Replication of yeast DNA and novel chromosome formation in mouse cells
Replication of yeast DNA and novel chromosome formation in mouse cells
Amanda
McGuigan
and
Clare
Huxley*
Department of Biochemistry and Molecular Genetics, Imperial College School of
Medicine at St Mary's,
London
W2 1PG,
UK
Received March 12, 1996;
Revised and Accepted April 25, 1996
ABSTRACT
To determine whether yeast DNA can replicate or segregate in mammalian cells, we
have transferred genomic DNA from the yeast
Saccharomyces cerevisiae
into mouse cells. Most of the lines contained stably integrated yeast DNA.
However, in two of the lines, the yeast DNA was maintained as numerous small
extrachromosomal elements which were still present after 26 cell divisions in
selection but which were lost rapidly out of selection. This indicates that,
although yeast DNA can replicate in mouse cells, the yeast centromere does not
function to give segregation. In one cell line we observed a large novel
chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably
comprise the functional centromere. The yeast DNA in the novel chromosome has a
compacted chromatin structure which may be responsible for the efficient
formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse
chromosomes forms constrictions at the point of integration. These features
have previously been presumed to be hallmarks of centromeric function in
transfection assays aimed at identifying putative centromeric DNA. Hence our
results suggest caution be exercised in the interpretation of such assays.
INTRODUCTION
The DNA sequences necessary for replication and segregation in mammalian cells
are poorly understood. In contrast, replication origins and centromeric
sequences in the yeast
Saccharomyces cerevisiae
are well defined. Replication origins were isolated by their ability to confer
replication on a plasmid and are called `autonomously replicating sequences'
(ARSs) (reviewed in
1
). Plasmids containing an ARS element are maintained extrachromosomally if
selection is maintained but are rapidly lost from the culture if selection is
dropped, due to unequal segregation. DNA with ARS activity, as determined by
the plasmid assay, generally correlates with replication origins located in the
yeast chromosomes. Centromeric DNA stabilizes ARS plasmids in yeast by
conferring equal segregation at mitosis (reviewed in
1
).
Similar experiments involving transfection of plasmid- or cosmid-cloned fragments of mammalian DNA into mammalian cells have not led
to the functional characterization of either origins of replication or
centromeres. A number of different origins of replication have been located
within mammalian chromosomes by a variety of methods. In particular, one
located ~17 kb downstream of the dihydrofolate reductase (
Dhfr
) gene in Chinese hamster chromosomes has been well characterized by a number of
different investigators (reviewed in
2
,
3
). However, when the DNA encompassing this putative origin of replication was
introduced back into mammalian cells it was not found to replicate any more
efficiently than neighbouring DNA known not to contain a chromosomal
replication origin (
4
) and it does not form extrachromosomal elements which can be maintained under
selection. One explanation for the observation that DNA containing a known
mammalian chromosomal replication origin does not generally form
extrachromosomal replicating elements in mammalian cells (similar to ARS
plasmids in yeast) is that a nuclear localization signal is needed in addition
to the ability to replicate (
5
). Thus, a plasmid carrying a 13.3 kb fragment containing the
Dhfr
replication origin replicates efficiently in a transient replication assay over 4 days, but is not stable over 15 days (
6
). However, if a nuclear retention signal (but not a replication origin) is
added from the latent origin of replication (OriP) of Epstein Barr virus, along
with the viral protein EBNA1, the plasmid is stable over 15 days (
6
).
Transient replication assays have been used to show that replication of
introduced fragments of DNA is dependent on the size of the DNA, with fragments of human DNA larger than ~12 kb replicating efficiently (
7
). In addition, yeast DNA has been shown to replicate only marginally less
efficiently than human DNA, while more CG-rich bacterial DNA replicates significantly less efficiently (
8
). This has led to the general hypothesis that replication in mammalian cells is
determined by chromatin context in the chromosomes but that extrachromosomal
DNA is replicated in a way that is dependent on size, but independent of
sequence (references above and reviewed in
9
).
DNA cloned in yeast artificial chromosomes (YACs) has been transferred into
mammalian cells and has given rise to extrachromosomal replicating elements in
mammalian cells. When the YAC yHPRT, which contains 660 kb of human DNA, was
introduced into mouse L A-9 cells, about half the resulting cell lines contained yeast and YAC DNA
as extrachromosomal elements in some of the cells (
10
). Similarly, when a YAC containing ~70 kb near the adenosine deaminase gene was transferred to either a Chinese
hamster ovary cell line or another mouse fibroblast line, extrachromosomal elements were observed in two out of nine cell lines (
11
). However, in each case, the extrachromosomal elements contained both human DNA
and yeast genomic DNA, making it impossible to conclude which DNA was responsible
for replication of the DNA in the mouse cells.
Transfection experiments aimed at determining whether putative centromeric DNA can function in mammalian cells have also led to results that are difficult to interpret. The mammalian centromere is located at the primary constriction where several megabases of repetitive
DNA are generally located and this has precluded the cloning of the centromeric
regions in an intact and unrearranged form. Interest has centred on the [alpha] satellite DNA in man and the sequence which is thought to be
functionally equivalent in mouse, minor satellite. The [alpha] and minor satellites are tandemly repeated sequences located at the
centromeres of all human or mouse chromosomes. Alphoid DNA is always retained
when truncations of the human Y chromosome are selected by retention of a
functional centromere (
12
). Also, natural deletions of the Y chromosome always carry alphoid DNA at the
functioning centromere (
13
), indicating that these sequences are probably necessary for Y centromere
function. Although transfection of alphoid DNA into mammalian cells has not led
to efficient centromere formation, it does give several features of centromeres. In one set of experiments, alphoid DNA was introduced into African green monkey
cells (
14
). After integration into a host chromosome, anaphase bridges were observed
which could be due to a dicentric chromosome. In addition,
de novo
chromosomes were observed which consisted largely of the input alphoid DNA, not host cell
alphoid DNA, and had a single functional centromere (
14
). In another set of experiments, alphoid DNA integrated into human chromosomes
was observed to form constrictions and anaphase bridges (
15
). A
de novo
chromosome has also been observed after transfection of a [lambda] clone containing putative centromere DNA (
16
).
DNA cloned in YACs has been transferred into mammalian cells to assay both
replication ability and centromere function. As some yeast genomic DNA is often
transferred along with the cloned DNA, it is important to know whether yeast
genomic DNA alone can replicate and whether the yeast centromeres function in
mammalian cells. It is also important to know how heterologous non-specific DNA behaves in mouse cells so that one can compare this with the
behaviour of DNA with putative replication and centromere function. In this
paper we have transferred yeast genomic DNA to mouse cells and determined the
fate of the yeast DNA.
MATERIALS AND METHODS
Yeast culture, transformation and DNA preparation
The yeast strain F9 is derived from the strain AB1380 (
17
) by integration of a mammalian selectable marker, neomycin resistance (
neo
r
), into the
ura3
gene on yeast chromosome V. First, a yeast strain with a YAC carrying the human
factor IX gene was isolated from the Washington University library (
18
). The retrofitting vector pLUNA was then introduced and integrated into the
chromosomal copy of the
ura3
gene rather than the
URA3
gene on the YAC (
19
). This strain was grown overnight without selection and single colonies grown
up and checked for loss of the YAC. F9 is a resulting colony which carries the
neo
r
marker on chromosome V and contains no human DNA. Agarose blocks of high molecular weight DNA were made by a previously published protocol (
20
) using the modifications previously described (
21
).
Mammalian cell culture and fusion with yeast spheroplasts
Mouse cell line L A-9 (GM00346B), which is negative for hypoxanthine phosphoribosytransferase
(HPRT) activity, was obtained from the NIGMS Human Genetic Mutant Cell Repository. The chromosomes in this mouse cell line are mostly metacentric and there are
also chromosomes with multiple centromeres. L A-9 cells were cultured in DMEM containing 10% fetal bovine serum at 37oC with 5% CO
2
. Fusion between L A-9 cells and yeast spheroplasts was carried out as previously described (
10
). After fusion, cells were grown with 600 [mu]g/ml active G418 (Gibco BRL) in order to select for the
neo
r
gene. Colonies arose at a frequency of ~2 * 10
-6
and only one colony was picked from each plate. Cells were tested regularly for
mycoplasma and were always found to be negative.
Each fusion colony was expanded to ~10
8
cells, at which point the rate of loss of the selectable marker was determined
(Table
1
), DNA was made (Fig.
1
) and cells were stored under liquid nitrogen. The cells were subsequently
thawed and grown with or without selection for all analyses by fluorescence
in situ
hybridization (FISH) and for Figure
5
.
Gels, DNA transfer and hybridization
DNA was transferred from agarose gels to Hybond N (Amersham) as recommended by the manufacturer. DNA probes were labelled using the
Megaprime kit (Amersham) and purified using commercially available push columns
(Stratagene NucTrap probe purification columns).
Prehybridization and hybridization of Southern blots was carried out in a
modified version of Church buffer (
22
) (16.8 g/l NaH
2
PO
4
[middot]H
2
O, 54.1 g/l Na
2
HPO
4
[middot]12H
2
O, 7% SDS, 100 [mu]g/ml denatured salmon sperm DNA) at 65oC. Filters were washed in 0.5* SSC, 0.1% SDS at 65oC for 30 min. To strip the filters, 500 ml 0.1* SSC, 0.01 M EDTA, pH 8.0, was boiled and then SDS
added to 0.1%. This mixture was added to the filter and left shaking at room
temperature until cool.
Probes
The following probes were used on Southern blots. The
neo
r
probe is a 1.1 kb
Xho
I-
Hin
dIII fragment from the plasmid PMC1Neo Poly A (
23
). The
Ty1
probe is a 1.2 kb
Xho
I-
Hin
dIII fragment from the plasmid pCS-X (
24
). The single copy mouse probe is a 1.7 kb
Bam
HI-
Sst
I fragment of the mouse
Utrophin
gene (a gift of Ms U. Gangadharan).
The following probes were used for FISH. Total yeast DNA was prepared from the
strain AB1380. Mouse minor satellite was prepared by PCR from total mouse DNA
using the primers 5'-AAATCCCGTTTCCAACGAATGTG-3' and 5'-GTAGAACAGTGTATATCAATGAG-3'. The major satellite probe
was a 200 bp
Pst
I fragment excised from the plasmid R531 (a gift of David Kipling). Total mouse
DNA was prepared from C57BL/6 tissue.
Fluorescence
in situ
hybridization
Rapidly dividing cells were incubated with a low concentration of colcemid
(final concentration 0.01 [mu]g/ml) for 1 h before fixation. Generally this gave a reasonable proportion of cells in metaphase. In one
pellet of cells from the line F9-12 (4 days in selection and 4 days with no selection; see Table
2
) there was quite a high percentage of anaphase cells in addition to metaphase
cells.
Preparation of slides and fluorescence
in situ
hybridization (FISH) were carried out largely as described elsewhere (
25
). Labelling with biotin-14-dATP was carried out by two methods. Yeast genomic DNA and mouse
genomic DNA were labelled using a Bionick kit (Gibco BRL), whereas the mouse
minor PCR product and the plasmid-derived fragment of major satellite DNA were labelled using a Bioprime kit
(Gibco BRL). For double detections, total yeast DNA was labelled with
digoxigenin-dUTP using a DIG DNA labelling kit (Boehringer Mannheim).
Aliquots of 100 ng probe, 10 [mu]g sonicated and denatured salmon sperm DNA and 10 [mu]g
Escherichia coli
tRNA were hybridized per slide. Washing and detection for single detection of
biotin-labelled yeast DNA was carried out as follows. Slides were washed for 20
min in 50% formamide, 2* SSC at 37oC, then for 20 min in 2* SSC at 37oC and then for 20 min in 1* SSC at room temperature. Avidin/FITC mixture
(200 [mu]l, 5 [mu]g/ml avidin/FITC, 4* SSC, 1% Marvel) was then added and a cover slip placed over the
solution followed by incubation in a moist chamber at 37oC for 1 h. The slides were then washed three times for 5 min in 4* SSC, 0.1% Tween-20 at 42oC and then placed in a propidium iodide counterstain
solution (0.2 [mu]g/ml propidium iodide, 2* SSC) for 15 min at room temperature and then destained (2* SSC, 0.05% Tween-20) for 1 min at room temperature.
Double detection of digoxigenin-labelled yeast DNA and another biotin-labelled probe was carried out as follows. The biotin was detected
as above up to the three washes in 4* SSC, 0.1% Tween-20 at 42oC. Then 200 [mu]l mouse monoclonal anti-digoxigenin antibody (Sigma, D8156) mixture (5 [mu]l/ml in 4* SSC, 1% Marvel) was added and the slides
incubated in a moist chamber at 37oC for 1 h. The slides were then washed three times for 5 min in 4* SSC, 0.1% Tween-20 at 42oC. Anti-mouse IgG TRITC conjugate (200 [mu]l) (Sigma, T2402) mixture (1 [mu]l/ml in 4* SSC, 1% Marvel) was then added and
incubated at 37oC for 1 h. The slides were then washed three times for 5 min in 4* SSC, 0.1% Tween-20 at 42oC. DAPI (25 [mu]l, 0.2 [mu]g/ml) antifade solution was applied to the slide.
In the case of the total mouse probe, 2 [mu]g of probe were used and the signal was amplified. The slide was washed with
50% formamide, 2* SSC for 20 min at 42oC, then 2* SSC for 20 min at 42oC and then 1* SSC for 20 min at room temperature. The biotin
was detected as above up to the three washes in 4* SSC, 0.1% Tween-20 at 42oC. Then 200 [mu]l of a mixture of 5 [mu]l/ml mouse anti-digoxigenin antibody (Sigma, D8156) and 5 [mu]g/ml Biotinylated Anti-Avidin D (Vector Laboratories) in 4* SSC, 1% Marvel was added and
incubated at 37oC for 1 h. The slides were then washed three times for 5 min in 4* SSC, 0.1% Tween-20 at 42oC. Then 200 [mu]l of a mixture of 5 [mu]g/ml avidin/FITC and 1 [mu]l/ml anti-mouse IgG-TRITC conjugate in 4* SSC, 1% Marvel was added
and incubated at 37oC for 1 h. The slides were washed once more and stained with DAPI as above.
Slides were visualized on a Leitz Aristoplan Microscope and photographed using
Fujichrome ASA 1600 slide film.
RESULTS
Transfer of yeast genomic DNA into mouse L A-9 cells by fusion with yeast spheroplasts
The yeast strain F9 used for these experiments is a derivative of AB1380. It
carries a mammalian selectable marker, resistance to the drug G418 (
neo
r
), integrated into the
ura3
gene on chromosome V. The yeast genomic DNA from the strain F9 was transferred
to mouse L A-9 cells (an established mouse fibroblast line) by fusion with yeast
spheroplasts followed by selection with G418 to select for cells which had
taken up yeast DNA. Seventeen independent cell lines (called F9-1 to F9-17) were grown up from colonies on separate plates.
The DNA content of the cell lines was determined by Southern blotting. All of
the cell lines were found to carry the
neo
r
gene, though at widely varying copy number, as shown in Figure
1
A (data for lines F9-1 and F9-17 not shown). They were also analysed for the yeast repetitive
element
Ty1
DNA, which is present at ~30 copies spread throughout the yeast genome.
Ty1
DNA was present in all the cell lines except F9-6 and F9-16, as shown in Figure
1
B (data for lines F9-1 and F9-17 not shown). Finally the blots were hybridized with a single copy
mouse probe to confirm roughly equal loading of the mouse DNA in each lane (data not shown). These results are summarized in Table
1
.
Fluorescence
in situ
hybridization (FISH) was then used to determine the fate of the yeast DNA in
the mouse cells. The probe was total yeast DNA labelled with FITC (green) and
the chromosomes were counterstained with propidium iodide (red), as shown in
Figure
2
. The cell lines F9-5, F9-13 and F9-15 were all found to contain a single, small integration of
yeast DNA into a mouse chromosome (Fig.
2
A and data not shown). In two of the cell lines the yeast DNA was found in a
number of different forms in different cells. Cell line F9-11 was found to be a mixture of cells containing small integrations of
yeast DNA (27% of cells), cells containing large numbers of small
extrachromosomal elements (32% of cells, Fig.
2
C) and cells containing no yeast DNA (40% of cells). Cell line F9-12 was found to contain cells with small integrations of yeast DNA (23% of cells), cells with large numbers of small extrachromosomal elements (17% of cells, Fig.
2
E), cells with very large integrations of yeast DNA (18% of cells), cells with a
large novel chromosome (14% of cells, Fig.
2
F) and cells with no signal (28% of cells). The results for these two cell lines
are summarized in Table
2
.
Integrations of yeast DNA are stable and form a constriction
In three cell lines, F9-5, F9-13 and F9-15, the yeast DNA was found as integrations into a mouse
chromosome. The position of integration of the yeast DNA could generally be
seen on the propidium iodide or DAPI stained chromosomes as a constriction of
the chromosome, as shown in Figure
2
B. The stability of the yeast DNA in the integrations was investigated by
growing the cells without selection for ~25 days and then determining the percentage of cells still resistant to
G418. The G418 resistance in the fusion cell lines F9-5, F9-13 and F9-15 was found to be fairly stable, with <23% of cells losing the G418 resistance in ~25 days of growth without selection (Table
1
). These cells divide every ~24 hours out of selection, so for F9-5, where 23% of cells lost G418 resistance over 27 divisions, this
would correspond to 1% of cells losing the G418 marker per cell division. Most
of the other cell lines were not analysed by FISH. However, in 11 of the 17
cell lines, the G418 resistance was lost from <50% of cells in ~25 days of growth without selection (Table
1
), which would correspond to 3% per cell division. These cell lines probably
have stable integrations of the yeast DNA.
Extrachromosomal elements consisting of yeast DNA replicate, but segregate
poorly
In two of the fusion cell lines, F9-11 and F9-12, the yeast DNA in some of the cells was present as
extrachromosomal elements (Fig.
2
C and E). The elements occur at quite high copy number, several hundred per
cell, and are scattered amongst the chromosomes. The elements are present in
the cells after the cell lines had been expanded to ~10
8
cells (at least 26 cell divisions), suggesting that the yeast DNA can replicate
efficiently in the mouse cells. It is possible that the extrachromosomal
elements have picked up DNA from the mouse host cells and it is this DNA which
allows them to replicate. However, when total mouse DNA was used as a probe in
FISH, it hybridized to the mouse chromosomes very strongly, but no signal was
seen over the extrachromosomal elements (Fig.
4
C and D). Mouse minor and major satellites were also investigated and neither
were detected on the extrachromosomal elements (Fig.
3
A and C).
The ability of the elements to segregate at cell division was investigated by
growing the cell lines without selection for 19-22 days followed by determination of the number of cells still resistant
to G418. Ninety nine per cent of cells of F9-11 and 98% of cells of F9-12 had lost resistance to G418 after 19 and 22 days respectively of
growth out of selection (Table
1
), which would correspond to at least 24 or 18% of cells losing the G418
resistance per cell division respectively. Filter hybridization confirmed that
the loss of resistance to G418 was due to loss of the
neo
r
gene rather than inactivation of the gene (Fig.
5
). This loss in the absence of selection was analysed in more detail for F9-12, which was grown for 13 days without selection and analysed by FISH at
various times (Table
2
). Over this time the percentage of cells with extrachromosomal elements fell
from 17 to 0%, and this is largely matched by the rise in the percentage of
cells with no detectable yeast DNA, which rose from 28 to 39%. Going from 17 to
3% in 11 days (Table
2
) would correspond to 16% of cells losing the elements per cell division.
Clearly these extrachromosomal elements are maintained inefficiently in the
mouse cells.
.
Stability of yeast genomic DNA as determined by FISH
Cell line
Days in
Days out of
Number of
Per cent
Per cent extra
Per cent
Per cent
Per cent
selection
a
selection
a
metaphases
small
chromosomal
large
novel
no signal
scored
integration
elements
integration
chromosomes
F9-11
9
0
62
27
32
0
0
40
F9-11
12
0
53
34
28
0
0
38
F9-11
18
0
55
55
5
0
0
40
F9-11
31
0
69
93
1
0
0
6
F9-12
5
0
153
23
17
18
14
28
F9-12
4
4
120
28
13
19
14
26
F9-12
4
8
75
35
6
21
11
27
F9-12
4
11
123
33
3
17
11
36
F9-12
4
13
100
32
0
19
10
39
a
Cells were brought up from liquid nitrogen and grown first in selection and then
out of selection for the number of days shown before analysis by FISH.
A possible mechanism for loss of the extrachromosomal elements during growth
without selection can be visualized by FISH. At anaphase the extrachromosomal
elements are more widely spread through the cytoplasm than the chromosomes (Fig.
3
F). During interphase, micronuclei containing large amounts of yeast DNA were frequently seen close to the main nucleus of a cell (which
also contains yeast DNA), as shown in Figure
2
D. The micronuclei could be caused by packaging into distinct micronuclei of
elements which are distant from cellular chromosomes at anaphase. Micronuclei
containing yeast DNA were present in ~15% of cells in F9-11 and loss of these from the cells could account for the rapid loss
of the yeast DNA.
The novel chromosomes segregate and have picked up mouse centromeric DNA
FISH analysis revealed that 14% of cells in the line F9-12 contained novel chromosomes which were large and occurred at one or two copies per
cell, as shown in Figure
2
F. The low copy number of the chromosomes suggests that, unlike the high copy
number extrachromosomal elements, the novel chromosomes are segregating. The ability of the chromosomes to segregate was measured by growing the cell
line for a period of time without selection followed by FISH analysis to
determine how many of the cells carried the chromosome. The percentage of cells
carrying the novel chromosome went down from 14 to 10% during 13 days growth
without selection (Table
2
), which would correspond to only ~3% loss per cell division. This slow rate of loss is in marked contrast to
the small extrachromosomal elements, which went from 17 to 0% during the same
experiment, or ~16% loss per cell division. Thus, although not completely stable, the novel
chromosomes segregate well.
The ability of the novel chromosomes to segregate, while the extrachromosomal
elements do not, could be due to the larger size of the chromosomes or to the
acquisition of mouse DNA from the host cell. FISH analysis with mouse minor
satellite DNA as the probe showed that the novel chromosomes carry a region of
mouse minor satellite DNA at one end (Fig.
3
B). In contrast, no minor satellite DNA was detected on the extrachromosomal
elements, which do not segregate (Fig.
3
A). Major satellite was also observed on some of the novel chromosomes, but this
was present in widely varying amounts and was often not detectable (Fig.
3
D), and it was also not detectable on the extrachromosomal elements (Fig.
3
C). Finally, total mouse DNA was used as a probe and a small region could be
detected corresponding to the minor satellite DNA, but none was detected along
the bulk of the novel chromosome, indicating that the arms of this chromosome
consist almost entirely of yeast DNA (Fig.
4
A and B). Figure
4
also shows the compacted structure of the yeast DNA in the novel chromosome.
The minor satellite DNA on the novel chromosome is probably located at the
functional centromere, as it is on normal mouse chromosomes. This was tested by
observing the novel chromosome at anaphase. Out of 167 anaphase spreads of F9-12 examined, 10 (6%) had bridges consisting of mouse DNA, while 18 (11%)
had bridges which consisted of yeast DNA. When the bridges were observed after
hybridization with both yeast and minor satellite probes, it was found that the
mouse minor satellite had separated into the two anaphase clusters along with
the normal mouse centromeres, while the yeast DNA formed the bridge (Fig.
3
E). This indicates that the minor satellite is located at a functional
centromere which has segregated, while the yeast DNA is holding the two
chromatids together, forming an anaphase bridge.
DISCUSSION
We have analysed the fate of
S.cerevisiae
genomic DNA present in a number of cell lines formed by fusion of mouse L A-9 cells with spheroplasts of a yeast strain which carries the
neo
r
gene on yeast chromosome V. In the majority of the cell lines, the yeast DNA
has probably stably integrated into a mouse chromosome. However, in two of the
cell lines, the yeast DNA was found to be maintained as extrachromosomal elements or novel chromosomes.
Our observations on the behaviour of extrachromosomal elements suggest that
S.cerevisiae
DNA can replicate in mouse cells. Efficient replication of the yeast DNA is
supported by the presence of the elements after the cell lines had been
expanded for at least 26 cell divisions. It is possible that the yeast DNA is
being repeatedly excised from the chromosomes, but this is not likely, as there
is selection for integration rather than excision on prolonged growth in
selection. It is also possible that the extrachromosomal elements had picked up
small amounts of mouse DNA, though none was detected by FISH. The conclusion
that
S.cerevisiae
DNA can replicate in mammalian cells is consistent with the previous
observation that a chromosome from
Schizosaccharomyces pombe
is capable of forming similar unstable extrachromosomal elements in mouse cells
(
26
). Also, in a transient replication assay over 4 days, fragments of
S.cerevisiae
DNA have been shown to replicate in human cells almost as efficiently as human
DNA (
8
). However, this is unlikely to be due to use of the yeast replication origins
directly, as yeast ARS sequences are not preferentially utilized to initiate replication in mammalian cells (
8
) or
Xenopus
oocytes (
27
). This suggests that yeast origins of replication were not mediating
replication in the extrachromosomal elements described here.
The elements occurred at a high copy number per cell and they were lost rapidly
in the absence of selection (~16% of cells lost the elements per cell division), suggesting that they do
not segregate. This is very similar to ARS plasmids in
S.cerevisiae
, which are known to replicate efficiently but to segregate poorly, and also to
double minutes (DMs) in cancer and drug-resistant cell lines (
28
), which replicate efficiently but segregate with variable efficiency and
generally do not have a functional centromere. Small (between ~12 and 40 kb in size) fragments of eukaryotic DNA replicate efficiently in
human cells but do not form stable elements over longer periods unless viral
elements for nuclear localization are provided (
5
,
7
,
8
). The yeast-derived elements seen here appear to segregate well enough to be
maintained in the presence of selection and this could be due to yeast
sequences which increase nuclear retention or to the relatively large size of
the elements.
A novel chromosome formed almost entirely of yeast DNA was observed in a
proportion of the cells of line F9-12. These chromosomes are far larger than the unstable extrachromosomal
elements described above and they appear to have a functioning centromere, as
they occur at one or two copies per cell and segregate quite well in the
absence of selection (~3% loss per cell division). Indeed, on FISH analysis they were always found
to carry mouse centromeric minor satellite DNA, whereas the unstable
extrachromosomal elements did not. The mouse minor sequences and the
functioning centromere have clearly come from the mouse host during formation
of the chromosome, which has also involved some form of amplification or co-ligation of the yeast DNA. Novel chromosome formation thus appears to be a
feature of heterologous DNA introduced into mammalian cells.
The
S.cerevisiae
DNA in the mouse cells forms a compacted chromatin structure in comparison with
the mouse chromosomes. This can be observed in the constrictions formed at the
site of integration of the yeast DNA and also in the narrow structure of the
novel chromosome, which could be detected even without FISH analysis.
Constrictions have previously been observed at the position of integration of alphoid DNA (
15
), of non-centromeric YACs (
10
,
11
) and of
S.pombe
DNA (
29
). The compacted chromatin may also be responsible for the anaphase bridges
formed by the yeast DNA in the novel chromosomes. A considerable amount of work
has been carried out to try and understand the basis of the constrictions
formed by
S.pombe
DNA and it appears that the frequent attachment of the yeast DNA to the rodent
cell nucleoskeleton may be responsible (
29
).
A number of other investigators have reported the transfer of YAC and yeast
genomic DNA to rodent cells by fusion. Generally, the YAC DNA, with varying
amounts of yeast genomic DNA, has been observed by FISH to be integrated into
the mouse genome (
30
-
32
). We have previously transferred YAC DNA to the mouse LA-9 cell line used in this report and have observed extrachromosomal
elements in some of the cells in ~50% of the cell lines and these elements behaved very similarly to the
elements described in this paper (
10
). Similarly, extrachromosomal elements have been observed by Nonet and Wahl (
11
) in Chinese hamster cells and mouse cells. The fact that most other
investigators have not observed extrachromosomal elements could be due to the
use of other cell lines which do not support such elements efficiently or to
the fact that fast growing cell lines have been preferentially investigated,
which would select with stable integrations.
YACs can carry large inserts and may be useful for cloning the functional
elements of mammalian chromosomes, including replication origins and
centromeres. In this paper we have introduced
S.cerevisiae
genomic DNA into mouse cells. We find that yeast DNA is able to replicate in
mouse cells. This means that care must be taken when assaying DNA cloned in
YACs for ability to replicate to make sure that yeast genomic DNA is not
responsible for the replication. The yeast DNA is also involved in the
formation of constrictions, novel chromosomes and anaphase bridges. As there is
no evidence of centromeric activity of yeast DNA in mammalian cells, these
features should be interpreted as possibly being non-specific results of the introduction of exogenous DNA. Thus these observations are of importance in
interpreting the results of assays for centromere function.
ACKNOWLEDGEMENTS
We thank Dr Nick Davies for the yeast strain carrying
neo
r
on yeast chromosome V, Dr David Kipling for the major probe and Ms U.
Gangadharan for the
Utrophin
gene probe. We thank Robert Williamson and Rodney Rothstein for useful
discussions. This work was supported by MRC grant PG9227430.
