Journal of Bacteriology, February 1999, p. 1005-1013, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Family of Telomere-Associated Autonomously Replicating
Sequences and Their Functions in Targeted Recombination in
Hansenula polymorpha DL-1
Jung-Hoon
Sohn,1
Eui-Sung
Choi,1
Hyun Ah
Kang,1
Joon-Shick
Rhee,2 and
Sang-Ki
Rhee1,*
Biotechnology Research Division, Korea
Research Institute of Bioscience and Biotechnology, Yusong, Taejon
305-600,1 and
Department of Biological
Sciences, Korea Advanced Institute of Science and Technology,
Yusong, Taejon 305-701,2 Korea
Received 29 June 1998/Accepted 23 November 1998
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ABSTRACT |
A family of multiple autonomously replicating sequences (ARSs)
which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a
chromosome, as a probe showed several homologues in the genome of
H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif
and ended with 18 to 23 identical repetitions of 5'-GGGTGGCG-3' which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids
containing the newly obtained telomeric ARSs almost exclusively
resulted in the targeted integration of a single copy or multiple
tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment
in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted
of the same ARS motif and highly conserved sequences observed in
HARS36. Even though the frequencies of targeted recombination were
varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through
homologous recombination at the telomeric repeats, which serve as
high-frequency recombination targets.
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INTRODUCTION |
The methylotrophic yeast
Hansenula polymorpha has attracted much attention as a host
for the production of recombinant proteins (for reviews, see references
12 and 20). One of the reasons might be its unusual property of genetic recombination between a
transforming plasmid and the chromosome. The multiple tandem integration of up to 100 copies of a nonlinearized plasmid has been
observed in the chromosome via homologous and nonhomologous recombination (2, 23, 35). Several unusual observations related to the recombination, such as plasmid reorganization by the
insertion of chromosomal DNA (4, 16) and the amplification of a gene copy number after integrative transformation, have also been
reported (14). No report, to date, has been made about the
natural plasmid in H. polymorpha, and as found in other
yeasts, autonomously replicating sequences (ARSs) of H. polymorpha that can act as the replication origin have been
obtained either from its chromosome (4, 35, 40) or other
sources (3, 42). The frequency of transformation was
significantly increased when an ARS element was employed. A
transforming plasmid, even one with an ARS, showed a very low mitotic
stability of less than 5% for 10 generations, but cells with extremely
high mitotic stability could easily be obtained after some dozens of
generations in a nonselective condition, resulting from the integration
of a plasmid into the chromosome (35, 40). During this step,
interestingly, multiple tandem copies of a plasmid are often integrated
into the chromosome. Due to the lack of intensive studies of
H. polymorpha at the molecular level, it is uncertain
whether an episomal plasmid with an ARS can be maintained consistently,
due to its higher frequency of recombination. Furthermore, the
mechanism of multiple tandem integration is also unknown.
From the biotechnological point of view, this characteristic of
H. polymorpha provides a valuable tool for the
development of industrial strains producing recombinant proteins.
Stable maintenance of high copy numbers of gene expression
cassettes could greatly improve the productivity of recombinant
proteins in long-term fermentation. Unfortunately, however, the
frequency of obtaining multiple integrants, up to 100 copies as
described above, appears to be quite low and unpredictable. We
previously suggested the importance of the ARS employed to the
increased frequency of multiple tandem integration (40). An
ARS of Hansenula, HARS36, was obtained from the genome of
H. polymorpha DL-1 by an enrichment procedure to
produce an ARS with characteristics of tandem repeat integration. HARS36 increased the frequency of transformation and multiple tandem
integration of plasmids into chromosomes. In a functional analysis of
HARS36, three important domains for the episomal replication and
integration into chromosomes were identified as regions A, B, and
C. Especially, region C of HARS36 was responsible for the enhanced rate of chromosomal integration (40).
Transformation of a plasmid containing HARS36 and a selection marker
facilitated the selection of an integrant with over 100 tandem copies
of a plasmid in chromosomes (2). Furthermore, the
integration copy number was elaborately controlled by the use of a
dominant selection marker (39). Strikingly, most of the
integration events with a plasmid containing HARS36 appeared to occur
near different ends of chromosomes, suggesting that the origin of
HARS36 related to the ends of chromosomes (39, 40).
Here, we demonstrated that HARS36 is a family of telomeric ARSs
residing in several ends of chromosomes in H. polymorpha. Region C of HARS36, which enhanced chromosomal
integration of a plasmid, was found to be a telomeric repeat of
H. polymorpha that has not been identified to date. Additionally, we also demonstrated that the ends, if not all, of
H. polymorpha chromosomes consisted of sequences
similar to HARS36 and served as high-frequency recombination targets
for a plasmid containing HARS36.
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MATERIALS AND METHODS |
Strains, media, and plasmids.
H. polymorpha DL1-L
(leu2) derived from DL-1 (ATCC 26012) (26) was
kindly provided by M. Y. Beburov (Moscow, Russia). Yeast cells
were grown on yeast extract-peptone-dextrose (YPD) medium (1%
[wt/vol] yeast extract, 2% [wt/vol] Bacto Peptone, and 2% [wt/vol] glucose). Transformants obtained with a plasmid containing LEU2 were selected in a minimal selective medium (0.67%
[wt/vol] yeast nitrogen base without amino acid and 2% [wt/vol]
glucose). Escherichia coli DH5
[F
lacZ
M15 hsdR17 (r m)
gyrA36] was used for the general recombinant DNA
techniques. The plasmid pCE36 containing HARS36 and LEU2 of
H. polymorpha (1, 40) was used as a control
plasmid for the measurement of the transformation frequency and the DNA
source of HARS36. Plasmids pGEM-CE120 and pGE-MARS286 containing
deletion derivatives of HARS36 (40) were used to obtain the
DNA fragments for labeling of hybridization probes. Plasmids
pBluescript KS+ and pBC KS+ (Stratagene, La Jolla, Calif.) were used
for the general DNA cloning and sequencing. Plasmids pBKS-TEL188, -135, and -61 contained one of three telomeric fragments that were obtained
in this study. Plasmids pCTEL188, -135, and -61 were derived from pCHLX
containing H. polymorpha LEU2 (40) after
subcloning of the HindIII-BamHI fragments of
pBKS-TEL188, -135, and -61, respectively.
General DNA techniques.
General DNA manipulations were
performed as described by Sambrook et al. (37). DNA
fragments required for subcloning and labeling experiments were gel
purified with a QiaEx kit (Qiagen, Valencia, Calif.). Total yeast DNA
was isolated with Novozym (Novo Biolabs, Bagsvaerd, Denmark) for
large-scale preparation and with glass beads for small-scale
preparation, as described by Johnston (24). Plasmid DNA
isolation from E. coli was done by the boiling method
described in the work of Sambrook et al. (37). Nucleotide sequencing was carried out by dideoxy sequencing reaction with an ABI
Prism dye terminator cycle sequencing ready reaction kit (Perkin-Elmer
Cetus, Foster City, Calif.), and the sequences were read in an
automatic DNA sequencer (model 373A; Applied Biosystems, Foster City,
Calif.). E. coli was transformed by the Simple and Efficient
Method (22).
Transformation of H. polymorpha and
stabilization of the transformant.
The transformation of
H. polymorpha was performed according to the lithium
acetate-dimethyl sulfoxide method described by Hill et al.
(19). Initial transformants showing heterogeneous colony
sizes due to different mitotic stabilities were stabilized with a
procedure to introduce a transforming plasmid into the chromosome. For
the stabilization, all transformants selected for LEU2
prototrophy in minimal selective medium were pooled with YPD broth and
inoculated into a 500-ml baffled flask containing 50 ml of YPD broth
medium. After 24 h, 1 ml of culture broth was transferred to 50 ml
of fresh YPD medium. This procedure was repeated until the cells had
reached 50 generations. Then, the cells were plated on minimal
selective plates to select for LEU2 prototrophy again.
Complete stabilization was monitored by spreading cells onto minimal
selective plates and checking for an even growth rate, judging from the
sizes of colonies. After 100% stability in the LEU2 phenotype of the
individual stabilized colony was confirmed by the replica plating of
cells on minimal and complex media, the colonies were stored under
selective conditions.
Cloning of telomeric fragments.
For the cloning of telomeric
fragments, total genomic DNA was first treated with T4 DNA polymerase
to fill in the 3' overhangs of chromosome ends, prior to digestion with
PstI. Treatment with T4 DNA polymerase was done in a mixture
of 50 mM Tris-HCl (pH 8.5), 5 mM MgCl2, 5 mM
dithiothreitol, 50 µg of bovine serum albumin per ml, and 0.1 mM
concentrations of deoxynucleoside triphosphates in a 50-µl volume for
1 h at room temperature. After digestion with PstI for
4 h at 37°C, the DNA sample was fractionated by 1% agarose gel
electrophoresis. DNA fragments of 300 to 600 bp isolated from the
agarose gel were ligated to the plasmid pBluescript KS+, which was
linearized with PstI and EcoRV to make libraries of E. coli. Among transformants, 50 to 100 subsets covering
0.5 × 103 to 1 × 103 colonies in
total were tested for hybridization with the P3 probe (Fig.
1). After the screening of subsets,
individual clones were isolated from positive subsets by the same
procedure.
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FIG. 1.
Physical map of HARS36 (A) and genomic Southern blots
(B). Each of the functional elements A, B, and C was described in Sohn
et al. (40). Four different probes, P1 to P4, were
digoxigenin-labeled from different parts of HARS36, as shown on the
map. Probe P1 was labeled from a DNA fragment containing the entire
(base pairs 1 to 1227) HARS36, P2 from nucleotide 239 to 700, P3 from
nucleotide 239 to 523, and P4 from nucleotide 746 to 1227. The
hybridization was performed as described in Materials and Methods. Lane
M, digoxigenin-labeled lambda HindIII size marker (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.6 kb); in other lanes, genomic DNA
digested with EcoRI (lane 1), with HindIII
(lane 2), with XbaI (lane 3), with XhoI (lane 4),
with BamHI (lane 5), and with PstI (lane 6).
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Bal31 digestion.
Thirty micrograms of genomic
DNA was digested at 30°C with 1 U of Bal31 nuclease (New
England Biolabs, Beverly, Mass.) in a total volume of 500 µl of 600 mM NaCl-20 mM Tris-HCl (pH 8.0)-12 mM CaCl2-12 mM
MgCl2-1 mM EDTA. A 3-µg sample was recovered before the
addition of the enzyme and at several time intervals after the enzyme
addition. Ten microliters of 0.25 M EGTA was added to stop the
reaction, and DNA was pelleted with 2 volumes of ethanol. This DNA was
used for further digestion with restriction endonuclease.
Southern hybridization.
Genomic Southern hybridization was
done as described by Sambrook et al. (37). Total chromosomal
DNA was isolated and digested with restriction endonucleases.
After electrophoresis, DNA was capillary transferred onto a nylon
membrane (Schleicher & Schuell GmbH, Dassel, Germany). The labeling of
probe DNA was performed with a non-radioactive DNA labeling and
detection kit (Boehringer Mannheim, Mannheim, Germany).
Digoxigenin labeling of probes P1 and P3 was carried out
with ARS fragments from pCE36 and pGE-MARS286 as templates,
respectively. Probes P2 and P4 were labeled with the
AvaI-KpnI fragment of pCE36 and the
HindIII fragment of pGEM-CE120, respectively. Probes pBC
and LEU2 were made from pBC KS+ (Stratagene) and the
BamHI-XbaI fragment of pCHLX containing
H. polymorpha LEU2 (1), respectively. All
hybridization was carried out at 42°C in a hybridization oven
(Hybaid, Middlesex, United Kingdom) with a hybridization solution (5×
SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1%
[wt/vol] N-lauroylsarcosine, 0.02% [wt/vol] sodium
dodecyl sulfate, 5% [wt/vol] blocking reagent, and 50% [vol/vol]
formamide) as recommended in the manufacturer's instructions.
Nucleotide sequence accession numbers.
The nucleotide
sequences of HARS36, TEL188, TEL135, and TEL61 have been
submitted to the GenBank database under the accession numbers U31858,
U82170, U82171, and U82172, respectively.
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RESULTS |
Genomic origin of HARS36.
Previously, we reported on an
analysis of several functional elements of an ARS of
Hansenula, HARS36 (Fig. 1A), which has the ability to
introduce multiple copies of a plasmid containing this sequence into
the chromosomal end (40). HARS36 has three important
functional elements: regions A and B are required for episomal
replication, and region C is responsible for the high tendency of
integration into a chromosome. The persistent telomeric localization of
the transforming plasmids with HARS36 strongly suggested that HARS36
may also originate from the end of a chromosome (40). To get
some information on the genomic origin of HARS36, we carried out
nucleotide sequencing of region C and found that region C contains 18 copies of a highly regular 8-bp G-rich repeating unit
(5'-GGGTGGCG-3'). The repeating unit resembled the telomeric repeat sequence of several other yeasts and other eukaryotic organisms, as shown in Table 1, supporting the
presumption that HARS36 originated from the end of a chromosome.
For the further analysis of the chromosomal origin of HARS36,
genomic Southern blots were carried out with HARS36 as a probe (Fig.
1B). Surprisingly, more than 10 bands were detected, even though
the signal intensities varied among the bands. Sequence homologues to
HARS36 appeared to exist in multiple copies in the genome, as in the
case of multiple ARS families found near the ends of chromosomes in
Saccharomyces cerevisiae (6). Telomeric and
subtelomeric regions of S. cerevisiae are composed of
conserved sequences such as Y' and X sequences which contain ARSs
(7, 46). Thus, it is conceivable that HARS36 is a family of
ARSs that reside near the ends of chromosomes in H. polymorpha. To determine which part of HARS36 is responsible for
the multiplicity, further genomic Southern blot analyses were done with
three probes, P2, P3, and P4, from different parts of HARS36 (Fig. 1A).
Probe P2, containing both ARS parts (regions B and A) and the repeated sequence (region C) of HARS36, and probe P3, containing only the ARS
part, were used. Probe P4 was labeled from region D of HARS36, which
has no effect on ARS activity after deletion (40). As shown
in Fig. 1B, P2 also hybridized more than 10 chromosomal DNA bands,
which is almost the same number as P1. Interestingly, the P3 probe,
which has no repeat sequence (region C), hybridized with only five
signals strongly detected with P1 and P2. Thus, weak bands detected
with P2 appeared to be detected by the repeat sequences in region C of
HARS36. In contrast to these, probe P4 hybridized with a unique
signal that was also detected with the P1 probe. These results
suggested that the multiplicity of HARS36 in the genome was largely
determined by the sequence between nucleotides 239 and 700 of HARS36
containing the whole ARS motif (40). Several homologues to
the ARS motif containing regions A, B, and C of HARS36 appeared to be
present in the chromosome of H. polymorpha. Furthermore, several additional homologues to region C of HARS36 but
without regions A and B also appeared in the chromosome.
Cloning of a family of multiple telomeric ARSs.
From the
results of the telomeric localization of a transforming plasmid
containing HARS36 (40) and the resemblance of the repeat
sequence of region C to other telomeric repeats, we assumed that the
chromosomal fragments of H. polymorpha hybridized with P2 might contain an identical telomeric repeat sequence, but only five
among them harbored a common ARS element (regions A and B). Depending
on the restriction enzymes used, P3 hybridized with one to five
chromosomal fragments (Fig. 1B). Only a single band of about 450 bp was
detected when genomic DNA was digested with PstI, suggesting
that all five telomeric fragments detected with P3 have a highly
conserved sequence including a PstI site near the end of the
chromosome. To check this, PstI-digested genomic DNA
fragments of 300 to 600 bp, which were hybridized with the probe P3,
were cloned into PstI-digested pBluescript KS+. About 103 colonies were screened with probe P3, but we failed to
get positive clones.
If all five PstI fragments of 450 bp represent the ends of
different chromosomes, an end of each PstI fragment would be
the physical end of a chromosome. Because chromosomes often end with a
3' overhang of about 10 nucleotides (46), the end of
chromosome fragments should be made blunt to be suitable for cloning
into a vector. Therefore, the total genomic DNA was first treated with T4 DNA polymerase and further digested with PstI, and
subsequently DNA fragments of 300 to 600 bp were subcloned into
PstI-EcoRV-digested pBluescript KS+. Of 500 colonies screened, three different DNA inserts were obtained from
Southern blots probed with P3. The sizes of the three inserts varied
from 400 to 450 bp. ARS activities of the three telomeric fragments
(TEL188, -61, and -135) were measured to confirm their nature as ARSs,
as previously described (40). The transformation frequencies
of three plasmids, pCTEL188, -61, and -135, which contain the
respective telomeric fragments in the backbone of pCHLX, were compared
to that of pCHLX as a control plasmid with no ARS activity. ARS
activities of two telomeric fragments, TEL188 and -61, were comparable
to that of HARS36 (40), whereas a small decrease (data not
shown) in ARS activity was observed for TEL135.
DNA sequences of the three different telomeric fragments were analyzed
and compared with that of HARS36 (Fig.
2). All three fragments possessed ARSs
which showed an extremely high number of sequence homologies to HARS36.
The 8-bp G-rich telomeric repeats (5'-GGGTGGCG-3') were
also found, as in region C of HARS36. The only notable differences were
in the junction between the subtelomeric sequence and telomeric
repeats. The numbers of telomeric repeats varied from 18 to 23 in
different telomeric fragments. Although a large part of the
subtelomeric sequence corresponding to a portion of the bent structure
(region B) was absent (40), TEL135 also showed a high
sequence similarity, especially in the ARS core part. The sequence
of TEL188 was completely identical to that of HARS36, even in the
junction, suggesting that TEL188 might originate from the same
end of a chromosome from which HARS36 was obtained. The telomeric
repeats (region C) in HARS36, however, were not at the end but were
flanked by a fragment (region D) whose deduced amino acid
sequence showed a high similarity of over 80% to the middle
domain (amino acids 398 to 593) of DNA polymerase III (1,097 amino
acids) of S. cerevisiae (30) (data not
shown). Furthermore, completely different genomic Southern blot
patterns were obtained with probes P2 and P4 (Fig. 1B). Finally, we
concluded that HARS36 was obtained as a chimeric fragment consisting of
a fragment from the same end of a chromosome from which TEL188 was
obtained and another fragment, the H. polymorpha
homologue of DNA polymerase III of S. cerevisiae obtained
during the construction of the Sau3AI-digested ARS library
(40). Taken together, these results indicated that the
repeat sequence (5'-GGGTGGCG-3') found in region C of HARS36
is the telomeric repeat of H. polymorpha. Furthermore,
the ARS domain of HARS36 is a member of a family of multiple ARSs found
in several, if not all, ends of chromosomes and surrounded with a
highly conserved sequence, at least up to the PstI site.
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FIG. 2.
Comparison of nucleotide sequences of three telomeric
fragments with that of HARS36 (nucleotides 239 to 691). The
DNA-directed bent motif
(AATTA-N7-AATTA-N6-AATTA) and putative ARS core
sequence of HARS36 (40) are boxed. Dashes indicate deleted
or missed sequences. The 8-bp G-rich telomeric repeat sequence and
number of repeats are also shown.
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Recombinational characteristics of three telomeric ARSs.
Previously, it was found that a plasmid containing HARS36
tended to be integrated into several different ends of chromosomes among different transformants. The recombinational characteristics of
three newly cloned ARSs were also tested. Three nonlinearized plasmids,
pCTEL188, -61, and -135, containing a telomeric ARS and H. polymorpha LEU2 as a selection marker were transformed into
H. polymorpha. Each pool of transformants was
completely stabilized to integrate a transforming plasmid into the
chromosomes, as described in Materials and Methods. After
stabilization, 36 individual colonies of each pool grown on minimal
selective plates were randomly selected to check their integration
patterns. Total genomic DNA was isolated from individual colonies and
digested with XbaI. Southern blotting was done with a P3
probe that could hybridize with the five XbaI-digested
genomic fragments of H. polymorpha (Fig. 1). Southern
blots of three sets of 36 integrants with the P3 probe are shown in
Fig. 3. Interestingly, most integrants showed different patterns of five P3 homologues (T1 to T5) with those
found in untransformed cells (Fig. 3, lane C). It indicated that the
patterns of five P3 homologues were modified by the integration of a
transforming plasmid. Judging from the change of Southern blot patterns
in five P3 homologues, we concluded that a transforming plasmid
containing a telomeric ARS integrated into one of the five P3
homologues with a considerably high frequency (72 of 108 integrants
tested).
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FIG. 3.
Integration patterns of transformants with plasmids
pCTEL188, -61, and -135. Three sets of 36 randomly selected integrants
were obtained by transformation with three plasmids, pCTEL188, -61, and
-135, containing a telomeric ARS. All genomic DNAs were digested with
XbaI and probed with P3 (Fig. 1). T1 to T5 are the five P3
homologues of the XbaI-digested genomic DNA of untransformed
H. polymorpha DL-1. The 1.8- and 5.2-kb bands are,
respectively, a common band that appeared when integration occurred
near the end of chromosomes and a band that appeared when integration
occurred in a tandem repeated array of a plasmid.
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If all five P3 homologues were different end fragments of chromosomes,
the possible patterns of integration into five P3 homologues would be
as it is depicted in Fig. 4. In the case
of a single-copy integration into one of five P3 homologues that is
located in the physical ends of the chromosomes, two modifications
might be expected in the XbaI-digested band patterns of P3
homologues. One modification could be the size upshift of about 3.4 kb
in a band corresponding to the homologue, and the other could be the appearance of a new band (1.8 kb) corresponding to the fragment containing H. polymorpha LEU2 and the telomeric
ARS. The size of the new XbaI band should be the same in all
integrants into P3 homologues, because an end of the band is the
physical end of the chromosome. As expected, a new band of about 1.8 kb
is common to all 72 integrants showing a size shift of a band
corresponding to one of the five P3 homologues (Fig. 3). Surprisingly,
the 1.8-kb band is also detected in the integrants without a size shift
of P3 homologues (32 of the remaining 36 integrants). This fact
suggested that the integration also occurred in end fragments other
than the five P3 homologues. Such end fragments were not detected with P3 but were weakly detected with P2 containing the telomeric repeated sequence (Fig. 1B). Small size variations of this 1.8-kb fragment in
the integrants appeared to be caused by the differences in positions of
integration or in numbers of the telomeric repeat. In the case of
tandem integration of a transforming plasmid into a single locus, an
additional 5.2-kb signal would be generated, which corresponds to the
internal copies of the transforming plasmid located inside the repeat
of plasmids (Fig. 4). The 5.2-kb band was detected in 44 integrants, in
which a 1.8-kb band also appeared (Fig. 3). These facts strongly
suggested that a single or multiple integration of the plasmids
containing newly cloned ARSs were exclusively targeted to the different
ends of chromosomes via homologous recombination.
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FIG. 4.
Schematic diagram of telomeric integrations into five P3
homologues (T1 to T5). The sizes of five P3 homologues were roughly
estimated from the Southern blot results shown in Fig. 3. Black boxes
and striped boxes indicate that the telomeric conserved region up to
the PstI site containing the ARS and telomeric repeats were
conserved in five different ends of chromosomes. Empty boxes and bold
lines represent the H. polymorpha LEU2 gene and pBC
KS+, respectively. Probes used in this study are shown under the
corresponding regions as two-headed arrows. An example of each
integrant showing a single-copy integration of pCTEL188 into one of the
five P3 homologues was selected from the Southern blot results shown in
Fig. 3. Numbers after Int188 to the right are lane numbers.
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Telomeric location of five P3 homologues.
To confirm the
telomeric integration of the plasmids and the telomeric origins of five
P3 homologues, five different integrants of pCTEL188 (Int188-6,
Int188-18, Int188-10, Int188-15, and Int188-1), which harbored a
single-copy integration into one of five P3 homologues (T1 to T5), were
selected and further characterized. As shown in Fig.
5, genomic Southern blots were done with
three different probes, P3, pBC, and LEU2. All five different
integrants showed a size-shifted P3 homologue that was not detected in
untransformed cells (Fig. 5A). Each size-shifted P3 homologue could
also be detected with probe pBC, indicating that the size shift of each P3 homologue was caused by the integration of a transforming plasmid (Fig. 5B). A common new signal of about 1.8 kb was detected with probes
LEU2 and P3 but not with pBC (Fig. 5C). A genomic single copy of
H. polymorpha LEU2 (6.5 kb) was also detected with
probe LEU2. To prove the terminal localization of the
XbaI-digested 1.8-kb band, possibly a new chromosomal end
fragment, the sensitivity of the 1.8-kb signal to Bal31 was
tested. Genomic DNA of Int188-1 was treated in time course with
endonuclease Bal31 and then digested with XbaI.
Southern blotting of the DNA was done with LEU2 as a probe (Fig.
6). The internal endogenous H. polymorpha LEU2 signal of 6.5 kb was not sensitive to
Bal31 digestion. In contrast to this, the size of the 1.8-kb
signal was progressively shortened by incubation with Bal31.
It indicated that the 1.8-kb fragment common to all integrants (except
4 of 108) is a new end of a chromosome. It also indicated that all five
XbaI-digested P3 homologues are different ends of
chromosomes.
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FIG. 5.
Confirmation of integrations of pCTEL188 into the five
P3 homologues (T1 to T5) with different probes. Five single-copy
integrants of pCTEL188 (lanes: 1, Int188-6; 2, Int188-18; 3, Int188-10;
4, Int188-15; and 5, Int188-1) were selected from the results shown in
Fig. 3 and analyzed with three probes, P3 (A), pBC (B), and LEU2 (C)
(Fig. 4). All genomic DNA was digested with XbaI. (C,
untransformed H. polymorpha; M, digoxigenin-labeled
lambda HindIII size marker [23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.6 kb]).
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FIG. 6.
Bal31 sensitivity of the common 1.8-kb
band. Genomic DNA of Int188-1, which harbored a single
integration of pCTEL188 into P3 homologue T5, was treated with
exonuclease Bal31 for various amounts of time. Subsequently,
DNA was digested with XbaI. Southern blotting was done with
probe LEU2. (lane 1, untransformed H. polymorpha
without Bal31 treatment; lane 2, Int188-1 without
Bal31 treatment; M, digoxigenin-labeled lambda
HindIII size marker [23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and
0.6 kb]).
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Frequency of targeted recombination.
The pattern and frequency
of each plasmid's integration into the different chromosomal ends are
presented in Table 2 by an analysis of
the Southern blot shown in Fig. 3. Though the transforming plasmids
integrated largely into the telomeric regions of chromosomes whose
sequences are highly homologous to P3 (T1 to T5), they also appeared to
integrate into the other telomeric fragments that have less homology to
P3. The number of chromosomes of H. polymorpha was
reported to be six, based on the number of DNA bands resolved by
pulsed-field gel electrophoresis (13). Thus, besides five P3
homologues, seven additional telomeric fragments should occur as a
group of less homology to P3. All plasmids containing one of the three
telomeric ARSs integrated into the telomeric locus with an overall
frequency of 96.3%, indicating that the integration occurred almost
exclusively via homologous recombination. The telomeric repeats
that exist in all chromosomal ends might be partly ascribed
to this high frequency of homologous recombination responsibility. In
addition, two telomeric ARSs, TEL188 and TEL61, introduced the plasmid
into P3 homologues (T1 to T5) with much higher frequencies than TEL135.
The frequency of targeted integration of TEL188 and TEL61 into P3
homologues was around 75%, but that of TEL135 was around 47%. The
striking difference between these two subtypes is the long deletion of
a part of the bent region (region B) of the ARS in TEL135. Thus, it is
conceivable that the bent regions present in TEL188 and TEL61 play a
role in the targeted integration into P3 homologues. Higher
frequencies of tandem integration with TEL188 (47.2%) and TEL61
(44.4%) than that with TEL135 (30.6%) (Table 2) also appeared to be
related to this sequence.
| |
DISCUSSION |
The Southern blot analysis with HARS36 as a probe suggested that
five telomeres of H. polymorpha chromosomes appear to
contain sequences highly homologous to HARS36. We have isolated and
characterized three telomeric fragments among the HARS36 homologues
that harbored a common ARS motif and telomeric repeats. In a functional
study, a derivative of HARS36 which lacks the telomeric repeats, region C, was more effective than the entire HARS36 for prolonged autonomous replication in an episomal state (40). The combination of an ARS domain and a repeated sequence domain greatly increased the potential of HARS36 for the multiple integration of plasmids containing these domains. It was noted that the integration events always occurred
near the ends of different chromosomes, suggesting that a highly
conserved sequence environment may also exist near the ends of
different chromosomes in H. polymorpha, as found in
other eukaryotic organisms (7, 9, 32, 36).
Telomeric regions of yeasts have been known to be highly
recombinogenic. There has been evidence of frequent recombination between telomeres (21) and between plasmids and chromosomes (10). Targeted recombination in this study suggested that
two high-frequency recombination targets exist, telomeric repeats (5'-GGGTGGCG-3') and a bent region in the ARS motif, in the
ends of chromosomes of H. polymorpha. It has been
observed that telomeric repeats serve as a recombinational hot spot
(5, 25). Repressor activator protein (RAP1) of S. cerevisiae is an essential nuclear protein that recognizes a
13-bp consensus sequence found in numerous upstream activating
sequences at the silencers of transcriptionally repressed mating-type
genes and in telomeric repeats (38). Interestingly, RAP1 was
found to stimulate the telomeric repeat-mediated recombination (15). Binding of RAP1 to a site of the HIS4 gene
also stimulates recombination at this locus (34, 45),
suggesting that the high frequency of targeted recombination in this
study could be mediated by the similar protein(s) of H. polymorpha. Investigation of the telomeric binding protein(s)
could provide insight into the nature of recombination in H. polymorpha. Involvement of intrinsically bent DNA elements in the
enhanced recombination has also been reported for other organisms
(11, 29). Though the exact biological function of the bent
structure in the DNA is still unclear, it has been speculated that it
modifies the chromatin structure of DNA and thus facilitates the
binding of related protein factors for DNA replication, transcription,
and recombination (17).
In the case of TEL135, which has a large deletion near the bent region,
a decrease in frequencies of both tandem array integration and targeted
integration into the five P3 homologues was observed, compared to the
frequencies for TEL61 and TEL188. TEL135 also showed decreased ARS
activity. This observation indicated that the bent structure of the
subtelomeric region in H. polymorpha might play a
role in replication or recombination, both of which might affect the
frequency of the tandem integration of plasmids. Two mechanisms
of plasmid integration in tandem array could be conceivable: either the
integration of plasmid in a single copy number and subsequent
integration of next copies of plasmid into the same site via homologous
recombination or the integration of preformed dimeric (or multimeric)
plasmid into the chromosome. Based on this, we suggest that the lower
frequency of tandem array integration for TEL135 could be the
consequence of either a lower recombination efficiency or a lower
proportion and/or copy number of autonomous multimeric plasmid before
stabilization. Further intensive studies are required to elucidate the
mechanism that produces the tandem repeated arrays in H. polymorpha.
The present study revealed that the chromosomes of H. polymorpha ended with identical repeats, an 8-bp G-rich sequence
(5'-GGGTGGCG-3'), as found in other eukaryotic chromosomes
(18). Generally, telomeric repeated sequences consist of a
simple sequence in tandem repeats of 5 to 9 bp and with a G-rich strand
running 5'
3' towards the end of the chromosome. Telomeric repeats
from several other yeasts, such as S. cerevisiae
(44), Schizosaccharomyces pombe (27), several Candida species, and Kluyveromyces lactis
(28), have been reported. The telomeric repeats
(5'-GGGTGGCG-3') of H. polymorpha started from 80 bp downstream of the putative ARS core of HARS36. This
repeating unit was extended by 18 to 23 repetitions to make up about
144 to 184 bp in total. It showed an unusually high G+C content
of over 87% and a G content of over 75%. It appeared
to be the highest G+C content among those of telomeric repeats
reported to date (18). The G+C content of telomeric repeats
is often inversely related to the overall G+C content of the genomes
(33). Such an extremely high G+C content for the telomeric
repeats appeared to be exceptional for H. polymorpha,
for which the genomic G+C content was 48% (31). Another
interesting feature of the telomeric repeat of H. polymorpha is the highly regular pattern of repeats compared with
those of conventional yeasts, such as S. cerevisiae [T(G)2-3(TG)1-6] and S. pombe [TTAC(A)G2-5]. Several Candida
species and K. lactis showed the regular and longer repeating units (up to 26 bp), except for the 8-bp regular repeat (ACTGGTGT) found in Candida guilliermondii
(28). Recently, another 8-bp regular repeat (TCTGGGTG)
was also found in different yeasts, such as Saccharomyces
castellii and Saccharomyces dairensis (8).
In addition to the simple repeats found at the very ends of
chromosomes, subtelomeric regions of chromosomes in many organisms often contain repetitive elements called telomere-associated sequences (18). Very little is known about the origin or function of
these elements (46). Structures of telomeres have been
studied in detail in S. cerevisiae (6, 7,
41). Most of the chromosomal ends of S. cerevisiae were found to be associated with two conserved elements, Y' and X. A repetitive ARS family and tandem repeats of a
short G-rich sequence [T(G)2-3(TG)1-6] were
also found to be associated with the Y' and X elements (7,
43). It is apparent from our results that the subtelomeric region
of several chromosomal ends of H. polymorpha also
contained such conserved elements containing a family of the ARS motif.
All single-copy integrants of a transforming plasmid into different
chromosomal ends in this study might be valuable for telomere analysis.
Plasmid rescues into E. coli after religation of
XbaI-digested genomic fragments from single-copy integrants
could make it possible to obtain the flanking fragments containing
subtelomeric regions from different chromosomes. Information about
individual chromosomes with a telomeric structure will provide us with
a tool for the genome mapping of H. polymorpha, which
has been little investigated.
| |
ACKNOWLEDGMENT |
This work has been funded by the Ministry of Science and
Technology of Korea. We deeply appreciate its financial support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biotechnology
Research Division, Korea Research Institute of Bioscience and
Biotechnology, Yusong, P.O. Box 115, Taejon 305-600, Korea. Phone:
82-42-860-4005. Fax: 82-42-860-4592. E-mail:
rheesk{at}kribb4680.kribb.re.kr.
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Journal of Bacteriology, February 1999, p. 1005-1013, Vol. 181, No. 3
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Abstract
Introduction
