From the Centre de Recherche en Infectiologie, Centre de Recherche
du CHUL et Département de Biologie Médicale, Faculté
de Médecine, Université Laval,
Québec G1V 4G2, Canada
Received for publication, October 3, 2000, and in revised form, January 3, 2001
We have used a telomere-associated chromosome
fragmentation strategy to induce internal chromosome-specific breakage
of Leishmania chromosomes. The integration of telomeric
repeats from the kinetoplastid Trypanosoma brucei into
defined positions of the Leishmania genome by homologous
recombination can induce chromosome breakage accompanied by the
deletion of the chromosomal part that is distal to the site of the
break. The cloned telomeric DNA at the end of the truncated chromosomes
is functional and it can seed the formation of new telomeric repeats.
We found that genome ploidy is often altered upon telomere-mediated
chromosome fragmentation events resulting in large chromosomal
deletions. In most cases diploidy is either preserved, or partial
trisomic cells are observed, but interestingly we report here the
generation of partial haploid mutants in this diploid organism. Partial
haploid Leishmania mutants should facilitate studies on the
function of chromosome-assigned genes. We also present several lines of
evidence for the presence of sequences involved in chromosome mitotic
stability and segregation during cell cycle in this parasitic
protozoan. Telomere-directed chromosome fragmentation studies in
Leishmania may constitute a useful tool to assay for
centromere function.
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INTRODUCTION |
Leishmania is a kinetoplastid protozoan parasite that
is endemic in several parts of the world and is responsible for
considerable mortality and morbidity. The structure of
Leishmania chromosomes is comparable to many other protozoa
with a central core of single copy or tandemly arrayed genes and a
large number of subtelomeric and telomeric repeats at chromosome ends
(1-4). The physical map of the Leishmania major
Friedlin genome has been recently established by cosmid fingerprinting
(5) and the smallest Leishmania chromosome has been
sequenced (4). The sequence of the rest of the parasite genome is
progressing steadily with almost half of it present in the data
bases. In eukaryotes, the functional elements known to be
essential for segregation and transmission of chromosomes during cell
division are origins of replication, centromeres, and telomeres.
Although the structure-function and organization of the telomeres are
relatively well known in Leishmania (3, 6) the presence of
centromere and origins of replication are not yet established in any
protozoan parasite. In Kinetoplastidae, telomeres are composed of
tandem repeated copies of a 5'-TTAGGG-3' hexamer organized in arrays of
variable length in which the guanine-rich strand runs 5'-3' toward the
chromosome end (7-10), hence resembling human telomeres (11). A
telomerase activity has been recently described in kinetoplastid
parasitic protozoa including Trypanosoma brucei, L. major, and Leishmania tarentolae (12). A
sequence that could resemble an autonomous replication origin has been documented in T. brucei (13). Artificial minichromosomes
lacking centromere-like sequences have been constructed in trypanosomes (14, 15). However, these minichromosomes were unstable and they were
rapidly lost when grown in the absence of selective pressure.
Modification of eukaryotic genomes by homologous targeting constitutes
an important tool toward understanding genome structure and function.
Several approaches including targeted gene disruption in Chinese
hamster ovary cells (16) and embryonic stem cells (17),
transposon insertions (18), rare cutting restriction endonucleases
(19), the Cre recombination system (20), telomere-directed chromosome
breakage (21-29), and the construction of yeast (30) and mammalian
(31, 32) artificial chromosomes have been used in the last decade to
manipulate eukaryotic genomes and assessing function and structure of
several chromosomes within these organisms. In Leishmania,
gene targeting is mediated almost exclusively by homologous
recombination and it has been widely used during the last decade to
study the function of individual genes (33-38). The use of homologous
recombination to generate gross alterations within the
Leishmania genome has not been explored yet.
We report here a telomere-mediated chromosome fragmentation approach to
engineer large chromosomal truncations at defined positions within the
Leishmania genome by homologous recombination. Our studies
show that cloned telomeric and subtelomeric repeats of the
kinetoplastid protozoan T. brucei are capable of inducing a
chromosome-specific breakage and the seeding of new telomeres. Using
this strategy we have addressed chromosome mitotic stability during
cell division in this kinetoplastid parasitic protozoan and have
generated large haploid deletions for facilitating functional studies
in this diploid organism.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
L. tarentolae
TarII and Leishmania donovani donovani MHOM/IN/80/DD8
strains have been described previously (39, 40). Cells were grown in
SDM-79 medium supplemented with 10% fetal bovine serum (Multicell,
Wisent Inc.) and 5 µg/ml hemin. Approximately ~4 µg of linearized
DNA fragments derived from the different targeting constructs were used
to transfect Leishmania cells by electroporation as
described (41). Transfectants were selected with 40 µg/ml G418
(Geneticin, Life Technologies, Inc.) or 160 µg/ml hygromycin B
(CalBiochem) on SDM-agar plates (1%). Colonies resistant to G418
and/or to hygromycin B were isolated after 10-15 days of growth at
29 °C.
Construction of Chromosome Fragmentation Vectors--
The
pTRYNEO-TEL and pPTR1YNEO-TEL fragmentation vectors (see Fig. 1) were
made as follows. The 1.8 kb1
PstI-EcoRI fragment (TEL) from vector pT4 (7)
containing 50 perfect tandem telomeric repeats of the hexamer
5'-CCCTAA-3' plus a ~1.5 kb of subtelomeric repeats of T. brucei was first cloned into the PstI-EcoRI
sites of pSP72 (Promega). Then, the NEO gene cloned
downstream of a 92-bp synthetic polypyrimidine stretch necessary for
the maturation of NEO transcript was extracted as a
SmaI-EcoRV from vector pSPYNEO (42) and inserted
into the EcoRV site of the vector. Vector pYNEO-TEL was
further digested with BglII and ligated either to a 1.2-kb
BamHI-BglII fragment carrying the L. donovani trypanothione reductase (TR) gene or to a
400-bp BamHI-BglII fragment containing the second
half of the L. tarentolae PTR1 gene to yield pTRYNEO-TEL and
pPTR1YNEO-TEL, respectively. The fragmentation vector pTR
NEO
-TEL
(Fig. 1) was constructed following three cloning steps. First, to
change the orientation of the (TG)n repeats, the 1.8-kb
fragment containing the telomeric and subtelomeric repeats of T. brucei was extracted by PvuII-EcoRV and
re-introduced into the EcoRV site of pSP72. Then the
L. donovani TR gene was introduced into the BamHI
site of this vector as a BamHI-BglII fragment.
Finally, the
NEO
expression cassette isolated from vector
pSL1180-
NEO
(43) as a EcoRV-SmaI fragment
was inserted into the EcoRV site of the above vector.
Maturation of the NEO transcript in this cassette is
undertaken by the intergenic region of the
-tubulin gene (
IR or
) (44). To construct the fragmentation vector pPTR1
NEO
-TEL
(see Fig. 1), we have replaced the TR gene in
pTR
NEO
-TEL by the L. tarentolae PTR1 gene derived from
vector pSPY21 in which the PTR1 coding sequence made by
polymerase chain reaction was cloned into the
EcoRI-BglII sites of pSP72. The PTR1
gene in pTR
NEO
-TEL was cloned as a
SmaI-SmaI fragment into the
XbaI-ApaI sites of the vector filled in with
Klenow DNA polymerase I. To construct vector pYHYG-SceI, the
18-bp recognition sequence of the yeast endonuclease I-SceI
was first introduced into the KpnI site of pSP72 and then
the hygromycin phosphotransferase gene (HYG) derived from
pSPYHYG (42) was cloned as a BamHI-BglII fragment
into the BamHI site of this vector. The I-SceI
recognition site was made as described by Tamar et al. (45).
Expression of the HYG gene in vector pYHYG-SceI
is driven by a synthetic polypyrimidine stretch of 92 bp including an
AG-spliced acceptor site (42). To make vector
pPTR1YHYG-SceI, the BamHI-BglII
HYG-SceI expression cassette was introduced into the unique
BamHI site of the L. tarentolae PTR1 gene, part
of vector p40.8 (41). To target sequence X located at 3.9 kb upstream
of the L. donovani TR gene, the
BamHI-BglII HYG-SceI cassette treated
with Klenow DNA polymerase I was introduced into the ClaI
site (filled in with Klenow) of a 7-kb XhoI fragment, part
of cosmid E7-2 (36), previously cloned into the XhoI site of
vector pSP72. To construct vector pB562YHYG-SceI, a 6.8-kb BamHI-BamHI fragment derived from cosmid B5-62
mapped on L. donovani infantum chromosome 5 (45) was first
cloned into the BamHI site of pGEM7Zf (Promega). The
BamHI-BglII HYG-SceI expression
cassette was then introduced into the unique BglII site of
this 6.8-kb fragment.
DNA Analysis--
Total genomic DNA of Leishmania was
prepared as described (47). Intact Leishmania chromosomes
were prepared from cells harvested during mid- to late log-phase,
washed, and lysed in situ in 1% low melting agarose plugs
as previously described (46). I-SceI digestion was performed
as described (45). Intact and I-SceI-digested Leishmania chromosomes were migrated through 1% ultrapure
agarose (Bio-Rad) gels in 1 × TBE using a Bio-Rad CHEF-DR III
apparatus at 5.9 V/cm, 120° separation angle and switch times varying
from 35 to 120 s for 25-30 h. Southern blot, hybridization, and
washings were done following standard procedures (48). The PTR1-, TR-, NEO-, and HYG-specific probes used in these studies were made by
polymerase chain reaction. A PGPA-specific probe was made as described
(49). A probe containing more than 50 tandem telomeric repeats of the
hexamer 5'-CCCTAA-3' and a ~1.5 kb of subtelomeric sequences of
T. brucei (TEL) isolated from plasmid pT4 (7) was used in
this study. A ~500-bp polymerase chain reaction fragment corresponding to the subtelomeric repeat of T. brucei was
used as a probe to distinguish between endogenous and cloned telomeric DNA. Probe B562 corresponds to a 6.8-kb
BamHI-BamHI fragment derived from cosmid B562
mapped on L. donovani infantum chromosome 5 (45). Probe X
corresponds to a 3.9-kb XhoI-ClaI fragment
located upstream of the L. donovani TR gene. Probe Y
corresponds to a 1.7-kb BamHI-HindIII fragment
located at ~10 kb downstream of the L. tarentolae PTR1 gene and it is part of vector pMAC40 (39). To evaluate genome ploidy in
the different transfectants subjected to chromosome fragmentation
events (see Figs. 8 and 9), we have quantified hybridization intensities of different chromosome-specific genes by densitometric analysis of the Southern blots and filters using a PhosphorImager with
the ImageQuant 3.1 software. Internal standards corresponding to single
copy Leishmania genes were used for this analysis.
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RESULTS |
Leishmania Chromosome Fragmentation Vectors--
The ability to
engineer large chromosomal alterations by gene targeting mediated by
homologous recombination in Leishmania cells would be useful
for analyzing chromosome function. To generate a defined chromosomal
breakage at internal genomic sites, we have therefore constructed a
series of chromosome fragmentation vectors that could direct a cloned
telomeric DNA to a specific position within the Leishmania
genome. All these fragmentation constructs are acentric (centromeres
have not been characterized yet in protozoan parasites) and contain
cloned telomeric DNA from T. brucei (7), the neomycin
phosphotransferase (NEO) marker for selection and a region
of homology to allow targeted integration into the parasite genome by
homologous recombination. The direction of chromosome breakage is
dependent on the orientation of telomeric repeats in the fragmentation
vectors, which are cloned in a 5' to 3' TTAGGG orientation to serve as
a template for the telomerase. The cloned telomeric DNA is part of a
1.8-kb fragment derived from the kinetoplastid protozoan parasite
T. brucei that contains 300 bp of the telomeric hexamer
repeat (TTAGGG)n and 1.5 kb of subtelomeric sequences (7) (Fig.
1). The maturation of the NEO
transcript in these vectors is driven either by a synthetic stretch of
90 pyrimidines with an AG-spliced acceptor site (Y) (42) or from the
intergenic region of the
-tubulin gene (
) (44) (see Fig. 1). In
all cases, the NEO gene is oriented toward the cloned
telomeric DNA to be better expressed as already reported for T. brucei artificial minichromosomes (14, 15). We have chosen to
target two distinct loci, the trypanothione reductase (TR)
gene of L. donovani (36, 50) mapped on chromosome 5 and the
L. tarentolae pteridine reductase 1 gene (PTR1),
part of the H locus (41, 51) mapped on chromosome 23. The fragmentation
vectors were designed in such a way to delete sequences from both parts
of the chromosome breakage site depending on the 5'-3' orientation of
the guanine-rich strand with respect to each chromosomal end (see Fig.
1).

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Fig. 1.
Schematic representation of chromosome
fragmentation vectors to induce a defined telomere-mediated chromosome
breakage within the Leishmania genome. A 1.8-kb
fragment from T. brucei harboring 50 copies of the
5'-TTAGGG-3' hexamer and 1.5 kb of subtelomeric sequences (black
box) was cloned at the end of these vectors. The 5' to 3'
orientation of the G-rich strand of the telomeric DNA is indicated by
an arrow. PTRYNEO-TEL and pTR NEO -TEL constructs were
made to induce a chromosome breakage at the level of the trypanothione
reductase (TR) locus of L. donovani yielding
different fragmentation products depending on the orientation of the
telomeric repeats. pPTR1YNEO-TEL and pPTR1 NEO -TEL constructs were
made to induce a break at the level of L. tarentolae PTR1
locus. The neomycin phosphotransferase gene (NEO) is cloned
in the same direction as the G-rich strand of the telomeric DNA.
IR corresponds to the intergenic region of the
-tubulin gene important for transcript maturation in
Leishmania (44). Y corresponds to a 92-nucleotide
synthetic polypyrimidine stretch with an AG-spliced acceptor site known
to provide the necessary signals for transcript maturation in this
parasite (42).
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Telomere-directed Chromosome Fragmentation in Leishmania--
We
first introduced the cloned telomeric DNA into the L. donovani
TR gene on chromosome 5 by transfecting pTRYNEO-TEL and pTR
NEO
-TEL linear fragmentation vectors (see Fig. 1) to delete either one or the other arm of chromosome 5 flanking the TR
target locus. The chromosomal position of the TR gene has
been estimated at 120 and 400 kb from the telomeres using an in
vitro chromosome fragmentation approach mediated by the
endonuclease I-SceI (45). The outcome of a fragmentation
event depends upon the orientation of the stretch of targeting DNA
along the Leishmania chromosome. If the TR gene
is oriented toward the long 400-kb arm of the chromosome, the
fragmentation event using the pTRYNEO-TEL linearized construct (Fig. 1)
should produce a 400-kb truncated chromosome hybridizing to the NEO
probe (Fig. 2A). If present in
the opposite orientation, the same construct should generate a
truncated chromosomal fragment of 120 kb (Fig. 2A).
Hybridization of Leishmania chromosomes resolved by CHEF
electrophoresis with a TR probe indicated a specific breakage at the
level of the TR locus and the generation of a 400-kb
truncated chromosome in the transfectants (Fig. 2A). This
engineered minichromosome hybridized to the NEO probe (Fig.
2A) strongly suggesting that the TR gene is
oriented toward the right telomere of chromosome 5. Only one allele has
been targeted upon G418 selection hence explaining the hybridization of
the intact 520-kb chromosomal allele with the TR probe in the
transfectant, as the Leishmania genome is diploid (Fig.
2A).

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Fig. 2.
Telomere-directed fragmentation of L. donovani chromosome 5 (520 kb). A, schematic
representation of the breakage of chromosome 5 upon integration of the
pTRYNEO-TEL vector at the level of the TR locus.
Asterisk, the localization of the TR gene within
this chromosome was established by in vitro fragmentation
studies (45). Depending on the orientation of the TR gene,
we could either generate a 400-kb truncated chromosome with the cloned
telomeres at the left end or a 120-kb chromosomal fragment
with the vector telomeres at the right end. In both cases,
the 120- and 400-kb fragments lacking telomeres at one of their ends
should also be generated. The obtained scenario is encircled by an
open box. The subtelomeric and telomeric repeats in the
fragmentation vectors are represented by the black box and
their orientation is indicated by an arrow. Cloned telomeres
integrated into the parasite genome are indicated by an open
arrow and pre-existent genomic telomeric repeats by a closed
arrow. Leishmania chromosomes were separated by CHEF
electrophoresis, transferred onto nylon membranes, and hybridized to
the TR and NEO probes. Lanes 1, L. donovani
wild-type; 2, L. donovani subjected to chromosome
fragmentation using the pTRYNEO-TEL vector. B,
telomere-mediated chromosome fragmentation to maintain the 120-kb
region of chromosome 5 using the pTR NEO -TEL vector in which
telomeric DNA was correctly oriented to induce a different
fragmentation event. Lane 2 corresponds to L. donovani transfected with pTR NEO -TEL vector.
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Once we determined the orientation of the TR gene on
chromosome 5, we made a fragmentation vector to generate a 120-kb
truncated chromosome. Upon the homologous integration of
pTR
NEO
-TEL vector into the TR locus, a specific
chromosome breakage has occurred, which resulted in a truncated 120-kb
chromosomal fragment hybridizing to the TR and NEO probes (Fig.
2B). Similar experiments were carried out with the second
target locus, the PTR1 gene, on chromosome 23 of L. tarentolae. The chromosomal position of PTR1 gene was estimated by the endonuclease I-SceI-mediated chromosome
fragmentation approach to be at 140 and 650 kb from the telomeres (45).
Homologous integration of the pPTR1
NEO
-TEL (Fig. 1) linearized
vector into the PTR1 locus resulted in chromosome
fragmentation as shown in the filter hybridization of Fig.
3A. The generation of a 650-kb truncated chromosomal fragment hybridizing to PTR1 and NEO probes suggests that the PTR1 gene is oriented toward the long
650-kb arm of chromosome 23 (Fig. 3A). As expected, the
integration of the pPTR1YNEO-TEL vector generated a 140-kb fragment
hybridizing to the NEO probe (Fig. 3B). Hence, our results
indicate that the integration of cloned telomeric DNA from T. brucei into defined internal chromosomal locations within the
Leishmania genome has produced specific chromosome breakage
and the generation of truncated minichromosomes carrying telomeric
repeats at both sides (Figs. 2 and 3). All the fragmentation vectors
used for the above studies carry a single region of homology from 0.4 to 1.2 kb and once linearized they generate one double-strand break
that could be used for targeting by homologous recombination. Targeting
most likely occurred through homologous recombination by a single
crossover event at the level of the targeted genomic sequence (Figs. 2
and 3). The T. brucei telomeric DNA did not integrate into
pre-existing internal telomeric repeats that could possibly surround
the TR and PTR1 loci, as suggested from sequence
data (data not shown).

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Fig. 3.
Fragmentation of L. tarentolae
chromosome 23 (790 kb) upon integration of cloned telomeric
repeats at the level of the PTR1 locus.
A, schematic representation of the breakage of chromosome 23 upon homologous integration of the pPTR1 NEO -TEL vector.
Asterisk, the chromosomal localization of the
PTR1 gene was established by the I-SceI in
vitro fragmentation approach (45). Depending on the orientation of
the PTR1 gene within the chromosome, we could either
generate a 650-kb truncated chromosome with the cloned telomeres at the
right end or a 140-kb chromosomal fragment with the vector
telomeres at the left end. The orientation of the cloned
telomeric DNA is as indicated in Fig. 2. CHEF blot hybridization of
Leishmania chromosomes with the PTR1 and NEO probes
(lower panel). Lanes 1, L. donovani
wild-type; 2, L. donovani subjected to chromosome
fragmentation using the pPTR1 NEO -TEL vector. B,
telomere-mediated chromosome fragmentation to maintain the 140-kb
region of chromosome 23 using the pPTR1YNEO-TEL vector. Lane
2 corresponds to L. donovani cells subjected to
chromosome fragmentation upon integration of vector
pPTR1YNEO-TEL.
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De Novo Formation of Telomeres in the Truncated Leishmania
Chromosomes--
Our results presented in Figs. 2 and 3 show that
linear fragmentation vectors carrying cloned telomeres at one end, once
integrated by homologous recombination into the targeted loci, can
induce defined chromosomal truncations and generate chromosomal
fragments with added telomeres at the break site. To determine whether
the cloned T. brucei telomeric DNA can seed the formation of
a new telomere, the length and heterogeneity of the terminal
restriction genomic fragments in the different classes of transfectants
(Figs. 2 and 3) were examined. Restriction fragments of known size that harbor either the T. brucei cloned 1.8-kb telomeric and
subtelomeric repeats (Fig. 4,
A and B) or the telomeric DNA together with the NEO marker and/or the targeted gene (Figs. 4,
A-C, and 5) were compared by
Southern blot hybridization. In all cases, the length of the telomeric
repeats at the end of the truncated chromosomal fragments was higher
than the one in the original fragmentation vectors extended from few
hundred to more than 3,600 nucleotides depending on the transfectant
and on the fragmentation event (Figs. 4 and 5). Indeed, XbaI
genomic fragments of 12 kb (Fig. 4A) and 9.5 kb (Fig.
4B), and a PstI 3-kb fragment (Fig.
4C) have hybridized to a NEO probe instead of the expected
10.5-, 8.0-, and 2.5-kb fragments, respectively (lower
panel, Fig. 4, A-C). Moreover, Southern blot
hybridization of the transfected clones digested with enzymes that
delimit the cloned telomeres using a telomere-specific probe indicated
fragments which were smaller in length than the average telomere size
observed in wild-type cells (Fig. 4, A-C). These results
suggest that the cloned telomeric DNA can be used as a substrate for
further telomere elongation upon chromosome truncations. Although
hybridization of the ClaI-digested fragments derived from
clones 1 to 3 with a telomere-specific probe has revealed a set of
heterogeneously sized fragments (Fig. 4, A and B), hybridization of the XbaI digests of the same
clones also harboring the telomeric repeats to the gene-specific TR,
PTR1 and NEO probes indicated a relatively discreet pattern, especially for clone 1 (Fig. 4, A and B). Further
hybridization of the same blots to a T. brucei subtelomeric
repeat probe which can distinguish added telomeres at the end of the
truncated chromosomes from the endogenous ones (Fig. 4, A-C)
showed that only the bands at the range of 3.8 kb (Fig. 4A)
and 3-3.4 kb (Fig. 4B) were recognized (data not shown)
hence supporting the results obtained with the gene-specific probes.
The remaining small telomere-hybridizing fragments correspond most
likely to spontaneously shortened telomeres derived from various
chromosomes (see "Discussion"). Similarly, no high variation in the
length of the de novo seeded telomeres was observed while
analyzing clones derived from the same transfectant (Fig. 5A
and data not shown). The lack of highly heterogeneous profile in the
length of the de novo seeded telomeres in clones 1 to 4 is
discussed below (see "Discussion").

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Fig. 4.
Cloned telomeres of T. brucei
can seed the formation of a new telomere in Leishmania
upon their integration at internal genomic positions.
A, Southern blot hybridization of L. donovani
wild-type (wt) and of clones (1 and 2)
transfected with the fragmentation vector pTRYNEO-TEL (see Fig. 1). The
genomic DNA of these strains was digested with XbaI
(X) and ClaI (C), respectively,
transferred onto nylon membranes and hybridized to the TR-, NEO-, and
telomere (TEL)- specific probes. B-C, Southern
blot hybridization of L. tarentolae wild-type and clones
(3 and 4) transfected independently with
fragmentation vectors pPTR1 NEO -TEL and pPTR1YNEO-TEL. The genomic
DNA of these strains was digested with XbaI (X),
ClaI (C), or PstI (P),
transferred onto nylon membranes and hybridized with PTR1, NEO, and TEL
probes. The lower panels represent schematic drawings of the
wild-type and targeted alleles in these three classes of transfectants
with relevant restriction sites and the size of the expected
restriction fragments.
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Fig. 5.
Growth of newly seeded telomeres in
Leishmania truncated chromosomes generated upon
telomere-mediated chromosome fragmentation. A, Southern
blot hybridization of L. tarentolae transfected with the
fragmentation vector pPTR1 NEO -TEL. Different clones
(1-5) were selected at mid-log phase, digested with
KpnI (K), transferred onto nylon a membrane and
hybridized with a NEO probe. B, L. donovani clone 1 transfected with the fragmentation vector pTR NEO -TEL and grown at
different stages within the parasite cell cycle. Genomic DNA extracted
from the different growth stages (log phase, mid-log, early
stationary, and late stationary) was digested with
XbaI (X) and transferred onto nylon filter for
hybridization with the NEO probe. Lower panels in
A and B indicate the position of the relevant
restriction sites at the ends of the truncated chromosomes.
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Telomere size can change depending upon environmental or developmental
conditions (for review, see Ref. 52). We have therefore investigated
whether the size of the newly seeded telomeres in the transfectants
varies throughout the parasite's cell cycle. L. donovani
pTR
NEO
-TEL cells harboring a truncated 120-kb chromosome (Fig.
2B) were selected at log-, mid-log, early stationary, and late stationary phase and their DNA was digested with XbaI
and analyzed by Southern hybridization using a NEO probe. The length of
the newly seeded telomeres was significantly increased by ~2 kb
when parasites were grown at early stationary phase and shortened back
to normal size when cells stopped dividing (Fig. 5B). The differences in size observed between the endogenous telomeres and the
construct-associated telomeres, the use of the cloned telomeres as
a template for telomere elongation as suggested from the systematic
increase in the length of telomeric repeats upon genomic integration
and chromosome breakage, and the demonstration that the cloned
telomeres are dynamic structures presenting an increase or a decrease
in length in response to culture conditions support that the latter
have seeded the formation of a new telomere at the site of the
chromosome break (Figs. 4 and 5).
Mitotic Stability of Truncated Chromosomes in Leishmania
Transfectants upon Telomere-associated Chromosome
Fragmentation--
So far, telomere-associated chromosome
fragmentation in yeast and in mammalian somatic cells was used mainly
for mapping studies and for generating artificial chromosomes to assay
centromere function (for reviews, see Refs. 31 and 53). To assess the mitotic stability of Leishmania-truncated chromosomes during
cell division, a clone from each individual transfectant (Figs. 2 and 3) was subdivided in two subclones, one maintained with G418 selection and the other grown for several generations in the absence of drug
selection. The stability of the truncated chromosomes was addressed by
pulsed field electrophoresis and hybridization studies. The integration
of the pTRYNEO-TEL fragmentation vector has induced a chromosome
breakage at the level of the TR locus resulting in two
chromosomal fragments, one of 400 kb containing the cloned telomeres at
the left end and the other of 120 kb with no telomeres at the right end
(Figs. 2A and 6A).
The 120-kb fragment was lost very rapidly as even after the first
passage (~8-10 generations) it was not detectable in the cells as
indicated by hybridization studies with probe X located upstream of the
TR gene (Fig. 6A). This was an expected result as
broken chromosomes without telomeres are often very unstable (53, 54).
The 400-kb truncated chromosomal fragment with functional telomeres at
both termini was, as anticipated, maintained in the presence of G418
selection. When cells were grown in the absence of drug pressure for
more than 200 passages (at least 1800 generations), the stability of
this 400-kb chromosomal fragment was not affected (Fig. 6A).
The extremely high fidelity of segregation of this truncated chromosome
strongly suggests that sequences required for maintenance and
segregation should be present within this part of chromosome 5. To
assess whether any chromosomal fragment harboring functional telomeric
repeats at both ends can also be stably maintained during the parasite cell division, we have analyzed the mitotic stability of the 120-kb truncated chromosome generated upon integration of vector pTR
NEO
into TR locus. This chromosomal fragment was maintained as
far as the G418 selection was present but, in contrast to what seen with the 400-kb fragment (Fig. 6A), it was rapidly lost upon
removal of the drug pressure (Fig. 6B). The 400-kb
chromosomal fragment without telomeric repeats at one chromosome end,
generated following fragmentation in this transfectant, was also lost
as confirmed by hybridization with probe B562 (Fig. 6B).
These data further support the presence of sequences important for
mitotic stability only within the long 400-kb arm of chromosome 5 (Fig.
6A).

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Fig. 6.
Mitotic stability of truncated L. donovani chromosomes generated upon telomere-mediated
chromosome fragmentation. A, integration of the
fragmentation vector pTRYNEO-TEL into the TR locus of
L. donovani chromosome 5 and stability of the resulted
truncated chromosomal 400- and 120-kb fragments. Rev A
corresponds to a transfectant with the 400-kb truncated chromosome
grown in the absence of selective pressure for long periods of time
(lanes 1-3 correspond to cells grown for 1, 2, and 3 years). CHEF blot of L. donovani wild-type (wt)
and RevA chromosomes hybridized to the TR probe (lower
panel). Genomic DNA derived from clone 1 (c1) of
L. donovani/pTRYNEO-TEL transfectant was hybridized after
transfer with probe X (see "Experimental Procedures") to confirm
the loss of the 120-kb chromosomal fragment. Asterisk, the
position of a putative centromere (CEN) is arbitrary.
B, integration of the fragmentation vector pTR NEO -TEL
into the TR locus and mitotic stability of the resulted
truncated chromosomal fragments. RevB corresponds to a
transfectant with the 120-kb truncated chromosome grown in the absence
of selective pressure for few passages (lanes 2 and
3). Lane 1 corresponds to the initial
transfectant prior to reversion. CHEF blot hybridization of L. donovani wt and RevB with TR probe (lower panel).
c2 represents clone 2 of L. donovani/pTR NEO -TEL transfectant hybridized to the B562
probe (see "Experimental Procedures") to confirm the loss of the
400-kb fragment.
|
|
We have further extended our studies to other chromosomes and
Leishmania species using the same telomere-mediated
chromosome fragmentation approach. We generated new truncated
chromosomal fragments derived from chromosome 23 of L. tarentolae and tested their mitotic stability in the absence of
drug selection. The integration of PTR1
NEO
-TEL cassette at the
PTR1 locus produced a 650-kb truncated chromosome with the
cloned telomeres at the right end, which was maintained in the presence
of G418 selection (Figs. 3 and
7A) and an unstable 140-kb
fragment missing telomeres at one end that was rapidly lost as shown by
hybridization studies using a telomere-specific probe (Fig.
7A). The 650-kb fragment, part of chromosome 23, has been
stably inheriting from cell to cell for more than 70 passages in
culture without any drug pressure (Fig. 7A). This suggests
that sequences important for mitotic stability are likely to be present
within this 650-kb fragment. The possibility that the remaining 140-kb
fragment of chromosome 23 might also contain such sequences was
examined by assessing its mitotic stability during a number of cell
divisions. However, this fragment was not maintained further in the
cell upon the removal of the drug as shown by hybridization studies
with the PTR1 probe (Fig. 7B). Moreover, the 140-kb fragment
was initially present in multiple copies (~5-7) (Fig. 7B
and data not shown) that were rapidly lost by culturing the parasite in
the absence of drug selection for ~10 passages. Chromosomal
instability is often associated, among other things, with gene
amplification (55, 56). These results indicate that specific parts of
Leishmania-truncated minichromosomes demonstrate a high
mitotic stability that ensures their retention and segregation during
cell division suggesting the presence of "centromere-like"
sequences within these chromosomes. Our results also show that when
these "centric" fragments lack telomeric repeats at one of their
termini they cannot be healed by the addition of new telomeres as has
been seen in other systems (see "Discussion").

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Fig. 7.
Mitotic stability of truncated L. tarentolae chromosomes generated by telomere-mediated
chromosome fragmentation. A, integration of the
fragmentation vector pPTR1 NEO -TEL into the PTR1 locus
of L. tarentolae chromosome 23 and stability of the resulted
truncated chromosomal fragments of 650 and 140 kb. RevC corresponds to
cells harboring the 650-kb truncated chromosome grown without selective
pressure for several passages (in order 10, 20, 40, 60, and 70). CHEF
blot of L. tarentolae wild-type and RevC chromosomes
hybridized to the PTR1 probe (right panel). Lane
3 corresponds to L. tarentolae/pPTR1 NEO -TEL
transfectant hybridized, after transfer, with a telomere-specific probe
(TEL) to confirm the loss of the 140-kb fragment. B,
integration of the fragmentation vector pPTR1YNEO-TEL and stability of
the resulted truncated chromosomal fragments. RevD
corresponds to cells harboring the 140-kb truncated chromosome grown
without selective pressure for few passages (in order 3, 5, 6, 10, and
15). The 140-kb chromosomal fragment was present in more than one copy
in the initial transfectant. CHEF blot of L. tarentolae wt
and RevD chromosomes hybridized to the PTR1 probe (right
panel). Lane 4 represents a clone of L. tarentolae/pPTR1YNEO-TEL transfectant hybridized with probe Y (see
"Experimental Procedures") to confirm the loss of the 650-kb
fragment.
|
|
Hybridization of CHEF and Southern blots of selected clones derived
from different transfectants subjected to chromosome fragmentation (Figs. 2 and 3) to a number of single copy chromosome-specific probes
suggest that the truncated chromosomes with the seeded telomeric DNA
are present at one copy per cell with the only exception of the 140-kb
truncated fragment of chromosome 23 shown to be present in multiple
copies (Fig. 7B and data not shown). The presence of the
stably inherited truncated chromosomal fragments at one copy per cell
further supports the possibility that they harbor "centromere-like" sequences.
Effects of a Telomere-mediated Chromosome Fragmentation on Parasite
Genome Ploidy: Generation of Partial Leishmania Haploid
Mutants--
In other eukaryotic systems, gross genomic alterations
upon chromosome fragmentation events often produce aneuploid cells (57). We have tested whether a similar situation is taking place in
Leishmania transfectants subjected to a telomere-associated chromosome fragmentation. Two approaches were used to assess ploidy in
these transfectants. These include an hybridization with a number of
single copy genes flanking the site of chromosome breakage combined to
a PhosphorImager analysis and an enzymatic approach based on the
genomic integration of the endonuclease I-SceI unique site
at regions surrounding chromosome truncations followed by in
vitro chromosome digestion permitting evaluation of the copy number of chromosomal alleles. These studies were done in transfectants grown in the absence of drug selection for several passages,
represented here as revertants (Rev), that have in principle maintained
or lost truncated chromosomal fragments depending on the experiment. We
have first examined whether chromosome fragmentation in pTRYNEO-TEL transfectant has yielded a partial haploid strain containing only one
copy of the 120-kb fragment of chromosome 5 (Fig.
8A, left panel). Comparative
hybridization of a PstI digest between pTRYNEO-TEL RevA and
wild-type using probe X suggested that this sequence is present at one
copy in the transfectant (see Fig. 8A, middle panel). By
hybridizing the same DNA with a TR probe, we depicted two fragments of
3.4 and 2.4 kb corresponding to the gene copies present in the intact
chromosomal allele and in the targeted allele, respectively (Fig.
8A, middle panel). The hybridization intensities of the
various digests suggest that sequence X in the deleted 120-kb fragment,
is present at one copy (Fig. 8A, middle panel). These
results were confirmed by PhosphorImager analysis using the
PTR1 single copy gene probe as an internal standard (legend of Fig. 8). Integration of the I-SceI recognition sequence
into sequence X just upstream of the TR locus and further
digestion with the I-SceI endonuclease resulted in two
fragments of 400 and 120 kb hybridizing to probe X (Fig. 8A,
right panel). Since we did not detect an additional band at 520 kb, we concluded that only one intact allele of chromosome 5 is present
in this mutant, which further supports the partial haploid genotype of
this strain. In RevB strain, we anticipate the presence of one allele
for chromosome 5 as both truncated chromosomal fragments were lost
after the removal of drug selection (Fig. 8B, left panel).
However, Southern blot hybridization using the single copy gene probes,
TR and B562, demonstrated the presence of two alleles for
this chromosome (Fig. 8B, right panel), which was further
confirmed by PhosphorImager analysis using the PTR1 single
copy gene probe as an internal standard (legend of Fig. 8). Moreover,
integration of the endonuclease I-SceI into sequence B562
(further downstream of the TR gene) of RevB strain and
further enzymatic digestion of chromosome 5 indicated that in addition
to the fragmented allele, one intact allele was also present (Fig.
8B, middle panel). The remaining intact allele is not due to
a partial I-SceI digestion as confirmed by the hybridization
with the HYG probe (Fig. 8B, middle panel).

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Fig. 8.
Alterations in the genome ploidy of L. donovani transfectants harboring large chromosomal deletions
as a result of experimentally induced chromosome fragmentation
events. A, the HYG-SceI cassette was
inserted by homologous recombination into sequence X of the RevA strain
(lane 1) (expected to be haploid for the 120-kb chromosomal
fragment; left panel) (see also Fig. 6A) and
chromosomes were digested with I-SceI endonuclease
(lane 3), resolved by CHEF electrophoresis and hybridized to
X and HYG probes (right panel). The genomic DNA of L. donovani wild-type (lane 1) and RevA (lane
2) was digested with PstI (P), transferred
onto nylon membranes and hybridized to X, TR, and
PTR1 probes (middle panel). The copy number of
TR (lanes 1/2: 62371/34942, for 3.4-kb
band) and X (lanes 1/2: 63242/25932)
genes was also confirmed by PhosphorImager analysis using the
PTR1 (lanes 1/2: 32921/35314) gene
(single copy gene) as an internal standard. Values between parentheses
are those measured by PhosphorImager analysis. B, the
HYG-SceI cassette was inserted into B562 locus of the RevB
strain (lane 4) (expected to be haploid for the whole
chromosome 5 after the loss of the 400- and 120-kb fragments)
(left panel; see also Fig. 6B) and chromosomes
were digested with I-SceI nuclease (lane 5 accounts for the whole transfectant population and lane 6 for a clone), resolved by CHEF electrophoresis and hybridized to B562
and HYG probes (middle panel). Southern blot analysis of
L. donovani wild-type (lane 7) and RevB
(lane 8) genomic DNA digested with PstI and
hybridized to TR, B562, and PTR1 probes (right panel). The
copy number of the TR (lanes 7/8:
15911/14418) and B562 (lanes 7/8:
19073/17931) gene probes was also confirmed by PhosphorImager analysis
using PTR1 (lanes 7/8: 15268/14666) as an
internal standard. Values between parentheses are those measured by
PhosphorImager (see "Experimental Procedures").
|
|
Similar studies were undertaken to assess ploidy in the different
transfectants following telomere-mediated fragmentation of chromosome
23. Genomic integration of the PTR1
NEO
-TEL cassette and growth of
the transfectants without G418 selection should result in a partial
haploid strain with one intact chromosomal allele and one truncated
allele of 650 kb (Fig. 7A). Comparative Southern blot
hybridization of RevC genomic DNA with the chromosome-specific PTR1 and PGPA probes indicated the presence of a
second intact allele of chromosome 23 to compensate for the deletion of
the 140-kb chromosomal fragment resulting hence in a partial trisomic strain (Fig. 9A, left panel).
Indeed, three copies of the PTR1 gene, two as part of a
9.8-kb fragment derived from the intact chromosomal alleles and the
third included in a 6.2-kb fragment derived from the targeted allele,
and two copies of the PGPA gene were detected by
hybridization (Fig. 9A, middle panel). Partial aneuploidy in
RevC was confirmed by PhosphorImager analysis using the
TR single copy gene probe as an internal standard (legend of
Fig. 9). In RevD, the 140-kb chromosomal fragment was lost after growth
in the absence of drug selection (Fig. 7B) and this should
yield a transfectant haploid for chromosome 23. I-SceI-mediated chromosome fragmentation and hybridization
with a PTR1 probe indicated the presence of a second allele for
chromosome 23, in addition to the one targeted and digested with
I-SceI endonuclease (Fig. 9B, right panel). These
data support a chromosome duplication event as seen also in RevB strain
(Fig. 8B).

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Fig. 9.
Alterations in the genome ploidy of L. tarentolae transfectants carrying large chromosomal
deletions mediated by experimentally induced chromosome fragmentation
events. A, RevC is expected to be partial haploid for
the 140-kb part of chromosome 23 (left panel) (see also Fig.
7A). Southern blot of L. tarentolae wild-type
(lane 1) and RevC (lane 2) genomic DNA digested
with XbaI (X)-BglII (Bg)
and hybridized to the PTR1 probe. Southern blot of L. tarentolae wild-type (lane 3) and RevC (lane
4) genomic DNA digested with HindIII and hybridized to
PGPA probe (right panel). The copy number of the
PGPA (lanes 3/4: 3215/4023) and
PTR1 (lanes 1/2: 14124/12854, for the
9.8-kb band) gene probes was also confirmed by PhosphorImager analysis
using TR (lanes 3/4: 934/870) as a
single copy gene standard. Values between parentheses are those
measured by PhosphorImager (see "Experimental Procedures").
B, integration of the HYG-SceI cassette into the
PTR1 locus of RevD strain (expected to be haploid for
chromosome 23 after the loss of the 650- and 140-kb fragments)
(left panel) (see also Fig. 7B). Chromosomes were
digested with I-SceI endonuclease, resolved by CHEF
electrophoresis, and hybridized to PTR1 and HYG probes (right
panel). The RevD transfectant became diploid for chromosome
23.
|
|
 |
DISCUSSION |
We have constructed a number of linear fragmentation vectors
carrying telomeric DNA at one end and a double-strand break at the
region of homology to induce internal chromosome-specific breakage and
the deletion of the chromosomal part that is distal to the site of
integration. We have shown here that the integration of cloned
telomeric DNA from the kinetoplastid T. brucei into defined
positions of the Leishmania genome can induce chromosomal breakage and seed for the formation of a new telomere. We have also
presented several lines of evidence for the presence of sequences involved in chromosome mitotic stability and segregation in this parasitic protozoan. Moreover, we have examined the maintenance of
ploidy in parasites harboring truncated chromosomes and reported the
generation of a partial haploid mutant for a defined chromosomal region. The use of telomere-mediated chromosome fragmentation to
generate gross alterations and large deletions within the parasite genome has not yet been explored. Targeted breakage of yeast and human
chromosomes mediated by the integration of cloned telomeric DNA has
been successfully reported previously (22-25, 27-29, 53). Introduction of a piece of a cloned telomeric DNA into a yeast or human
chromosome seeds the formation of a new telomere and causes the
chromosome to break (21-23). Gene targeting in Leishmania occurs exclusively by homologous recombination (58, 59). Directed targeting of the linear fragmentation vectors, designed for this study,
into the Leishmania genome has probably occurred by a
single-strand repair model involving the formation of one homologous
junction at the level of the region of homology with one double-strand break recombinogenic free end and another that involves an illegitimate junction at the level of the cloned telomeric DNA. One-sided invasion mechanisms for homologous recombination have been previously described in other systems (60-62). However, we cannot exclude the possibility of a double reciprocal recombination mechanism involving a concatamer of linear vector molecules.
De Novo Telomere Formation upon Chromosome Fragmentation Events in
Leishmania--
Work using cloned human telomeric DNA containing
0.5-1 kb of 5'-TTAGGG-3' repeats can seed telomere formation when
re-introduced into a variety of mammalian cells, including mouse
embryonic stem cells (23, 25, 26). It seems that the mechanism by which correctly oriented (5'-TTAGGG-3')n arrays seed new telomeres involves their recognition and extension by telomerase (63, 64). A
telomerase activity has been recently identified in kinetoplastid parasitic protozoa (12). Here we demonstrate that homologous integration of cloned T. brucei telomeric DNA into defined
positions of the Leishmania genome could provide a substrate
for the elongation reaction, hence seeding the formation of a new
telomere. The significant increase in the length of the cloned
telomeric DNA, its regulation upon culture conditions, and its capacity
of forming functional telomeres that can ensure the stability of the
truncated chromosomes (Figs. 6 and 7) support the formation of new
telomeres with hexamer repeats added during telomere replication (Figs.
4 and 5). The estimated growth rate of the seeded telomeric DNA on
these truncated chromosomes agrees with previous data on the growth of
natural chromosomes in T. brucei (7, 15, 65, 66). As much as 3.5 kb was seen to be added to telomeres of T. brucei in a
process that appears gradual and continuous and was calculated to
result in the addition of 6-10 bp per end per cell division (7, 65, 66). We have obtained similar extension from 0.5 to 3.6 kb upon integration of the cloned telomeric DNA into the Leishmania
genome (Figs. 4 and 5 and data not shown). Although the size range of telomeres in most organisms appears fixed, in some organisms it can
change depending upon environmental or developmental conditions (65,
67). Our studies suggest that telomerase activity in Leishmania is probably higher at early stationary phase of
growth as we have observed a significant increase in the telomere size at this stage of the parasite's cell cycle (Fig. 5B). It is
well known in other systems that telomerase activity is regulated by the cell cycle and that telomere elongation appears to start in late
S-phase coinciding with the action of telomerase (68-70).
Our studies show a lack of highly heterogeneous profile in telomere
length upon telomere seeding (Fig. 4). This could be explained by the
gradual shortening of telomeric DNA in clones 1 to 4 due to the high
number of cell divisions in these transfectants (more than 100 passages). Indeed, comparative hybridization studies between a
transfectant with low number of passages and clone 1 demonstrate a
clear shortening of the telomere length by ~1.5 kb in the latter
strain (data not shown). It has been reported in Tetrahymena
thermophila that in cells subjected to hundreds of cell divisions,
less heterogeneity in telomeric DNA length was observed due to gradual
shortening of telomeric repeats (67). Shorter telomeres grow faster and
can take over in continuous growing cells. Although telomere shortening
has been often associated to aging and to limiting cell proliferation,
recent data support that other factors with the presence or absence of
active telomerase being the critical one could also be involved
(71).
Mitotic Stability of Truncated Leishmania Chromosomes--
For the
stable maintenance and segregation of chromosomes during cell cycle,
functional elements such as telomeres, centromeres, and origins of
replication are essential. In yeast as well in mammalian cells, a
chromosome becomes unstable after loss of a telomere (53, 72).
Normally, the absence of a centromere causes acentric fragments that
are lost immediately during division in a wide variety of organisms
(73, 74). Several reports on telomere-directed chromosome breakage to
produce functional minichromosomes in yeast (22, 30) and in humans (25,
27-29) also support the above requirements for chromosome stability
and none of these studies have detected acentric fragmentation
products. Our studies have shown that in Leishmania,
chromosomal fragments lacking telomeres at one end were lost very
rapidly even when they contained putative sequences ensuring their
retention and segregation during cell division (Figs. 6 and 7). An
acquired telomere from another chromosome by gene conversion was not
seen in our system. Indeed, no telomeric repeats were added de
novo by telomerase onto nontelomeric Leishmania chromosomal truncated fragments. This healing process although relatively rare, has been seen in other systems, such as in
Tetrahymena and human chromosomes (75-77).
The extremely high stability of the 400- and 650-kb truncated
chromosomal fragments, part of chromosomes 5 and 23, respectively, that
have been maintained in the absence of selective pressure for more than
1800 and 700 generations strongly suggests the presence of sequences
that are involved in chromosome replication, retention, and segregation
when the cell divides. All fragmented chromosomal products that lack
these "putative functional sequences" were lost in the absence of
drug selection, even when flanked by functional telomeres (Figs. 6 and
7). In Trypanosomes, artificial minichromosomes of 10 and 13 kb containing a selectable marker, a number of tandemly linked genes,
and telomeric and subtelomeric repeats at both ends have been
constructed (14). However, these were only maintained stably in the
absence of drug selection for ~20 generations. Patnaik and
collaborators (15) have also reported the construction of artificial
minichromosomes for T. brucei by adding telomere and subtelomeric sequences to pT13-11 plasmid known to contain sequences important for its replication (13). These minichromosomes were also
rapidly lost after 7 generations in more than 50% of the transfected
population in the absence of selective pressure (15). These previous
reports support also the thesis that for a chromosome to be mitotically
stable in Leishmania, it should contain additional sequences
likely to act as centromeres. In other systems, if the newly seeded
chromosomal end is associated with a centromere, then a stably
truncated chromosome will be the end result with the displaced acentric
fragment being lost from the cell (31). This is indeed what we have
observed in the case of the 400- and 650-kb fragments of chromosome 5 and 23, respectively (Figs. 6A and 7A). These
truncated centric minichromosomes were stably inherited at one copy in
100% of the cells tested (Figs. 6 and 7, and data not shown). We are
currently working on the 400-kb region of L. donovani
chromosome 5 for identifying the sequences conferring mitotic
stability. Our goal is to use artificially induced chromosome truncations as a mean to define a minimal size chromosome that retains
the mitotic properties of a normal chromosome. The knowledge gained
from such systematic analysis should allow the identification of
components important for proper segregation and replication of
Leishmania chromosomes and the construction of
Leishmania artificial chromosomes to assay for centromere
function as it has been used in other systems (32, 78) and to also
study the regulation of gene expression in these organisms.
Ploidy Alterations in Leishmania Cells Subjected to Targeted
Chromosome Fragmentation--
To address ploidy alterations in cells
submitted to targeted chromosome fragmentation, the karyotypes of the
different transfectants with respect to loss or gain of genetic
material distal or proximal to the sites of break were analyzed by
Southern blot hybridization using a number of chromosome-specific
single copy markers and by CHEF analysis using the
I-SceI in vitro-mediated chromosome fragmentation
approach. Our results indicated that generally large chromosomal
deletions of 400-650 kb or deletion of the whole chromosomal allele
were not permissive (Figs. 8B and 9B) probably
because some of their gene content is essential for parasite survival and needs to be preserved in two copies. The maintenance of ploidy is
essential for the survival of a given species. Mechanisms exist to
ensure that during mitosis daughter cells receive identical sets of
chromosomes. Mechanisms should also exist to ensure maintenance of
ploidy in Leishmania after DNA damage, such as, a
double-strand break generated upon chromosome fragmentation. In the
case where smaller chromosomal deletions of 120-140 kb were generated,
two types of response with respect to ploidy maintenance were obtained. To compensate for the loss of the 140-kb region of chromosome 23, the
parasite became trisomic with two intact chromosomal copies in addition
to the 650-kb truncated fragment (Fig. 9A). The stable maintenance of the 650-kb truncated chromosome despite the fact that
the cell is diploid for chromosome 23 is striking. Aneuploidy is often
the result of abnormal chromosome segregation and trisomy is common in
human embryos and plays a major role in human health problems (79). It
has long been known that most tumors are aneuploid as a result of
chromosomal instability (80). Although aneuploidy was observed to
compensate for the loss of the 140-kb region of chromosome 23, this was
not the case, however, when an equivalent in size deletion (120 kb) was
generated for chromosome 5 (Figs. 8A and 9A). In
the latter case, we have obtained a partial haploid strain for
chromosome 5. It is interesting to notice that this partial haploid
mutant showed a significant decrease in survival within murine
macrophages compared to wild type cells (data not shown). The
possibility of generating partial haploid parasites upon
telomere-mediated chromosome fragmentation has numerous advantages for
studying genome function in this parasite either by simplifying gene
knockout strategies for this diploid organism and/or for assessing the
function of more than one genes within large chromosomal regions. The
mechanism(s) by which the parasite maintains ploidy upon chromosome
breakage events has not been investigated in the present study.
However, it has been shown that introduction of a chromosome
fragmentation vector into the budding yeast S. cerevisiae has altered the strain's karyotype by a "break copy duplication" mechanism (57). Chromosome duplication occurs both in a centromere distal and in a centromere proximal direction of the fragmentation vectors. We suggest that this mechanism might be important for maintenance of ploidy following a double-strand chromosomal break.
In this study, we have shown that the ability of engineering large
defined chromosomal fragments by homologous targeting of cloned
telomeric DNA into Leishmania has proved useful for defining genomic regions required for replication and segregation of chromosomes during cell division. It has been also helpful for producing
haploid mutants, which could facilitate studies on
parasite genome function. Telomere-mediated chromosome
fragmentation provides a powerful method for manipulating the
Leishmania genome and possibly the genome of other
kinetoplastid parasitic protozoa.
We thank Dr. Piet Borst (Netherlands Cancer
Institute) for providing us with plasmid pT4 containing the T. brucei telomeric and subtelomeric sequences, Dr. Marc Ouellette
for useful suggestions and critical reviewing of this manuscript, and
Carole Dumas for technical assistance.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M009006200
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