156 sequence, although IES 168ψG-11 is fully excised. Therefore, IES 156ψG-11 does not appear to cis-determine any fragmentation event.
ACKNOWLEDGEMENTS
We thank Rick Willet and Anne Baroin for correcting the
manuscript. This work was supported by the Centre National
de la Recherche Scientifique (grant no. 97N63/0016), the
Ministère de l’Education Nationale, de la Recherche et de la
Technologie (Programme de Recherche Fondamentale en
Microbiologie et Maladies Infectieuses et Parasitaires), the
Groupement de Recherches et d’Etudes sur les Génomes (grant
no. 22/95) and the Association pour la Recherche sur le Cancer
(grant no. 1374). K.D. was the recipient of a doctoral fellowship from the Association pour la Recherche sur le Cancer.
REFERENCES
1. Prescott,D.M. (2000) Genome gymnastics: unique modes of DNA
evolution and processing in ciliates. Nature Rev. Genet., 1, 191–198.
2. Klobutcher,L.A. and Herrick,G. (1997) Developmental genome
reorganization in ciliated protozoa: the transposon link. Prog. Nucleic
Acid Res. Mol. Biol., 56, 1–62.
3. Betermier,M., Duharcourt,S., Seitz,H. and Meyer,E. (2000) Timing of
developmentally programmed excision and circularization of
Paramecium internal eliminated sequences. Mol. Cell. Biol., 20,
1553–1561.
4. Duharcourt,S., Butler,A. and Meyer,E. (1995) Epigenetic self-regulation
of developmental excision of an internal eliminated sequence on
Paramecium tetraurelia. Genes Dev., 9, 2065–2077.
5. Duharcourt,S., Keller,A.M. and Meyer,E. (1998) Homology-dependent
maternal inhibition of developmental excision of internal eliminated
sequences in Paramecium tetraurelia. Mol. Cell. Biol., 18, 7075–7085.
6. Mayer,K.M. and Forney,J.D. (1999) A mutation in the flanking 5 ′-TA-3′
dinucleotide prevents excision of an internal eliminated sequence from the
Paramecium tetraurelia genome. Genetics, 151, 597–604.
7. Dubrana,K., Le Mouel,A. and Amar,L. (1997) Deletion endpoint allelespecificity in the developmentally regulated elimination of an internal
sequence (IES) in Paramecium. Nucleic Acids Res., 25, 2448–2454.
8. Mayer,K.M., Mikami,K. and Forney,J.D. (1998) A mutation in
Paramecium tetraurelia reveals functional and structural features of
developmentally excised DNA elements. Genetics, 148, 139–149.
9. Klobutcher,L.A. and Herrick,G. (1995) Consensus inverted terminal
repeat sequence of Paramecium IESs: resemblance to termini of Tc1related and Euplotes Tec transposons. Nucleic Acids Res., 23, 2006–2013.
10. Ku,M., Mayer,K. and Forney,J.D. (2000) Developmentally regulated
excision of a 28-base-pair sequence from the Paramecium genome
requires flanking DNA. Mol. Cell. Biol., 20, 8390–8396.
11. Dubrana,K. and Amar,L. (2000) Programmed DNA under-amplification
in Paramecium primaurelia. Chromosoma, 109, 460–466.
12. Caron,F. (1992) A high degree of macronuclear chromosome
polymorphism is generated by variable DNA rearrangements in
Paramecium primaurelia during macronuclear differentiation. J. Mol.
Biol., 225, 661–678.
13. Amar,L. (1994) Chromosome end formation and internal sequence
elimination as alternative genomic rearrangements in the ciliate Paramecium.
J. Mol. Biol., 236, 421–426. [Erratum, 1997, J. Mol. Biol., 265, 465.]
4662 Nucleic Acids Research, 2001, Vol. 29, No. 22
14. Xu,L. and Kleckner,N. (1995) Sequence non-specific double-strand breaks
and interhomolog interactions prior to double-strand break formation at a
meiotic recombination hot spot in yeast. EMBO J., 14, 5115–5128.
15. Rocco,V. and Nicolas,A. (1996) Sensing of DNA non-homology lowers
the initiation of meiotic recombination in yeast. Genes Cells, 1, 645–661.
16. van Houwelingen,A., Souer,E., Mol,J. and Koes,R. (1999) Epigenetic
interactions among three dTph1 transposons in two homologous
chromosomes activate a new excision-repair mechanism in petunia.
Plant Cell, 11, 1319–1336.
17. Deak,J.C. and Doerder,F.P. (1998) High frequency intragenic recombination
during macronuclear development in Tetrahymena thermophila restores the
wild-type SerH1 gene. Genetics, 148, 1109–1115.