Download Unusual chromosome structure of fission yeast DNA in mouse cells

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Journal of Cell Science 107, 469-486 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Unusual chromosome structure of fission yeast DNA in mouse cells
John McManus1,*, Paul Perry1,*, Adrian T. Sumner1, Diana M. Wright2, Eric J. Thomson1, Robin C. Allshire1,
Nicholas D. Hastie1 and Wendy A. Bickmore1,†
1MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland
2Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Edinburgh
EH9 3JR, Scotland
*These two authors contributed equally to this work
†Author for correspondence
SUMMARY
Chromosomes from the fission yeast Schizosaccharomyces
pombe have been introduced into mouse cells by protoplast
fusion. In most cell lines the yeast DNA integrates into a
single site within a mouse chromosome and results in
striking chromosome morphology at metaphase. Both light
and electron microscopy show that the yeast chromosome
region is narrower than the flanking mouse DNA. Regions
of the yeast insert stain less intensely with propidium iodide
than surrounding DNA and bear a morphological resemblance to fragile sites. We investigate the composition of the
yeast transgenomes and the modification and chromatin
structure of this yeast DNA in mouse cells. We suggest that
the underlying basis for the structure we see lies above the
INTRODUCTION
Ease of genetic manipulation, and small chromosome size (0.15 Mb), has allowed chromosome components such as centromeres, telomeres and replication origins to be examined in
the budding yeast Saccharomyces cerevisiae and the fission
yeast Schizosaccharomyces pombe (Clarke, 1990; Blackburn,
1991; Matsumoto et al., 1987; Brewer and Fangman, 1987;
Maundrell et al., 1988). Attempts to define these elements on
mammalian chromosomes have proved limited, due to the
large (102 Mb) and complex nature of the chromosomes and
the limited ability to manipulate them genetically. Compounded with this, suitable assays for some aspects of
mammalian chromosome structure and function are not
obvious (Tyler-Smith and Willard, 1993). One exception to
this is our knowledge of the structure and behaviour of
mammalian telomeres.
However, mammalian chromosomes are amenable to cytological analysis. They can be visualised at metaphase by light
and electron microscopy. The chromosomal position of
specific DNA sequences can be detected directly by in situ
hybridisation. Yeast chromosomes are generally too small to
see cytologically and only recently has the technique of chromosomal in situ hybridisation been applied to the study of
yeast chromosomes (Uzawa and Yanagida, 1992). DNA in
metaphase chromosomes is compacted by a factor of about
10,000 over that of a simple double-helical thread. This is
achieved through a series of packing or folding events, which
level of DNA modification and nucleosome assembly, and
may reflect the attachment of the yeast DNA to the rodent
cell nucleoskeleton. The yeast integrant replicates late in S
phase at a time when G bands of the mouse chromosomes
are being replicated, and participates in sister chromatid
exchanges at a high frequency. We discuss the implications
of these studies to the understanding of how chromatin
folding relates to metaphase chromosome morphology and
how large stretches of foreign DNA behave when introduced into mammalian cells.
Key words: chromosome condensation, methylation, nucleoskeleton,
Schizosaccharomyces pombe, sister chromatid exchange
are still the subject of some debate. At the first level of
packaging, DNA is assembled into nucleosomes, which fold to
form the 30 nm fibre, a compaction factor of approximately 40.
It is believed that these fibres are then folded into a series of
loops, reducing the length by another factor of 25. The final
compaction factor of 10 results from condensation of the interphase chromosome to the metaphase chromosome, which is
thought to occur through the formation of compact coils
(Rattner and Lin, 1985; Earnshaw, 1988).
Our understanding of the principles that govern these steps
in the compaction process is rudimentary, and it is not known
whether DNA sequence itself can influence the folding parameters. For example, we know that the DNA in a yeast chromosome is less compacted than the endogenous DNA in a
mammalian chromosome (Umesono et al., 1983). Is this a
reflection of the DNA sequence itself or the environment in
which the DNA is placed?
Mammalian chromosomes are not morphologically homogeneous. Firstly, constrictions on metaphase chromosomes are
apparent. The primary constrictions correspond to the location
of centromeres, and nucleolar organiser regions (NORs) participating in nucleoli formation form secondary constrictions
on the chromosome arms. There are large blocks of chromatin
that show a higher compaction and staining than other parts of
the genome throughout the cell cycle (heterochromatin) and
that replicate late in S phase (Drouin et al., 1990). Constitutive
heterochromatin is a permanent feature of the genome, whilst
facultative heterochromatin (for example, the inactive X chro-
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mosome) is developmentally regulated and appears to take part
in inactivating large parts of the genome. Therefore, different
DNA sequences and their epigenetic modifications have
various cytological expressions in mammalian cells. DNA at
primary constrictions and constitutive heterochromatin is
composed of large arrays of tandem repeats, and NORs contain
tandem arrays of rDNA genes. The cytogenetic phenotype of
the fragile X mutation correlates with expansion and methylation of a simple trinucleotide repeat and delayed DNA replication of the surrounding chromosomal region (Hansen et al.,
1993). A more subtle form of differentiation of mammalian
euchromatin is that manifest by chromosome banding. The biochemical characteristics of different chromosome bands
suggest that they reflect differences in gene and interspersed
repeat content, base composition, replication timing and
chromatin condensation along euchromatin (Craig and
Bickmore, 1993).
We are only just beginning to understand how the primary
DNA sequence and its activity affect mammalian metaphase
chromosome morphology, because of the complex and largely
undefined nature of the DNA involved. One way to resolve this
problem would be to introduce more defined, but still cytologically visible, pieces of DNA into mammalian cells and
examine the ensuing affects on chromosome structure. Allshire
et al. (1987) introduced chromosomes of the fission yeast S.
pombe into mouse cells by fusion, to examine the functional
capacity of yeast chromosomes in mammalian cells. Two
classes of transfectants were obtained: the stability and growth
characteristics of the first were consistent with linear
autonomous yeast chromosomes being present in the mouse
nucleus, while the other class of transfectants were stable and
several megabases of yeast DNA appeared to be integrated into
mouse chromosomes. We noted that in several of these cell
lines the site of integration of the fission yeast DNA looks
different under the light microscope: it appears to be narrower
and to stain less intensely with propidium iodide (PI) than
flanking mouse DNA.
In this paper we analyse features of S. pombe integrants that
are amenable to cytological and molecular analysis. We show
that, at the level of the light and the electron microscope, the
yeast DNA adopts features resembling those seen at primary
and secondary constrictions in normal metaphase mammalian
chromosomes, at induced fragile sites and in prematurely
condensed chromosomes (PCCs), and undercondensed regions
of chromosomes after treatment of cells with 5-azacytidine
(Takayama and Hiramatsu, 1993; Goessens, 1984; Harrison et
al., 1983; Gollin et al., 1984; Haaf and Schmid, 1989). We have
tried to find a molecular explanation for the unusual cytological appearance of the yeast DNA in these mouse cells. Our
results suggest that this may lie in the higher-order packaging
of the DNA, probably at the level of attachment of the yeast
DNA to the nucleoskeleton of the mouse nuclei. We investigate whether the S. pombe DNA replicates as a unit, and the
time in S phase when this occurs, and whether the S. pombe
DNA is more susceptible to breakage and sister chromatid
exchange (SCE) than the flanking mouse DNA.
MATERIALS AND METHODS
Cell culture, fixation and slide preparation
Mammalian cells were grown in DMEM, 10% foetal calf serum. 400
µg/ml G418 was added to the medium of hybrid cell lines. S. pombe
Int 5 was grown on EMM (Moreno et al., 1991) containing 75 mg/l
leucine. Mammalian cells harvested by mitotic shake-off were resuspended for 10 minutes in hypotonic solution (0.5% trisodium citrate,
0.25% KCl), fixed in 3:1 (v/v) methanol:acetic acid and stored at
−20°C. Chromosome spreads were stored in a desiccator for 5 to 12
days before FISH.
Fluorescence in-situ hybridisation (FISH) and staining
Total yeast DNA was nick translated with biotin-16-dUTP. Hybridisation and detection were by standard procedures (Pinkel et al., 1986).
Slides were treated with RNase (100 µg/ml in 2× SSC) for 1 hour at
37°C before use. Slides were hybridised at 37°C overnight with 20 µl
hybridisation mix (50% formamide, 2× SSC, 500 µg/ml salmon sperm
DNA, 1-2 µg/ml biotinylated yeast DNA, 10% dextran sulphate) per
slide, and washed at 45°C successively in 50% formamide 2× SSC,
2× SSC and 4× SSC 0.1% Triton X-100. Probe was detected with
1:500 avidin-FITC (Vector), followed by 1:100 biotinylated antiavidin (Vector) and 1:500 avidin-FITC. BrdU was detected with
mouse anti-BrdU conjugated to TRITC (Amersham). Slides were
counterstained in AFT10 (Citifluor) containing 2 µg PI/ml and/or 20
µg DAPI/ml. Slides for SCE and replication analysis were counterstained with DAPI only. The confocal A1/A2 dual filter set of the BioRad MRC-600 laser scanning confocal microscope was used to
capture PI (or TRITC) and FITC fluorescence in separate channels.
The two images were independantly scaled and merged in pseudocolour. In SCE experiments, only a single chromatid equivalent was
labelled with anti-BrdU/TRITC but the position of the unlabelled
chromatid could be discerned.
Microdensitometry and chromosome banding
Chromosome width and DNA content were measured with a scanning
microdensitometer. Feulgen-stained chromosomes were scanned with
a ×100 objective and a 0.2 µm spot at 90° to the chromosome axis
(Sumner et al., 1990). The ‘area’ and integrated optical density (IOD)
therefore represent (in arbitrary figures) the chromosome width, and
the IOD across that width. DNA concentration was calculated as
IOD/area2, since the chromosome cross-section is two-dimensional
but the measurement is one-dimensional. Chromosome width (µm)
was obtained by measuring the area of an object whose length was
measured using a stage graticule. C banding was according to the BSG
method of Sumner (1972). Ag-NOR staining was carried out as
described by Howell and Black (1980).
SEM
Chromosomes were prepared for SEM using the OTOTO procedure
(Sumner, 1991). SEM in situ hybridisation was as for FISH, except
that sites of hybridisation were detected with avidin-horseradish peroxidase and DAB followed by gold and silver intensification (Burns
et al., 1985). The OTOTO procedure, without trypsin treatment, was
then carried out.
Genomic DNA preparation and analysis
Mammalian DNAs were prepared, analysed by restriction endonuclease digestion, Southern transfer and DNA hybridisation by modifications of standard procedures (Allshire et al., 1987). Preparation of
yeast and mammalian DNAs for pulsed-field gel electrophoresis
(PFGE) were as described by Niwa et al. (1986) and Allshire et al.
(1987), respectively.
S. pombe protoplasts were prepared by modifying the method of
Beach et al. (1982). 107 cells/ml Int 5 cells were washed in 50 mM
citrate-phosphate, 1% β-mercaptoethanol and resuspended in protoplasting solution (50 mM citrate-phosphate, 1.2 M sorbitol, 40 mM
EDTA, 0.2% β-mercaptoethanol, 2 mg/ml Novozyme, 0.4 mg/ml
Zymolyase) at 4×108 cells/ml. Protoplasting at 37°C was assayed by
phase-contrast microscopy. Protoplasts were pelleted through a
sucrose cushion (0.8 M sucrose, 0.2 M KCl) at 400 g for 5 minutes.
Yeast chromosomes in mouse cells
S. pombe nuclei were pelleted at 8000 g, 20 minutes, 4°C and lysed
in 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS,
50 µg/ml RNase A. After 30 minutes at 37°C, 50 µg/ml proteinase K
was added for 4-5 hours. DNA was then extracted.
DNA probes
Preparation of ade6, stb and SV2neo probes is described by Allshire
et al. (1987). The ura4 probe was a 1.7 kb HindIII fragment of
pura4/SV2neo. Arg1 was a gift from S. Uzawa (Kyoto). Ade5 was a
5.4 kb PstI fragment from pNS2 (gift from O. Niwa, Kyoto) and m23
a 2.7 kb HindIII fragment from pSAm23 (Niwa et al., 1989). ars1 was
a 1 kb EcoRI fragment from pIRT-2 (Moreno et al., 1991) and cyh1
a 4.3 kb EcoRI fragment from pCY1 (gift from Y. Nakaseko, Kyoto).
Lys1 was a gift from S. Itaru (Kyoto). His1 was prepared from pMN1
(gift from P. Schuchert, Bern). A nuc2 probe was derived by PCR
(Hirano et al., 1988). cdc13 and cdc2 probes were gifts from P. Fantes
(Edinburgh). Nda3 and dis2 were gifts from M. Yanagida (Kyoto). dh
was a 2.3 kb BglII fragment from pSS242 (Chikashige et al., 1989).
tm is described by Takahashi et al. (1992). Nsu 21 was an 8 kb EcoRI
fragment from pNsu21 (Sugawara, unpublished observations).
Cosmids cade6-I, cura4-II were obtained by screening an S. pombe
genomic library constructed in pSS10 (Nakaseko et al., 1986).
Micrococcal digestion of nuclei
Mammalian nuclei were prepared from cells harvested by scraping.
After washing in ice-cold PBS cells were resuspended in lysis buffer
(10 mM Tris-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2,1 mM PMSF,
0.5% Triton X-100) and broken open by passing through a needle,
monitored by phase-contrast microscopy. Nuclei were pelleted at
1500 g, 5 minutes, 4°C and washed 3 times in lysis buffer without
Triton X-100. Nuclei were resuspended at 107/ml in 25% glycerol, 50
mM magnesium acetate, 0.1 mM EDTA, 5 mM DTT, 50 mM TrisHCl, pH 8.0 and stored at −70°C. Before digestion nuclei were resuspended at 107/ml in digestion buffer (15 mM Tris-HCl, pH 7.5, 60
mM KCl, 200 mM NaCl, 3 mM MgCl2, 0.5 mM DTT, 0.25 M
sucrose, 1 mM CaCl2).
S. pombe nuclei were prepared by the method of Polizzi and Clarke,
(1991) and stored at a concentration of 109/ml; 2 units/ml microccocal nuclease (Pharmacia) was added at 37°C, and left for various
lengths of time. Reactions were stopped with an equal volume of 1
M NaCl, 20 mM EDTA, 1% SDS, incubated with 1 mg/ml Proteinase
K at 37°C, and DNA was purified by extraction and precipitation.
Nucleosome ladders were electrophoresed and transferred to 0.2 µm
pore nitrocellulose.
Analysis of nuclear skeleton attachment
F1:1 cells were encapsulated in agarose beads, permeabilised and
digested by the method of Jackson and Cook (1988). Unattached
chromatin was removed by electrophoresis. The beads, now depleted
of unattached chromatin, were digested with 0.1% SDS, 50 mg/ml
proteinase K for 4 hours. After melting at 69°C for 1 hour the beads
were extracted with an equal volume of hot phenol followed by
phenol/chloroform, and the DNA was precipitated and passed through
an Elutip-D (Schleicher and Schuell). The gel was deproteinised in
the same way as the beads, then the position of the unattached
chromatin in the gel was determined by ethidium bromide staining of
a gel slice. The region of the gel containing the unattached DNA was
excised and the DNA eluted from it by electrophoresis inside dialysis
tubing. The DNA was concentrated with an Elutip-D. DNAs from
both attached and unattached fractions were then redigested with the
appropriate restriction enzyme, and analysed by electrophoresis and
Southern blotting.
Reverse transcriptase PCR
RNAs from F1.1, C127 and S. pombe cells were analysed by reverse
transcriptase (rt) PCR with M-MuLV reverse transcriptase (BCL) as
described by Kawasaki (1990). Primers for the ura4 gene were those
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described by Allshire et al. (1994). Ade6 primers E495 and E497 were
derived from nucleotides 764-784 and 1375-1355, respectively, of the
sequence described by Szankasi et al. (1988). Reactions were cycled
at 94°C, 1 minute; 50°C, 1 minute; 72°C, 1 minute for 35 cycles.
Amplified products were resolved on 2% Nusieve agarose (FMC).
Cell cycle analysis
Twelve F1.1 cultures were set up at 2.1×106 cells per 80 cm2 flask.
After 16 hours flasks received a 30 minute pulse of 10-5 M BrdU at
1 hour intervals from each other. Cells were rinsed twice with
prewarmed phosphate buffered saline (PBS), then prewarmed conditioned medium containing 10−5 M thymidine was added. Mitotic cells
were harvested simultaneously from all flasks exactly 12 and 24 hours
after the first BrdU addition. To analyse replication of the yeast insert
BrdU remained in the culture medium until harvest. Thus BrdU was
present for periods ranging from 1 to 12 hours before harvest.
Micronucleus analysis
Cells (1.7×106) were irradiated with 0, 100, 200 and 400 cGy at 37
cGy/min. Cytochalasin B (3 µg/ml) was added for 26 hours. Cells
were then washed with PBS, trypsinised, resuspended in hypotonic
medium diluted 1:3 with deionised water for 10 minutes and fixed
twice in 20:1 (v/v), methanol:acetic acid. Air-dried slides were made
the next day, and desiccated before use. This protocol was developed
because standard 80% methanol-fixed cytocentrifuge preparations
gave variable FISH signals. Micronuclei were scored with a standard
UV microscope. Binucleate cells were identified from DAPI fluorescence using a ×25 oil immersion objective, then checked at high
power using the Leitz I3 filter block to ensure that both main nuclei
were within the same cytoplasm, and to identify the FITC-labelled
insert.
SCE analysis
An anti-BrdU antibody was used to distinguish unifilar from unsubstituted chromatids after incorporation of BrdU during the penultimate
S phase before harvest, since the FPG procedure was not compatible
with FISH. The concentration of mitomycin C (MMC) that gave a
significant, yet easily scorable, increase in SCE without concomitant
increase in the cell cycle was established. Flasks were set up with
1.1×106 cells and 18 hours later treated with 10-5 M BrdU alone or
with either 10−7 M or 3×10−8 M MMC. After 7 hours the cells were
rinsed twice with prewarmed PBS and conditioned medium containing 10−5 M thymidine added. Flasks were harvested by mitotic shake
off 24.5 hours after the start of BrdU treatment and following a 15
minute colcemid treatment. The SCE frequency per cell was established by the FPG technique (Perry and Wolff, 1974) and analysis of
the SCE within the insert chromosome was conducted using the
combined FISH/anti-BrdU method.
RESULTS
Formation of mouse-S. pombe hybrids
S. pombe Int5 was constructed by introducing the SV2neo
gene into the ura4 locus on chromosome III to confer G418
resistance (Allshire et al., 1987). Int 5 chromosomes were
introduced into a mouse mammary tumour cell line (C127),
by either PEG-mediated fusion of spheroplasts or DNAmediated transfection, to generate fusion hybrids or transfectants, respectively. In this paper we analyse fusion hybrids
F1.1 and F48, and DNA transfectants DC1 and 2. The growth
characteristics of F1.1, in the absence of selection, suggested
that the yeast DNA was stably integrated into a mouse chromosome.
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1a
1b
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2b
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3b
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9
Yeast chromosomes in mouse cells
The S. pombe insert has a characteristic
chromosome morphology at the level of the light
microscope
The presence of yeast DNA integrated into a mouse chromosome was confirmed on metaphase spreads from fusion hybrid
cell lines. Fig. 1 shows metaphase chromosomes from two independent fusion lines, F1.1 and F48. Panels 1a to 7a show PIstained chromosomes and in the accompanying panels 1b to 7b,
and in panels 8 and 9, the site of yeast DNA integration is also
detected by fluorescence in situ hybridisation to biotinylated S.
pombe DNA. In all cases FISH signal corresponds with a site
of constriction on a mouse chromosome arm. The cell line that
has been the subject of most of our studies, F1.1, has only one
site of integration of yeast DNA in the majority of cells (Fig.
1, panels 1 to 5). Note the narrow appearance of the yeast DNA
relative to the flanking mouse chromosome and the reduction
in PI staining intensity. FISH signal extends throughout the constriction, suggesting that the bulk of the DNA in this region is
of yeast origin. In some cases there are one or two regions
within the S. pombe insert that are not stained at all by PI (Fig.
1, panels 1 to 4). The waisted aspect is a feature of the whole
yeast insert whereas the gaps in staining are confined to specific
regions of the transgenome. The degree of difference between
the S. pombe insert and the mouse DNA varies from cell to cell
depending on the level of chromosome condensation - the
abnormal character of the S. pombe region is more apparent at
prometaphase when chromosomes are less condensed. On
average the constricted site of yeast integration occupies 20%
(1.1 µm) of the total length of the chromosome in which it
resides. The mean width of the insert (1.20±0.28 µm, n=33),
measured by scanning microdensitometry, is 0.69±0.13 of that
of the adjacent mouse chromosome arms, whose width is
1.74±0.17 µm. The DNA concentration in the yeast insert is
similar to the rest of the chromosome arms in chromosomes
where the constriction is less pronounced. However, in chromosomes where the width of the yeast insert is less than half
that of the arms, the DNA concentration at the constriction is
up to 1.6 times that of the mouse euchromatin (Fig. 2).
In a small percentage of F1.1 cells there are two or more
yeast inserts, sometimes on the same chromosome (Fig. 1,
panels 6 and 7), each of which has the characteristic reduced
staining and waisted appearance. In <1% of the cells a small
chromosome fragment is seen with a terminal hybridisation
signal (Fig. 1, panel 8) and other interstitial sites of hybridisation. We conclude that there has been a chromosome break
within the yeast insert, and that the S. pombe insert may be
prone to breakage.
To show that the unusual character of the S. pombe
chromatin is not peculiar to the F1.1 hybrid, we analysed the
yeast insertion in an independent fusion line, F48. In these cells
Fig. 1. Light microscopy and FISH analysis of yeast transgenome
metaphase chromosome structure. (1a to 7a) F1.1 metaphase
chromosomes stained with PI. Arrows indicate the site of the
constricted and understained region. (1b to 7b) Corresponding FISH
analysis with biotinylated yeast DNA detected with avidin-FITC,
merged with the PI image. Note the correspondence between the in
situ signal and the site of constriction in all cases, even where
rearrangement of the S. pombe insert has occurred resulting in
multiple integration sites (6 to 8). (9) PI and FISH image of
metaphase chromosomes from an independent fusion hybrid (F48).
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Fig. 2. Microdensitometry of the F1.1 chromosome. Graph of the
ratio of DNA concentration in the yeast insert to that in the mouse
chromosome arms, against the ratio of the width of the insert to that
of the arms measured by microdensitometry. Note that although the
ratio is close to 1.0 in most cases, the most constricted inserts have a
significantly higher DNA concentration, up to 1.6 times that of the
mouse chromosome arms.
there are proximal and distal terminal sites of S. pombe integration on the same chromosome (Fig. 1, panel 9), both
showing the characteristic waisted appearance and reduced
staining. Hence these features are intrinsic to S. pombe DNA
and independent of the site of integration or the cell clone.
The constriction associated with the yeast insert is not stained
distinctively by C-banding or Ag-NOR staining. In contrast a
different, apparently dicentric, mouse chromosome with a constriction half-way along its length shows C-banding at that site,
characteristic of heterochromatin (Sumner, 1972), and silver
staining immediately distal to it (Fig. 3). Thus the fission yeast
transgenomes adopt a metaphase chromosome structure morphologically distinct from the bulk of the mouse genome.
Examination of the S. pombe chromosome domain
at the EM level reveals that the packaging is
different
To study the S. pombe domain at high resolution F1.1
metaphase chromosomes were analysed by scanning electron
microscopy (SEM). In situ hybridisation confirmed that the
constrictions seen were indeed the yeast integration sites (Fig.
4). The yeast transgenome is dramatically narrow relative to
flanking mouse DNA and, unlike the mouse centromeric constrictions, has separated sister chromatids (Fig. 4A,B). In Fig.
4C sister chromatids throughout the chromosome length are
unresolved. The longest constrictions (up to 1.7 µm in length)
occur in the longest (7 µm) chromosomes, and both condense
together such that in the most condensed chromosomes the
constriction is no longer visible (Fig. 5). The mean length of
the constriction, measured by SEM, is 0.8 µm and the mean
diameter 1.30±0.23 µm (n=38), compared with 1.89±0.30 µm
for other parts of the chromosome arms. The average width of
the constriction is thus 0.69±0.09 of that of the chromosome
arms, in excellent agreement with scanning microdensitometry, considering the different methods of preparation and measurement, and the swelling that may be induced by the OTOTO
method (Sumner and Ross, 1989).
Content of cell lines
Since the constriction in F1.1 comprises 20% of the length of
the mouse chromosome into which the yeast DNA has inte-
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Fig. 3. C-banding and Ag-NOR staining of the F1.1 chromosome.
(A) C-banded F1.1 chromosomes. The short arrow indicates a
dicentric chromosome, with a C-banded constriction half-way along
its length. By contrast the constriction at the site of yeast integration
(confirmed by FISH) two-thirds along the length of another
chromosome (long arrow) does not C-band. (B) Ag-NOR staining of
F1.1 chromosomes. The chromosome with the median constriction
(short arrow) has silver staining just distal to the constriction. The
site of yeast integration (distal constriction on the chromosome
indicated with the long arrow) is not labelled.
grated we anticipated that F1.1 may contain several megabases
of yeast DNA. This was investigated by Southern analysis of
F1.1 DNA with S. pombe probes. As expected, probes from the
region of chromosome III under selection (pura4/SV2neo)
were unrearranged in F1.1, as were other chromosome III
markers, including the ade6, arg1 and ade5 genes and the stb
locus (Allshire et al., 1987; data not shown). The m23 locus
and the major sup9-5S RNA bands are absent from F1.1, and
a probe (0.7 kb) from the non-transcribed spacer of the rDNA
complex shows that some copies of this are rearranged in F1.1
(Allshire et al., 1987). Of the two regions of chromosome III
absent or rearranged in F1.1, one (m23) is close to the centromere (Niwa et al., 1989) and the other (rDNA) maps
adjacent to both telomeres (Maier et al., 1992). To assess the
integrity of yeast DNA in F1.1, cosmids for the ade6 and ura4
loci were isolated from an S. pombe cosmid library. Most
bands hybridising to these cosmids in S. pombe Int 5 are unrearranged in F1.1 (Fig. 6A,B), confirming that large stretches of
chromosome III are intact. We also analysed chromosome III
in F1.1 by pulsed field gel electrophoresis (PFGE) of NotIdigested DNA and hybridisation to SV2neo. S. pombe chromosome III has no NotI sites (Fan et al., 1988). However,
instead of an expected NotI fragment of at least 3.5 Mb, a
smaller band migrating at 1.4 Mb is seen. A larger fragment
migrating at 3.5 Mb may be a partial digestion product or due
to variable methylation of NotI sites in the yeast transgenome
(Fig. 6F). Thus long-range rearrangement of chromosome III
must have occurred during the formation of F1.1.
To determine whether other yeast DNA was present in F1.1,
markers from chromosomes I and II were analysed. Only one
chromosome I marker, ars1, mapping close to rad4, was
detected in F1.1 (Moreno et al., 1991; Bickmore and Maier,
unpublished observations). All other chromosome I markers
tested; cyh1, lys1, his1 and nuc2, were absent (data not shown).
On chromosome II, dis2 and nda3 loci are intact, but the cdc13
and cdc2 loci are absent (data not shown). Thus parts of S.
pombe chromosomes I and II are present in F1.1 at the same
integration site as chromosome III, the chromosome under
direct selection. For both chromosomes I and II only markers
from one of the two chromosome arms could be detected,
whereas, markers from both arms of chromsome III are present
(Fig. 6G). The intensity of signal in F1.1 with S. pombe
markers indicates that multiple copies of the yeast markers are
not present in F1.1. Together, these analyses suggest that 5-10
megabases of yeast DNA is present in F1.1.
The presence of S. pombe centromeric sequences was investigated with probes specific for a repetitive element (dh)
present at all three centromeres (Murakami et al., 1991) and
for a single-copy sequence (tm) found only in centromeres I
and III (Takahashi et al., 1992). The pattern and intensity of
hybridisation of dh to F1.1 DNA was identical to that seen in
yeast DNA (Fig. 6C). The predominant EcoRI band (6.2 kb)
is derived from the large tandem arrays of truncated dh/dg
elements at cen3. The slightly larger fragment probably comes
from the innermost repeat motif at cen3, whereas the 9.5 kb
fragment corresponds to full-length repeat motifs present at
low copy number at cen1 and cen2 (Murakami et al., 1991).
Similarly, the pattern of hybridisation of tm indicates that this
is present in F1.1 (Fig. 6D). Therefore, the centromeres from
all three S. pombe chromosomes appear to be intact in F1.1.
By contrast, a probe for S. pombe telomeres, nsu21, is absent
from F1.1 (Fig. 6E). The presence of DNA from all three yeast
chromosomes in F1.1 suggests that co-ligation and rearrangement of yeast chromosomes occurred prior to integration into
the mouse chromosome.
Although some long-range rearrangement of yeast DNA has
occurred in F1.1, Fig. 6A and B suggests that substantial
stretches of the yeast genome are not rearranged in this fusion
hybrid. S. pombe DNA was also introduced into C127 cells by
DNA-mediated transfection (Allshire et al., 1987). Hybridising with cade6-I and cura4-II shows that the transgenomes of
Yeast chromosomes in mouse cells
475
Fig. 4. Scanning electron micrographs of F1.1 chromosomes. (A, B and C) Secondary electron images. Bar, 1 µm. Note the ‘knobs’ or
projections on both the chromosome arms and the site of constriction (which is two-thirds of the way down the chromosome arms). The mouse
centromeres are visible at the right-hand end of the chromosomes in (A) and (B) and the sister chromatids are unresolved at these sites whilst
they are clearly separated along the chromosome arms, including at the site of yeast integration. In (C) sister chromatids along the entire
chromosome length are not yet resolved. (D) Backscattered electron image of chromosomes hybridised with a yeast probe showing that the
constriction two-thirds of the distance along the chromosome (bottom left) is the site of the yeast insertion. Another chromosome with a
constriction half-way along its length (top right) is not labelled. Bar, 3 µm.
two of these transfectants, DC1 and 2, are highly scrambled
and rearranged (Fig. 6A,B).
Anaphase chromatid separation
Since apparently intact S. pombe centromeres are present in
F1.1, anaphase separation of F1.1 chromosomes was
examined. In some chromosome spreads mouse chromosomes
appeared to be in C-anaphase, as their sister chromatids were
clearly separated from each other, but in a small percentage of
cases the chromatids of the constriction-bearing chromosome
were still associated with each other at one point. FISH with
total yeast DNA confirms that the point of association
coincides with the site of yeast integration (Fig. 7). However,
we found no lagging of the yeast-insert-bearing chromosome
on the mitotic spindle (data not shown), in anaphase preparations made by incubating mitotically selected cells for 5-15
minutes in the absence of colcemid before harvest. Even
though there appears to be chromatid association at the site of
yeast integration the chromatids are still resolved from one
another by both light (Fig. 7) and electron (Fig. 4) microscopy
Fig. 5. Yeast insert length in SEM. Plot of yeast insert length against
that of the rest of the chromosome (µm). Although there is a linear
relationship between the two, the intercept of the calculated
regression line (y=0.251x−0.397) is not at the origin. This may
indicate that the insert is contracting faster than the rest of the
chromosome, or that there is an ascertainment problem in detecting
the insert in shorter chromosomes.
J. McManus and others
476
a
b
c
d
g
e
f
Fig. 6. S. pombe content of yeast/mouse fusion hybrids and DNA
transfectants. DNA was prepared from S. pombe/mouse fusion hybrid
F1.1, DNA transfectants DC1 and DC2, C127, the parental mouse cell
line, and from the S. pombe donor strain Int5. Genome equivalents of each
DNA were digested with EcoRI and transferred to hybridisation membrane
after electrophoresis. Southern blots were hybridised with a series of S.
pombe probes: (a) cura4-II cosmid from the ura4 locus; (b) cade6-I
cosmid from the ade6 locus; (c) the centromere-derived probe dh; (d) the
centromere-derived probe tm; (e) nsu21 a probe for S. pombe telomeres.
(f) F1.1, C127 and S. pombe Int 5 high molecular mass DNAs were
prepared in agarose blocks. F1.1 and C127 DNAs were digested with NotI
and subject to PFGE. S. pombe Int5 DNA was run undigested, since
chromosome III contains no NotI sites. After transfer, the blot was
hybridised to SV2neo. (g) Diagram of the S. pombe genome showing DNA
markers from chromosomes I, II and III shown by Southern blotting to be
present (+) or absent (−) in F1.1.
even on chromosomes where the host centromeres are still
unresolved.
DNA methylation
What characteristics of the S. pombe genome could account for
F1.1 chromosome morphology? The first obvious difference
between yeast and mouse genomes is DNA modification.
Mammalian DNA has CpG methylation, and depletion of this
dinucleotide. There is no detectable DNA methylation or CpG
suppression in S. pombe. CpG methylation influences chromosome structure and function; hence, if the yeast DNA fused
into the mouse genome remained unmethylated, it would constitute a large open chromosome region lacking methylated
DNA-binding proteins, contrasting with the bulk of the host
genome. This would not be inconsistent with the cytological
appearance of the yeast DNA in F1.1.
Some mouse cell lines methylate both endogenous and introduced DNA de novo (Antequera et al., 1990; Jahner and
Jaenisch, 1984). If C127 cells have de novo methyltransferase
activity we might see increasing methylation of the F1.1 yeast
insert in culture. Similarly, if 5meCpG-binding proteins play
an important role in chromosome morphology we might expect
to see a change in the appearance of the F1.1 insert accompanying DNA methylation. We therefore analysed methylation
of the S. pombe DNA using the MspI/HpaII isochizomer pair
in F1.1 cells sampled at different times after the initial fusion
Yeast chromosomes in mouse cells
477
Fig. 7. Anaphase sister chromatid separation. FISH of F1.1 anaphase chromosomes with biotinylated total yeast DNA. The chromatids of all
the mouse chromosomes have clearly separated from each other. In both (A) and (B) the site of yeast insertion (white signal) corresponds with
the point on these chromosomes where the sister chromatids are still held together. In (B) it is clear that the two sister chromatids are resolved
from each other.
event. F1.1 cells were retrieved from storage and arbitrarily
designated as passage (P) 0. Cells were cultured and harvested
at different passages up to P>40 (approximately 150 passages).
DNA and metaphase chromosomes were prepared from these
cells to allow a parallel analysis of methylation and cytological appearance of the yeast transgenome over time. Fig. 8
shows hybridisation of cura4-II to BamHI/HpaII and
BamHI/MspI digests of F1.1 DNAs at P20 through to P>40. A
progressive increase in the methylation state of the yeast DNA
is apparent. Little methylation is detected in early passage (P2P10) cells (data not shown). By P20 clear differences between
the HpaII and MspI lanes are seen, and this increases substantially through passage P40 until after approximately 150
passages (P>40) the yeast DNA is so heavily methylated that
virtually none of the MspI hybridising fragments is visible in
the HpaII digest. Similar results were obtained with other yeast
probes. Hybridisation with a probe for the mouse contrapsin
gene family and densitometry analysis of the HpaII and MspI
digests showed complete digestion of all DNA samples and no
change in the gross methylation levels of the mouse genome
over the time course of this study (data not shown). This was
not unexpected, since methylation of endogenous sequences in
mammalian cells in culture is a slow process (Antequera et al.,
1990).
A parallel cytological analysis of F1.1 metaphase chromosomes, by FISH with total S. pombe DNA, showed that the
yeast integration site is still marked by an abnormal appearance, even in cells where substantial methylation of the
transgenome had occurred. Therefore the lack of methylation
of the yeast DNA at early passages is not primarily responsible for the constriction, and increased levels of CpG methylation do not affect the cytological appearance of F1.1
metaphase chromosomes.
Fig. 8. Methylation of the yeast transgenome of F1.1. DNA was
prepared from F1.1 cells harvested at passage numbers P20, 40 and
>40 (approximate P150). These DNAs, together with those from
C127 and S. pombe Int 5 cells, were digested first with BamHI and
then with either HpaII (H) or MspI (M). After electrophoresis the
DNAs were transferred to hybridisation membrane and hybridised
with cura4-II.
Nucleosome assembly
After examining DNA modification we looked to the lowest
level of DNA packaging, assembly into nucleosomes, to
explain the cytological appearance of the yeast chromatin in
F1.1. The distance between the start of one nucleosome and
the next varies both between species and between tissues
within a species. This distance is 185 bp in mouse cell lines
(van Holde, 1989) and 160 bp in S. pombe, which also appears
to lack histone H1 (Chikashige et al., 1989; Bernardi et al.,
1991; Polizzi and Clarke, 1991). If the yeast nucleosome repeat
478
J. McManus and others
Fig. 9. Nucleosome pattern of
yeast DNA in S. pombe Int 5
and F1.1 cells. S. pombe Int 5
or F1.1 nuclei were digested
with micrococcal nuclease for
between 0 and 6 minutes. After
electrophoresis, nucleosome
ladders were transferred to 0.2
µm pore size nitrocellulose and
hybridised to dh and ade6
probes. Size markers were
φX/HaeIII. The relative
positions of the core (C)
nucleosome and increasing
numbers of linked nucelosomes
(C + 1N, etc.), in both yeast
and mouse cells, are indicated.
In both cases the probes
hybridise to a ladder with a
spacing typical of bulk
chromatin of the host cells. No
hybridisation to C127
nucleosomal ladders was
detected (data not shown).
were preserved in F1.1, this could set up a different chromatin
structure between the yeast and mouse DNAs in the building
block for higher-order packaging, the 30 nm fibre. The
diameter of chromatin fibres increases with nucleosomal repeat
length (Alegre and Subirana, 1989), S. pombe chromatin in
F1.1 might thus have a smaller fibre diameter than mouse
chromatin.
The spacer linking nucleosomes in S. pombe Int 5, F1.1,
DC1 and C127 nuclei was cleaved by limited micrococcal
nuclease digestion. The 30 bp difference in nucleosome repeat
lengths of S. pombe and mouse chromatin becomes exaggerated higher up nucleosome ladders. A multimer of four nucleosomes (C + 3N) from S. pombe is 626 bp (146+(3×160))
in length, whereas that from mouse chromatin is 701 bp.
Similarly, five linked yeast nucleosomes are 786 bp long, and
five mouse nucleosomes are 886 bp. Fig. 9A shows nucleosome ladders from S. pombe hybridised with dh and ade6. A
typical yeast nucleosome ladder is seen; for example, the C +
3N nucleosome array migrates at 600 bp, and C + 4N at <872
bp. The same was seen with other Int5 probes, including
SV2neo (data not shown), which, although not endogenous to
the yeast genome, is nonetheless packaged into typical yeast
nucleosomes. Fig. 9B shows hybridisation of dh and ade6 to
F1.1 nucleosomes. All yeast probes analysed in F1.1 hybridise
to a nucleosome ladder typical of C127 bulk chromatin, i.e.
four linked nucleosomes (C + 3N) migrate at > 603 bp and five
nucleosomes (C + 4N) at > 872 bp. There is no non-specific
cross-hybridisation to ladders from C127 cells (data not
shown). Thus, the yeast chromatin in the F1.1 genome has lost
its endogenous nucleosome pattern and adopted that of the host
genome. This is not specific to whole yeast chromosomes
introduced by fusion, but was also seen in the DC1 DNA transfectant (data not shown).
The nucleoskeleton
Since the packaging of yeast DNA into nucleosomes is indistinguishable from that of the surrounding mouse DNA in F1.1
we must look to higher-order chromatin packaging to explain
the anomalous cytological appearance of the alien DNA in
F1.1 cells. It has been suggested that eukaryotic chromatin is
gathered into loops, the bases of which are tethered to a proteinaceous nuclear matrix, scaffold or skeleton. Various biochemical approaches are used to assay for these attachments
and their structural and functional relevance and relationship
with each other are the subject of much controversy. We have
analysed attachment of the yeast DNA in F1.1 to the mouse
nucleoskeleton. Nucleoskeleton preparation attempts to keep
chromatin in close to physiological conditions, maintaining
many of the biochemical processes of the intact nucleus
(Jackson et al., 1988). Average mammalian loops sizes in such
preparations (80 kb) appear to be larger than those (15 kb)
obtained using scaffold preparation techniques (Jackson et al.,
1990). It is proposed that transcription and replication involve
attachment to the nucleoskeleton. An 80 kb average loop size
is consistent with the estimated average frequency of genes or
replication origins in mammalian cells. The size of nucleoskeleton loops has not been directly measured in yeasts, but
it is likely that the close packing of genes and replication
origins in yeast genomes means that the average loop size will
be considerably smaller than that of the mammalian genome
(Amati and Gasser, 1990). If such frequent attachments were
retained in F1.1 cells, this would produce a yeast transgenome
with a higher-order packaging different from surrounding
mouse chromatin.
Nucleoskeletons were prepared from F1.1 cells. After
HaeIII digestion and electrophoresis to remove unattached
chromatin, both the retained chromatin, remaining in the
beads, and the free chromatin, which had run into the gel, were
recovered. This allows a direct comparison between DNA
released from and retained on the nucleoskeleton, rather than
extrapolating from the hybridisation intensity between
retained and total DNAs (Jackson et al., 1990). Densitometry
of DNA recovered in both free (F) and retained chromatin (R)
fractions indicated that the retained fraction constitutes < 25%
Yeast chromosomes in mouse cells
479
Fig. 10. Retention of S. pombe sequences on the F1.1 nucleoskeleton. Total (T) F1.1 DNA and DNA released from (F) and retained on (R) the
F1.1 nucleoskeleton after HaeIII digestion were electrophoresed and transferred to hybridisation membrane. Gel scanning indicated that the
total amount of retained DNA (R) present on the gel is less than quarter that in the released fraction (R). Hybridisation was carried out with
probes derived from S. pombe Int 5: SV2neo, pUC9, ade6 and cura4-II.
of the DNA in the released fraction. This is an overestimate
of the percentage of the genome associated with the nucleoskeleton, since the efficiency of recovery of released
chromatin (spread across a large gel slice after electrophoresis) is poor relative to that of the DNA retained in the beads
(a relatively small volume of agarose). These DNA fractions
were hybridised to probes from the yeast transgenome (Fig.
10). Variable levels of nucleoskeleton association were
detected. ade6, showed no detectable hybridisation to the
retained (R) DNA fraction and thus no association with the
mouse nucleoskeleton. Hybridising bands of equal intensity
are seen in the released (F) and retained (R) fractions with
both a probe for SV2neo, a region of the yeast transgenome
being transcribed in F1.1 cells grown under G148 selection
(Fig. 10) and with ars1 (data not shown). This suggests some
weak or transitory association between these sequences and
the host nucleoskeleton. Two regions of the F1.1 transgenome
were found to be very highly enriched in the retained fraction.
One of these, detected by pUC9, is the pura4/SV2neo locus,
which is present as a tandem array of five to ten copies in S.
pombe Int5 (Allshire et al., 1987), it thus appears to be
strongly associated with the F1.1 nucleoskeleton. Using
cura4-II we were able to examine the associations of a contiguous 42 kb region of the yeast genome to the F1.1 nucleoskeleton. There are two hybridising bands (2.5 and 0.7 kb)
that are much more intense in the retained than in the released
fraction, suggesting a strong association with the nucleoskeleton (Fig. 10). Further examination of the cura4-II
cosmid revealed that the 2.5 kb band is part of the coding
region of the ura4 gene. Hybridising these same blots with
five probes from the mouse genome detected no host
sequences as enriched as these two yeast loci in F1.1 nucleoskeleton preparations. This suggests that regions of the yeast
transgenome of F1.1 can be intimately associated with the
rodent nucleoskeleton and that the frequency of attachment of
the yeast transgenome may be higher than that of the endogenous mouse genome.
Fig. 11. Transcription of S. pombe ade6 and ura4 genes in F1.1 cells.
Samples (1 µg) of total RNAs from F1.1, C127 and S. pombe were
reverse transcribed and the resulting 1st-strand cDNA was amplified
for 35 cycles by PCR with primers specific for the S. pombe ade6
and ura4 genes (+ lanes). To control for DNA contamination of
RNAs amplification was also carried out in the absence of revese
transcriptase (− lanes). Transcripts for both of these genes are
detected in RNA from F1.1 cells. The S. pombe RNA contains
substantial DNA contamination as evidenced by amplification
products in the − lanes, there is a very small amount of DNA
contamination in the F1.1 ade6 − lane, but this is clearly distinct
from the signal in the + lane. Size markers in bp are shown.
Transcription and replication of the yeast
transgenome
Nucleoskeleton attachment has been associated with transcription and replication (Jackson and Cook, 1993; Hozak et al.,
1993). We have investigated whether any of the yeast genes
present in F1.1 are capable of being transcribed. We examined
the expression of the ura4 and ade6 genes in F1.1 cells (Fig.
5A and B) by reverse transcriptase-PCR. Fig. 11 demonstrates
that transcripts from both of these genes are detectable in F1.1
cells after 35 cycles of amplification.
We determined the time of replication of the yeast insert
with respect to the mouse genome. A cell cycle analysis was
performed on exponentially replicating cells, and the replication of the insert, as determined by combined FISH and antiBrdU labelling, was matched to the replication pattern.
F1.1 cultures, pulsed at intervals with BrdU, were analysed
480
J. McManus and others
after 12 and 24 hours. The earliest samples, which were in G2
at the time of the BrdU pulse, had no labelled mitoses, as determined by immunofluorescence with an anti-BrdU antibody,
while the proportion of labelled divisions in successive
harvests rose as the cells that were in S phase when BrdU was
added came through to mitosis. The total F1.1 cell cycle time,
S phase and G2 period are approximately 14, 7.5 and 2 hours,
respectively (Fig. 12A).
To determine the relative time of replication of the yeast
transgenome, FISH with total yeast DNA (green signal) and
anti-BrdU labelling (red signal) were studied simultaneously
(Fig. 12B). When BrdU was present for the last 2 hours before
metaphase, a late labelling pattern was seen in the mouse chromosomes, but the yeast insert was unlabelled with BrdU (Fig.
12B, panels 1 and 2). Extending the exposure to the 3 hours
before metaphase labelled 17/23 yeast insert regions with BrdU
(Fig. 12B, panels 3 and 4). Thus, the yeast genome replicates
late in S phase, but it is not the last F1.1 DNA to undergo
synthesis. The time in S phase when replication of the yeast
transgenome is detected corresponds to a period when some of
the G bands of the mouse chromosomes are also being replicated. In panels 3 and 4 of Fig. 12B, the BrdU is spread
throughout the yeast insert, therefore the replication of this 510 Mb stretch of yeast DNA is temporally controlled in the
Fig. 12. Replication analysis of the S. pombe insert in F1.1 cells. (A) Percentage of F1.1cells labelled with an anti-BrdU antibody after
exposure to a pulse of BrdU for varying lengths of time prior to mitosis. The total cell cycle time (TCT) of these F1.1 cells is 14 hours of which
S phase comprises 7.5 hours. The point of the cell cycle during which the
S. pombe transgenome replicates is indicated by the large arrow.
A
(B) Combined BrdU detection and FISH with yeast DNA. F1.1 cells were
exposed to BrdU for 2 (panels 1 and 2) or 3 (panels 3 and 4) hours prior
to mitosis. Replicating DNA is detected with TRITC- anti-BrdU antibody
(red). Unreplicated portions of the genome were detected with DAPI. The
yeast transgenome was visualised by FISH with biotinylated yeast DNA
detected with avidin-FITC (green). Panels (a) show the combined
FITC/TRITC signals; panels (b) show the TRITC signal alone to indicate
the BrdU incorporation at the site of yeast insertion (arrowed). In panels 1
and 2 the yeast insert has not incorporated any BrdU. In panels 3 and 4
BrdU is incorporated throughout the yeast transgenome. In none of the
panels is the mouse DNA immediately adjacent to the insertion site
labelled by BrdU.
B
1a
1b
2a
2b
3a
3b
4a
4b
Yeast chromosomes in mouse cells
481
Fig. 13. Distribution of S. pombe insert signal among main nuclei
and micronuclei following X-irradiation. The numbers of
micronuclei containing FISH signal to total yeast DNA were scored
at increasing radiation doses (cGy). Large circles represent main
nuclei; small circles micronuclei. Black dots indicate the presence of
FISH signal.
context of a mouse chromosome and occurs in a defined period
of S phase. Mouse chromatin adjacent to the insertion site does
not incorporate BrdU at the same point in S phase as the yeast
transgenome (Fig. 12B, panel 4), suggesting that replication of
the yeast DNA is not occurring by invasion of replication forks
from flanking mouse DNA. Thus the yeast transgenome is
a
b
c
d
e
f
g
h
Fig. 14. SCE analysis of F1.1 chromosomes
following MMC treatment. BrdU was
incorporated into one chromatid of each pair and
detected with TRITC anti-BrdU (red). The sister
chromatid is not visible here but could be seen
with DAPI. FISH with biotinylated yeast DNA
(green) indicates the site of yeast integration.
Panels (a), (b) and (c) show SCEs occurring at
the site of the yeast transgenome. Panels (d), (e)
and (f) show S. pombe DNA not associated with
SCEs. Panel (g) shows a double SCE occurring
within the yeast insert. The presence of the
double exchange is more clearly seen when the
TRITC label alone is examined (panel h).
482
J. McManus and others
replicated in a temporally controlled manner, in the latter part
of S phase, using multiple origins of replication from within
the yeast transgenome itself.
Analysis of radiation-induced breakage within the
yeast insert
Fragile sites are induced if DNA synthesis or the time between
the end of replication and chromosome condensation is interfered with (Laird et al., 1987). The resulting gaps in chromosome structure are prone to breakage. The chromosome gaps
and constrictions in our yeast transgenomes, seen without
inducing agents, and the frequency of chromosome rearrangement at the site of S. pombe insertion (Fig. 1) suggest that this
region may be susceptible to chromosome damage and
breakage.
We were unable to analyse X-ray-induced breaks in F1.1
chromosomes, due to the severe reduction in mitotic index
following irradiation. However, X-rays also induces micronuclei, which arise from the failure of acentric fragments to attach
to the spindle and segregate during mitosis (Heddle et al.,
1991). If the yeast insert is a ‘hot-spot’ for radiation breakage,
FISH-positive micronuclei will be over-represented following
irradiation. Fig. 13 shows the results of FISH with total yeast
DNA on F1.1 nuclei and micronuclei following X-irradiation.
Hybridisation efficiency is generally high, since <12% of binucleate cells have no yeast DNA signal, except in the 100 cGy
sample, where 29% had no signal. The absence of signals in
some main nuclei in these cytocentrifuge preparations may be
because signal is lost in the focal depth of the specimen. This
is less of a problem with micronuclei, which, being smaller,
can be imaged in a single focal plane. Cells with signal in both
daughter nuclei and in a micronucleus may result from a
breakage within the insert producing both centric and acentric
fragments. The incidence of micronuclei was high, rising from
23% in controls to 55% at the highest radiation dose, yet FISHpositive micronuclei were found in < 2% of binucleate cells at
all doses (Fig. 13), indicating either that radiation causes no
substantial increase in breakage at the insert region, or that
yeast centromeric sequences present in the transgenome attach
broken fragments to the mitotic spindle.
Susceptibility of the S. pombe insert to DNA damage
as assayed by sister chromatid exchange
Fragile sites are recombinogenic; they are associated with high
frequencies of meiotic recombination and an increased number
of sister chromatid exchanges (SCEs) (Laird et al., 1987;
Hirsch, 1991). The frequency of mutagen-induced SCEs within
the yeast insert of F1.1 cells was measured using the DNA
alkylating agent mitomycin C (MMC). Table 1 shows the SCE
frequency in control and MMC-treated F1.1 cells, using the
FPG method (Perry and Wolff, 1974). To enable both SCEs
and the yeast insert to be identified simultaneously anti-BrdU
antibody was used to identify the unifilarly substituted
chromatid following BrdU incorporation during the penultimate S phase before harvest, following FISH with a yeast DNA
probe. Fig. 14 shows examples where SCEs have occurred
within (panels a, b and c) the yeast transgenome. In panels d,
e and f, there are no SCEs at the insertion site, but in panel e
SCEs are seen in other mouse chromosomes. In panels g and
h, the yeast insert has participated in a double SCE, no other
SCEs are present in this chromosome. The mean length of the
Table 1. Control and MMC-induced SCE frequency in
F1.1 cells in both the insert-bearing chromosome and
within the insert region itself
SCE/insert chromosome
Expected†
MMC (M)
0
3×10−8
10−7
SCE/cell
10.9
25.5
76.6
0.177
0.414
1.245
Observed
n
/chr
/insert
100
96
79
0.16
0.55
1.19
0.02
0.27
0.42
†The expected SCE/insert chromosome was corrected for chromosome size
and calculated from FPG analysis in column 2 on the basis of 80
chromosomes/cell.
yeast-insert-bearing chromosome in in situ preparations is 5.45
µm compared with 4.18 µm (n=312) for the rest of the mouse
karyotype, i.e. it is 30% longer than the average. The mean
length of the yeast insert in these same preparations was 1.1
µm (20% of total chromosome length). Since SCEs are
assumed to be distributed among chromosomes according to
length, the expected SCE/insert chromosome values in Table
1 are corrected for the length of the insert-bearing chromosome. FPG analysis showed that the average SCE
frequency/cell rose from 11 in untreated cells to 26 and 77 in
cultures treated with 3×10−8 M and 10−7 M MMC, respectively. The expected numbers of SCEs in the insert chromosome agree quite well with the data obtained by combined
FISH/anti-BrdU analysis (Table 1). In controls only 2/16 SCE
in 100 chromosomes were within the insert, consistent with
this region occupying 20% of the physical chromosome length.
At low, 3×10−8 M MMC, 26/53 SCEs in 96 chromosomes were
clustered at the insert, and the corresponding values at the
higher (10−7 M) MMC concentration were 33/94 SCE in 79
chromosomes. Thus, while the induced SCE/chromosome
accords with expectations, the SCE cluster within the insert
region, whereas the rest of that mouse chromosome is underrepresented. It would appear that the insert region is a hot-spot
for the formation of SCE, and also interferes with SCE
formation in the rest of that chromosome.
DISCUSSION
The metaphase morphology of S. pombe DNA in
mouse cells is distinctive
Our understanding of higher-order chromosome structure is
poor, largely due to the complex nature of mammalian
genomes and the absence of tractable experimental systems.
Introducing a large cytologically visible stretch of defined
yeast DNA into a mammalian chromosome allows us to
analyse facets of chromatin structure that affect the morphology of the metaphase chromosome. We have shown that a
stretch of S. pombe DNA several megabases long, inserted
independently at various places in the mouse genome, exhibits
very unusual properties at metaphase compared with flanking
mouse DNA, under both the light and electron microscopes.
The yeast DNA adopts a metaphase chromosome morphology
distinct from the bulk of the mouse genome: it is much
narrower and stains less intensely with propidium iodide than
mouse euchromatin (Figs 1, 4). Similar cytological features are
Yeast chromosomes in mouse cells
not seen when similar lengths of mammalian chromosomes are
inserted into mouse cells by fusion (Porteous et al., 1989), but
have been documented where mouse cells have been fused
with S. cerevisiae spheroplasts (Featherstone and Huxley,
1993; Nonet and Wahl, 1993).
It has been suggested that the S. pombe DNA compaction
ratio at metaphase (at least in mitotically arrested S. pombe
mutants) is about five times less than for mammalian DNA.
The width of S. pombe metaphase chromosomes is estimated
to be 0.2-0.4 µm, and the smallest chromosome (3.5 Mb) has
a length of just under 1 µm (Umesono et al., 1983). The width
of the yeast insert in F1.1 metaphase chromosomes (harvested
by mitotic shake off) is just over two-thirds of that of the mouse
chromosomal regions measured by either light microscopy or
SEM, with an average width of 1.2 µm under the light microscope and 1.3 µm under EM. Thus the width of the yeast
transgenome appears to be much greater than that of endogenous S. pombe chromosomes, but less than the width of a
normal mouse chromosome arm. The length of the yeast insert
in F1.1 is approximately 1 µm, by both light and electron
microscopy. Since we estimate that the F1.1 yeast transgenome
contains 5-10 Mb of DNA the packaging of S. pombe DNA in
the F1.1 mouse cell may be intermediate between that of the
normal fission yeast chromosome and a mammalian chromosome. However, the DNA concentration in the yeast insert
appears similar to that in the host mouse chromatin, except in
the most constricted chromosomes, where the yeast DNA
seems to be more concentrated (Fig. 2).
Composition of the F1.1 transgenome
Large stretches of the S. pombe chromosome under selection
(III), are present in F1.1. We also detect some DNA from the
other two yeast chromosomes (Fig. 6). Similarly, retention of
DNA non-syntenic with the locus under selection is seen when
human chromosomes are introduced into mouse cells by fusion
(Porteous et al., 1989). The yeast DNA in F1.1 has undergone
some rearrangement. Although conventional Southern blotting
shows that the integrity of the yeast DNA has been largely
maintained at the kilobase level, PFGE analysis indicates that
long-range rearrangement has occurred (Fig. 6). By contrast,
yeast transgenomes generated by DNA transfection show
extensive rearrangement at the kilobase level, consistent with
other studies where the integrity of transgenomes generated by
fusion and transfection have been compared (Bickmore et al.,
1989).
All three S. pombe chromosomes are present in F1.1 (Fig.
6). We do not know whether S. pombe centromeric sequences
retain any function in mammalian cells. No aberrant interaction of the yeast transgenome with the mouse mitotic spindle
has been detected but persistent association of sister chromatids at the site of yeast integration has been seen in F1.1 at
anaphase, even though the chromatids apprear to be fully
resolved (Fig. 7). Thus, mouse proteins involved in holding
chromatids together at centromeres may interact inappropriately with the yeast centromeric sequences in F1.1.
No yeast telomeric sequences were detected in F1.1 (Fig. 6).
We do not know whether S. pombe telomeres function in
mouse cells but their loss may have contributed to the coligation of yeast chromosomes and the rearrangement of the
rDNA loci at the ends of chromosome III (Allshire et al.,
1987). In total we estimate that 5-10 Mb of yeast DNA is
483
present in F1.1, at the site of constriction. Whilst FISH analysis
suggests that this site is composed solely of yeast DNA we
cannot exclude the presence of small, cytogenetically subvisible, pieces of mouse DNA interspersed with the yeast
transgenome.
The yeast transgenome is progressively methylated
We have shown that the introduced yeast DNA is efficiently
methylated by de novo DNA methyltransferase activity in F1.1
cells (Fig. 8); however, the progressive methylation of the
yeast DNA does not appear to affect the metaphase chromosome structure of the yeast transgenome. Methylation
generally correlates with condensed inert chromatin bound by
methylated DNA-binding proteins, whereas unmethylated
DNA has an open chromatin structure (Lewis and Bird, 1991).
DNA hypomethylation is associated with undercondensation
of both constitutive heterochromatin (Mitchell, 1992) and
euchromatin (Haat and Schmid, 1989). However, in the F1.1
yeast transgenome the presence or absence of methylation and
associated DNA-binding proteins does not seem to be an
important determinant of the level of metaphase chromosome
structure that we are examining.
S. pombe DNA in F1.1 is assembled into
nucleosomes typical of mouse chromatin
Yeast chromatin has a nucleosome repeat distinct from that of
mammalian chromatin. However, we have shown that the F1.1
yeast transgenome adopts a nucleosome pattern typical of bulk
mouse DNA (Fig. 9). We conclude that the positioning of
nucleosomes on the yeast DNA in F1.1 is not dictated by either
the DNA sequence or the previous position of nucleosomes on
the introduced chromatin. This suggests that after DNA replication the yeast DNA is packaged into nucleosomes as if it
were mouse DNA with no apparent reference to the position
or frequency of nucleosomes on the incoming yeast chromosomes. This may have implications for models of chromatin
assembly onto newly synthesised DNA in mammalian cells
(Adams and Workman, 1993). Similar results have been
reported by Bernardi et al. (1992), who observed that nucleosome position was not maintained when either the URA3 gene
of S. cerevisiae was analysed in S. pombe, or when the S.
pombe ade6 gene was analysed in S. cerevisiae.
S. pombe DNA associates with the mammalian
nucleoskeleton
The only level of chromatin packaging of the yeast
transgenome in F1.1 cells that appears to be unusual is its association with the mouse nucleoskeleton. Several regions of the
F1.1 yeast transgenome are associated with the mouse nucleoskeleton (Fig. 10). The frequency and strength of association
appears to be high and may be analogous to the small scaffoldloop size seen in yeast (Amati and Gasser, 1990). A small loop
size may also explain the poor staining of the F1.1 yeast insert
with intercalating dyes such as propidium iodide, which are
sensitive to the degree of supercoiling (Fig. 1).
The S. pombe genome can be transcribed in mouse
cells
Little is known about scaffold or skeleton attachment sites in
mammalian or yeast chromosomes, but association with the
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J. McManus and others
processes of replication and transcription have been suggested.
Gene and replication origin density in the S. pombe genome is
much higher than in mammalian genomes. The transcription
machinery of fission yeast has similarities to that of
mammalian cells (Jones et al., 1988) and S. pombe genes can
be transcribed in a HeLa cell extract (Kleinschmidt et al.,
1990). We have shown that both mouse DNA polymerases (see
below) and RNA polymerases (Fig. 12) are able to act on the
F1.1 yeast transgenome. The frequency of these interactions
with the S. pombe DNA may translate into a high frequency of
association with structures such as the nucleoskeleton.
The behaviour of S. pombe DNA in mouse cells
shares features in common with induced fragile
sites
The S. pombe F1.1 insert superficially resembles centromeric
heterochromatin, secondary constrictions, gaps in PCCs and 5azacytidine-treated chromosomes, and induced fragile sites.
However, there are far more similarities to fragile sites than to
any of these other phenomena. At the light microscope level,
we see more dramatic constriction of the chromatids and
understaining than that seen at centromeres or nucleolar organisers (Fig. 1). The yeast insert is also negative for both Cbanding and Ag-NOR staining (Fig. 3). Under the EM, the S.
pombe insert does superficially resemble the centromeric heterochromatin (Fig. 4), yet it does not consist of repetitive DNA
and, unlike the centromeres, it is clearly resolved into separate
chromatids at metaphase.
Undercondensed chromosome regions can be induced by
demethylation of mammalian DNA with 5-azacytidine (Haaf
and Schmid, 1989). However, we have shown that the appearance of the F1.1 transgenome does not vary with its methylation state (see above).
In some of cases, chromosome constriction has been linked
to delayed DNA replication or replication occurring just prior
to entry into metaphase. Fragile sites can be induced by agents
that interfere with DNA replication or which shorten G2 so that
late-replicating DNA regions are prematurely advanced into
metaphase (Laird et al., 1987). The fragile X syndrome is associated with delayed replication of DNA around the FMR1 gene
(Hansen et al., 1993). The undercondensed regions of prematurely condensed chromosomes (PCCs) correspond to regions
where DNA was in the process of being replicated immediately prior to forced chromosome condensation (Gollin et al.,
1984). The constriction in the F1.1 transgenome might result
if the S. pombe DNA replicated unusually late in the cell cycle,
perhaps because of inappropriate interactions between yeast
DNA replication origins and the mouse DNA replication
machinery. We have established that the whole S. pombe insert
in F1.1 cells replicates towards the end of, but within the
normal limits of, S phase (Fig. 12), corresponding to a time
when some of the G bands of the mouse euchromatin are also
being replicated, but before replication of the very latest
regions of mouse euchromatin (Vogel and Speit, 1986). Mouse
chromatin adjacent to the insertion site appears to complete
replication before the yeast transgenome begins, suggesting
that replication of the yeast DNA initiates within the yeast
DNA itself. BrdU labelling of the yeast transgenome appears
simultaneously throughout the integration site (Fig. 12), suggesting that multiple origins of replication within the S. pombe
DNA are being used and are coordinately regulated.
The narrowness, reduced staining and occasional gaps in the
S. pombe insert are all features reminiscent of fragile sites.
Consistent with this, in a small percentage of cells there is a
small chromosome fragment with a portion of the yeast insert
at one end (Fig. 1, panel 8). The length of the fragment corresponds to the expected distance from the telomere of the S.
pombe-containing chromosome to the site of yeast insertion,
suggesting that the chromosome has broken within the
transgenome. In addition to constriction, the yeast insert also
has gaps in the chromosome structure under the light microscope (Fig. 1). The gap in the fragile X chromosome is
connected by a 25 nm diameter chromatin fibre (Harrison et
al., 1983). PCC chromosomes also have gaps at the sites where
DNA replication was still occurring when condensation took
place and the size of these chromatin fibres is similar to those
seen in fragile X (Nussbaum and Ledbetter, 1986). However,
we have not seen such gaps by SEM in F1.1 (Fig. 4).
Fragile sites show increased frequencies of induced SCEs
(Laird et al., 1987; Hirsch, 1991). In F1.1 cells, 35-50% of
mitomycin C-induced SCEs occurring on the chromosome harbouring the yeast transgenome are at the site of S. pombe integration itself. This was shown dramatically in several cases
where there were two exchanges within the S. pombe insert
(Fig. 14, panels g and h). The mode of action of MMC is
unclear; monoadducts on DNA can interfere with the interaction with enzymes such as DNA polymerase (Basu et al.,
1993), bisadducts can result in inter- or intrastrand cross-links
(Bizanek et al., 1992). Both of these mechanisms could
influence the frequency of SCEs and the specific increase
within the S. pombe insert region could reflect increased susceptibility to alkylation or decreased susceptibility to repair of
DNA damage, compared to the rest of the mouse genome.
Metaphase chromosome condensation
Undercondensation or constrictions in mammalian metaphase
chromosomes have been interpreted as a failure of the final
level of coiling that normally takes place in euchromatin
(Rattner, 1992). However, this interpretation is controversial
and highlights our general ignorance of chromosome
packaging. This final coiling may be absent in normal yeast
chromosomes, which are less condensed than their mammalian
counterparts (Umesono et al., 1983), and aspects of this may
persist in S. pombe/mouse fusion hybrids. Similar structural
features have been noted where S. cerevisiae chromosomes are
integrated into mammalian chromosomes (Fetherstone and
Huxley, 1993; Nonet and Wahl, 1993). Why might yeast
DNAs in mammalian cells have unusual higher-order chromosome packaging? We have shown that the DNA modification
and lower-order chromatin structure of the yeast transgenome
are similar to those of the host mouse genome. At higher levels
of packaging we detect association of the yeast DNA with the
mouse nucleoskeleton at a frequency that may reflect the
attachments in endogenous yeast genomes (Amati and Gasser,
1990). Nucleoskeleton attachment may have a direct or an
indirect impact on the metaphase chromosome structure. It has
been suggested that the ‘knobs’ seen on chromosomes under
the EM are the tips of chromatin loops anchored at the chromosome core (Marsden and Laemmli, 1979), consistent with
the idea that mitotic chromosomes are organised into a series
of radial loops, which are then coiled and condensed (Rattner
and Lin, 1985). The projections of the yeast insert in F1.1 chro-
Yeast chromosomes in mouse cells
mosomes (Fig. 4) do indeed appear to be shorter than those of
the surrounding mouse chromatin.
Constriction of metaphase chromosomes may occur
wherever condensation is sterically inhibited by persistent
association with protein complexes. For example, nucleolar
components persist in the metaphase chromosome at the sites
of nucleolar organisers (Goessens, 1984). Condensation may
be hindered where DNA polymerase complexes remain at sites
of DNA synthesis in S phase chromosomes forced into
premature condensation (Gollin et al., 1984). Since fragile sites
have delayed replication (Hansen et al., 1993), Laird et al.
(1987) proposed that their chromatin is incompletely
condensed because it is too closely associated with the nuclear
skeleton, perhaps through the DNA replication machinery
(Hozak et al., 1993), and that this may produce the force for
breakage at fragile sites. Similarly, S. pombe DNA in mouse
cells is constricted, associated with the nucleoskeleton and
prone to breakage.
On a practical note, this study illustrates the fate of large
stretches of yeast chromosomes introduced into mammalian
cells by fusion. This route will be one that will be increasingly
taken by those introducing YACs into mouse or human cells
to complement mutations or establish genotype/phenotype
relationships (Pavan et al., 1990; Jakobovits et al., 1993).
J. McM. was supported by an MRC Research Studentship, D.M.W
was a Research Associate of the CRC. N.D.H. is an International
Scholar of the Howard Hughes Medical Institute. W.A.B is Lister
Institute Research Fellow. We thank the MRC HGU photography
department for photographic assistance and Judy Fantes for her FISH
expertise and advice, and Daryll Green for gel scanning. The laboratory of Prof. M. Yanagida (Kyoto) kindly provided the S. pombe
cosmid library and DNA probes. S. pombe DNA probes were also
obtained from N. Sugawara (Boston), P. Schuchert, (Bern) and P.
Fantes (Edinburgh). We also thank E. Maier (London) for assistance
in mapping of ars1.
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(Received 5 October 1993 - Accepted 10 December 1993)