ABSTRACT
The development of a system for shuttling DNA cloned as yeast artificial
chromosomes (YACs) between yeast and mammalian cells requires that the DNA is
maintained as extrachromosomal elements in both cell types.
We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (
oriP
) are maintained as stable, episomal elements in a human kidney cell line
constitutively expressing the viral transactivator protein EBNA-1.
Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple
alkaline lysis procedure and shuttled back into
Saccharomyces cerevisiae
by spheroplast transformation.
In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells.
The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.
Shuttle systems exist which allow cloned DNA to be transferred between bacteria
and yeast (
1
) and between bacteria and mammalian cells (
2
-
4
). Bacterial-yeast shuttle vectors have permitted the cloning of yeast genes by
complementation of yeast mutants. This has included identification of genes
important to the yeast cell cycle, metabolism (
5
), and repair and recombination systems (
6
) and has facilitated characterisation of
cis
-acting DNA elements such as promoters (
7
) and autonomously replicating sequences (ARSs) (
1
). Genes encoded on bacterial-mammalian shuttle plasmids have been shown to be stably expressed in
mammalian cells (
4
) and such vectors have been used for the efficient expression of cDNA libraries
(
2
). The
oriP
-EBNA-1 shuttle vectors allow cloning of genes by direct complementation
of mutant phenotypes and have been shown to be 100-fold more efficient than vectors lacking the EBV elements in the isolation
of low abundance sequences (
8
).
The term `shuttle system' reflects the ability to move a vector back and forth
between two hosts, hence a means of selecting the vector in both hosts is
required as well as a means of propagating it in both organisms. The original
Saccharomyces cerevisiae
-
Escherichia coli
shuttle vectors, YEp and YCp (reviewed in
1
) have different systems for autonomous replication in yeast. Both vectors contain pBR322 sequences, allowing replication and selection in
E.coli,
and both carry a gene for selection in yeast. Replication of YEp plasmids in
yeast initiates from within the 2[mu]m origin of replication and the plasmid is stably maintained at a copy
number of 30-50 per cell. In contrast, YCp vectors contain yeast centromere sequences and an origin of replication which allows
autonomous replication of the single copy plasmid in yeast cells.
Escherichia coli
-human cell line shuttle vectors contain a mammalian selectable marker
gene and rely on interaction between the Epstein-Barr virus latent origin of replication,
oriP,
and the viral transactivator protein EBNA-1, for stable, extrachromosomal maintenance of the plasmids in human cells (
9
,
10
).
oriP
consists of two components: a family of repeats which comprises 20 tandem
copies of a 30-bp repeat, and the dyad symmetry element which contains four copies of the
repeat (
11
,
12
). Both sets of repeats are necessary for stable, extrachromosomal maintenance
of plasmid DNA in human cells (
12
), and both bind the EBNA-1 protein. The dyad symmetry element is the site of initiation of episomal
DNA replication (
13
), while the family of repeats acts as an EBNA-1-dependent enhancer of transcription (
14
) as well as a replication fork barrier and termination site (
13
). In addition, the family of repeats promotes nuclear retention by the specific binding of EBNA-1 (
15
), followed by non-specific association of EBNA-1 with host cell chromosomes (
16
,
17
).
Plasmids carrying
oriP
are present as multiple copies within the human cells (
2
,
8
) permitting easy isolation by Hirt extraction (
2
,
18
), which can be followed by reintroduction into
E.coli
by transformation of competent bacterial cells. The ability to isolate the extrachromosomal DNA
leads to possibilities in addition to expression cloning. For example,
in vitro
mutagenesis in
E.coli
can be followed by reintroduction of mutated clones into mammalian cells and screening for altered phenotypes. The extrachromosomal DNA can then be isolated from the human cell lines, and returned to bacteria
for analysis and further manipulation, thus facilitating the functional
analysis of mammalian genes and their regulatory elements.
Although highly efficient, one major limitation of plasmid based bacterial-mammalian shuttle systems is the size of DNA which can be accommodated in
the vector. This problem could be overcome by the use of shuttle cloning systems with a larger capacity for DNA, e.g. P1 artificial chromosome (PACs), bacterial artificial chromosomes
(BACs) or yeast artificial chromosomes (YACs). The use of YACs is particularly advantageous because the yeast host allows efficient characterisation and manipulation of cloned sequences by homologous recombination, prior to introduction into mammalian cells.
The inability to maintain YACs as stable, extrachromosomal elements in mammalian cells has, however, hindered the development of a large cloning capacity yeast-mammalian shuttle vector system. We have recently made circular YACs
carrying the EBV
oriP
domain, termed OriPYACs, as shown in Figure
1
. Two YACs of 90- and 660-kb were used in the initial study. After circularisation and addition of
oriP
, the OriPYACs were introduced into human 293 cells constitutively expressing EBNA-1 by spheroplast fusion. A total of six cell lines were generated: three
from fusion with yeast containing OriPYAC90 (F90-1, -2 and -3) and three from fusion with yeast containing OriPYAC660
(F660-1, -2 and -3). All six cell lines contained replicating, extrachromosomal
elements. The 90-kb OriPYAC was found to be intact and unrearranged in all of the cell
lines analysed, whereas the intact form of the 660-kb OriPYAC was present in two out of three cell lines. The episomal
elements were found to be highly stable, with loss rates of 1-3% per cell division in the absence of selection (
19
).
A map of the final OriPYAC construct is shown in Figure
1
. Construction of the OriPYACs and generation of human kidney fusion cell lines
containing the OriPYACs has been described elsewhere (
19
). Fusion cell lines F90-2 and F90-3, containing an average of 1.3 and 18 intact copies of the 90-kb circular molecule respectively, and F660-3, containing three intact copies of the 660-kb circular molecule per cell were used for the
isolation of episomal elements.
OriPYAC episomes were isolated from the human fusion cell lines by a
modification of a previously described alkaline lysis procedure (
20
-
22
). 1 * 10
6
cells were washed once in phosphate buffered saline (without calcium or
magnesium), pelleted by centrifugation at 1000 r.p.m. and resuspended in 100 [mu]l of lysis buffer (50 mM NaCl, 2 mM EDTA, 1% SDS, adjusted to pH 12.45 with
2 M NaOH). The cells were lysed by vortexing at highest speed for 1 min followed by incubation at 30oC for 30 min. The lysis buffer was neutralised by addition of 0.2 vol of 1 M Tris pH
7.0, 0.11 vol of 5 M NaCl and 0.01 vol of 10 mg/ml proteinase K. The cellular
proteins were partially degraded by incubation at 37oC for 30 min. The cell lysates were then cooled to room temperature before
extraction with 1/3 vol phenol (saturated with 0.2 M NaCl, 0.2 M Tris-HCl pH 8.0). Extraction was carried out by very gentle inversion of the
tube several times. The tubes were then chilled slowly to 4oC and the phases separated by centrifugation at 5000 r.p.m. for 20 min at 4oC. The aqueous phase was recovered using a cut-off tip and 1/3 the volume of 24:1 chloroform-isoamylalcohol added. The phases were separated as before
and episomal DNA precipitated overnight at -20oC in the presence of 25 [mu]g of glycogen (in the case of OriPYAC90 elements) or 2 [mu]g of sheared salmon sperm DNA (for purification of
OriPYAC660) and 2 vol of ethanol. The DNA was recovered by centrifugation at 10
000
g
, 4oC, for 30 min, washed once in 70% ethanol and resuspended overnight in 10 [mu]l H
2
O (for yeast transformation) or 50 [mu]l H
2
O (for [gamma] irradiation) at 4oC.
Purified OriPYAC90 DNA from the human fusion cell lines was used to transform
the YAC library host strain
S.cerevisiae
AB1380 (
23
). Yeast cells were grown up in the rich medium YPD (1% yeast extract, 2%
bactopeptone, 2% dextrose) to a density of ~3 * 10
7
cells/ml. Spheroplast transformations were carried out as described previously
(
24
). Episomal DNA isolated from 10
6
human cells was used in each transformation reaction. An aliquot of 5 [mu]g of sheared salmon sperm DNA was added to act as carrier and 1 mM
polyamines (0.75 mM spermidine trihydrochloride-0.30 mM spermine tetrahydrochloride) (
25
) were included to help maintain the high molecular weight DNA intact.
Transformed spheroplasts (7.5 * 10
7
) were plated onto five 90 mm sorbitol agar plates lacking histidine.
Yeast clones containing OriPYAC90 or OriPYAC660 were grown to a density of ~3 * 10
7
cells/ml. 1.5 * 10
9
yeast cells were then washed once in 20 ml of water and once in 20 ml of 1 M
sorbitol and resuspended in 20 ml of SCEM (1 M sorbitol, 0.1 M sodium citrate
pH 5.8, 10 mM EDTA). Yeast lytic enzyme (ICN) (1.3 U) was added and the cell
suspension was incubated at 30oC until the yeast cells were 95% spheroplasted, ~25 min. The spheroplasts were washed twice in 20 ml of STC (1 M
sorbitol, 10 mM Tris pH 7.5, 10 mM CaCl
2
) before resuspension in 1 ml of STC. OriPYAC DNA was prepared from aliquots of
1 * 10
6
spheroplasts by the same alkaline lysis procedure as that described for human cells. Carrier DNA was included during precipitation of the purified
OriPYAC DNA, in an attempt to reduce shearing of the DNA.
Agarose plugs of high molecular weight yeast or mammalian cell DNA were prepared
by modification of a previously described protocol (
26
). [gamma] irradiations were carried out in a Gammacell 1000 Elite. Plugs were equilibrated twice in TE at 4oC prior to [gamma] irradiation in 2 ml of TE. Purified episomal DNA was irradiated in
solution, using the same irradiation conditions. Restriction enzyme digests of
plug DNA were carried out overnight in a volume of ~200 [mu]l. Plugs were equilibrated twice in TE at 4oC, followed by two washes for 30 min in restriction enzyme buffer
on ice, prior to overnight digestion with 40-60 U of enzyme.
Pulsed-field-gel electrophoresis (PFGE) was carried out on a BioRad CHEF DRII or
DRIII apparatus, using a 1% agarose gel cast and run in 0.5* TBE. Samples of irradiated DNA in solution were loaded onto a pre-chilled gel and run for 15 min without circulation of the buffer. The gels were then run at 11oC and 200 V, with the switching times given in the figure legends.
Southern transfer, onto Hybond N+ membranes (Amersham), was carried out using the conditions described by the manufacturer. All prehybridisations and hybridisations were carried out in modified Church buffer (16.8 g/l NaH
2
PO
4@
H
2
O, 54.1 g/l Na
2
HPO
4@
12H
2
O, 7% SDS, 100 [mu]g/ml salmon sperm DNA) at 65oC. Filters were washed with 2* SSC for 5 min, followed by two washes in 0.1* SSC- 0.1% SDS for 30 min, all at 65oC. The 0.9 kb
Sma
I fragment from pHEBo was used as the
oriP
probe (
27
).
In this study, we wanted to establish whether the OriPYAC episomal elements
could be easily isolated from the human fusion cell lines, and thus used as the
basis of a shuttle system for transferring large DNA molecules between
mammalian and yeast cells. Fusion cell lines containing the 90-kb OriPYAC rather than 660-kb OriPYAC were used first, because of the problems associated with
handling very large, purified DNA.
The 90-kb episomal OriPYAC90 molecules were isolated from the human fusion cell
lines F90-2 and F90-3 by a straightforward alkaline lysis procedure (
21
), as described in Materials and Methods, which is similar in principle to a
bacterial plasmid preparation. This method involves alkali treatment to
selectively denature host linear DNA, followed by shearing to allow efficient
strand separation of the denatured DNA. The lysate is then neutralised and a
limited protease step carried out before phenol extraction at high salt
concentration (
28
). This method was developed for isolation of the 172-kb EBV episomal molecules from human cells. Episomal DNA purified from the
human fusion cell lines was resuspended in TE containing 100 mM NaCl and the
integrity of the purified episomal DNA was tested by [gamma] irradiation followed by PFGE and hybridisation to an
oriP
probe. As can be seen from the last lane in Figure
2
, the OriPYAC90 DNA purified from the human cell line gives a clear band of 90 kb after [gamma] irradiation, suggesting that OriPYAC90 has been isolated intact from
human cells.
We then determined whether the purified OriPYAC90 DNA could be transformed
intact into yeast. Purified OriPYAC90 DNA isolated from 1 * 10
6
F90-2 or F90-3 cells, was used to transform the
S.cerevisiae
YAC library host strain AB1380, by a standard protocol using polyethylene
glycol (PEG) (
24
), as described in Materials and Methods. Sheared salmon sperm DNA was included as carrier in all reactions, to reduce shearing forces and increase
transformation efficiency. Polyamines (1 mM) were included in some
transformation reactions because they have been shown to reduce shearing of
high molecular weight DNA (
25
). However, these were found to be unnecessary to maintain molecules of this
size intact. A total of 138 colonies (42 with OriPYAC90 DNA prepared from F90-2 and 96 with OriPYAC90 DNA purified from F90-3) grew on media lacking histidine. The larger number of colonies
which were produced with DNA prepared from F90-3 may be a result of the higher copy number of the OriPYAC episomes
(average 18 per cell) in this cell line, in comparison with F90-2 (1.3 episomes per cell) (
19
).
Figure
2
shows analysis of three randomly chosen yeast clones which were positive by
this genetic selection. S1 and S2 were generated by transformation with
episomal DNA purified from F90-2, S3 by transformation with DNA purified from F90-3. Agarose plugs of high molecular weight DNA from AB1380, the
original OriPYAC90 yeast clone, the human cell line F90-2 carrying OriPYAC90, and transformation yeast clones S1, S2 and S3 were
treated with [gamma] irradiation and resolved on a PFG. Hybridisation to an
oriP
probe demonstrated the presence of a 90-kb band after irradiation of all samples except non-transformed AB1380 cells, suggesting that OriPYAC90 has been transferred intact
to all yeast clones analysed. The slight shift in mobility between yeast and mammalian bands has been observed frequently (
29
,
30
) and is due to the 200-fold greater complexity of the mammalian genome in comparison with the
yeast genome. OriPYAC90 is sufficiently small that the circular forms of the
YAC can resolve into the gel under certain PFGE conditions, the positions of
these circular molecules are indicated by arrows. Analysis of six further
positive yeast transformation colonies by gamma irradiation demonstrated that OriPYAC90 was intact in nine out of nine clones (data not shown).
Transfer of YAC DNA into mammalian cells by spheroplast fusion is convenient and does not impose the same size constraints as lipofection and microinjection. However, some cell types, particularly human
cell lines, appear to be refractory to spheroplast fusion. In addition, gene therapy and
in vivo
transgenic applications of OriPYACs would require introduction of purified DNA. Therefore, we wanted to determine whether OriPYAC90 DNA could be isolated intact from
yeast cells.
Conventional purification from low melting point agarose gels is difficult,
because circular molecules run anomalously on PFGE and bands cannot generally
be seen under UV light after ethidium bromide staining. Also, isolation of
episomal DNA by alkaline lysis is more difficult from yeast than from mammalian
cells, due to the high concentrations of degradative enzymes in yeast cells. Yeast carrying OriPYAC90 were first spheroplasted and the alkaline lysis procedure was then carried out as described for the human cells. Carrier DNA was included in both precipitation and resuspension of the OriPYAC DNA, in an attempt to reduce nicking or degradation of the
circular molecules. The purified OriPYAC DNA was then analysed by [gamma] irradiation and PFGE.
Figure
4
shows analysis of OriPYAC90 purified from yeast cells. In the unirradiated
sample of purified OriPYAC DNA, most of the DNA is in the circular, supercoiled
form of the molecule. A very small amount of linearised DNA is also present,
suggesting that some degree of nicking of the circular molecule has occurred
during the isolation. Treatment with [gamma] irradiation results in a band of 90 kb for both yeast carrying OriPYAC90
and the purified OriPYAC90 DNA.
In order to see whether these methods could be applied to a 660-kb molecule, in addition to the 90-kb one, we attempted to isolate OriPYAC660 from yeast and human
cells, by the same alkaline lysis procedure. Carrier DNA was included during
precipitation of the isolated episomal elements, in order to reduce shearing forces, and both polyamines and high salt (100 mM NaCl) were included during resuspension of the DNA. The DNA was irradiated, resolved
on a PFG and hybridised to an
oriP
probe (Fig.
5
). The human cell line F660-3 contains intact, 660 kb episomal elements (Fig.
5
A) which are present at a copy number of approximately three per cell (
19
), but no signal was seen from DNA purified from this cell line (Fig.
5
B). The very large size of the OriPYAC may contribute to the inability to purify
intact episomes.
The system described here allows DNA cloned as YACs to be shuttled between human
and yeast cells. We have previously transferred circular YACs containing the
EBV
oriP
domain from yeast cells into a human kidney cell line by spheroplast fusion. The
OriPYACs were found to be maintained as stable, episomal elements in all fusion
cell lines analysed (
19
). Here we show that episomal OriPYACs of 90 kb can be isolated intact from the
human cells by a straightforward alkaline lysis procedure. Ability to isolate
OriPYAC DNA by this method demonstrates that the OriPYACs are being maintained
as covalently closed, supercoiled molecules within the human cells, as has been
demonstrated for the latent viral genome (
34
,
35
). The isolated episomal elements were then reintroduced by yeast spheroplast
transformation into the
S.cerevisiae
YAC library host strain, AB1380 (
23
), where they were found to be maintained intact and unrearranged in nine out of
nine clones analysed.
The OriPYAC shuttle system has two major advantages over conventional EBV-based bacterial-human shuttle systems; ease of manipulation of the cloned DNA by homologous recombination, and large cloning capacity.
Saccharomyces cerevisiae
is an excellent host for manipulation of DNA by homologous recombination allowing fragmentation (
36
-
38
), generation of internal deletions (
39
,
40
), mapping of exons (
41
), integration of yeast or mammalian selectable markers (
42
-
44
) and introduction of defined mutations via gene replacement (
45
,
46
).
The large cloning capacity of YACs allows them to carry both intact genes and
their long range controlling elements which give full levels of controlled
expression. YAC DNA has been successfully introduced into mammalian cells by a
variety of methods including microinjection, lipofection and spheroplast fusion
(reviewed in
47
,
48
). In most cases the YAC DNA has given full levels of gene expression,
proportional to the copy number of the YAC and independent of the position of
integration in transgenic mice (
44
,
49
,
50
). YACs have also been used to complement mutations in mammalian cells (
51
,
52
) and have recently been used in the localisation of a hereditary disease gene
to a 500-kb interval (
53
).
The OriPYAC system should give the same high levels of controlled expression and
could be used to directly clone genes by complementation of mutant phenotypes.
OriPYACs spanning the critical region for a particular gene could be introduced
into cell lines demonstrating the mutant phenotype. OriPYACs could then be
isolated from cells showing complementation and reintroduced into
S.cerevisiae
for further manipulation, such as exon trapping or cDNA selection.
We also demonstrate here that the 90-kb OriPYAC can be isolated intact from yeast cells which should allow
extension of the OriPYAC system to applications where spheroplast fusion cannot
be used. Successful spheroplast fusion has only been described for two human
cell lines (
19
,
54
) and is clearly not suitable for many applications. Purification of the OriPYAC DNA from yeast will
permit introduction of OriPYACs into mammalian cells by other methods, including lipofection, which are applicable to a wide variety of cell types and which can be used for
in vivo
gene therapy applications (
55
-
57
).
An EBV-based system of gene therapy would have the advantage of long term
maintenance of the DNA, without the risk of mutagenesis by random integration.
In addition, the large insert size of OriPYACs should permit efficient expression of the introduced gene. Such an approach may be particularly suitable for syndromes which can be
corrected through the release of diffusible therapeutic factors from
lymphoblastoid cells, such as haemophilia A or B, or chronic granulomatous
disease. EBV is carried latently in the lymphoblastoid cells (asymptomatically)
by >90% of the world-wide adult human population (
58
) and recent work has demonstrated that a helper-dependent mini-EBV vector can stably transduce B lymphoblastoid cells from a Fanconi anaemia group C patient (
59
). The mini construct showed
in vitro
correction of the FA phenotype, with episomal expression persisting with a half-life of 30 days.
The ability to transfer OriPYACs between human cells and yeast thus provides the basis for a large cloning capacity yeast/human shuttle vector
system which should be of use in gene therapy applications as well as for
functional analysis of large fragments of mammalian DNA.
We thank B. Griffin, D. Huertas and A. McGuigan for helpful suggestions. K.S. is
a Wellcome Trust Prize Student. This work was partly supported by CF Trust
grant PJ387.
*To whom correspondence should be addressed at: Cold Spring Harbor Laboratory,
PO Box 100, Cold Spring Harbor, NY 11724, USA.
REFERENCES
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