ABSTRACT
A method for linking any standard yeast artificial chromosomes (YAC) is described. YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially. The
linking vectors also contain the
[beta]
-galactosidase gene as an expression reporter in mammalian cells.
Yeast artificial chromosome (YAC) clones have been essential tools in the
physical mapping of large regions of the mouse and human genomes (
1
) and have also been important for functional studies. They have been used to
introduce genes into cultured mammalian cells and mice (
2
) and to investigate less well defined chromosomal elements such as origins of
replication (
3
) and centromeres (
4
). However, YACs isolated directly from libraries may not contain the entire
region required, or may be chimeric or unstable. Some of these problems can be
overcome by using meiotic or mitotic recombination in yeast to reconstruct a
contiguous region of the genome from a set of incomplete but overlapping YACs and simultaneously remove chimeric sequences; the CFTR (
5
) and 2.4 Mb (
6
) dystrophin genes have been assembled in this way. In other cases it may be
desirable to join regions that are not contiguous in genomic DNA, or insert
sequences into the region being constructed. For example, a gene could be
linked to a centromere to form the basis of a mammalian artificial chromosome
(MAC) for gene therapy. Therefore, we have developed a set of linking vectors
that allows any pair of YACs to be joined. The YACs are initially introduced
into the same yeast cell and linked by a vector carrying a yeast marker gene
that targets the right arm of one YAC and the left arm of the other. In
principle, up to four YACs can be joined in this way.
Unique sequences from the right arm (RA) and left arm (LA) of pYAC4 were
constructed by PCR using a modification of primers previously described (
7
). The LA sequences were made by amplification with the primer pairs YAC XBA1-LP 5'-CCGTCTAGAATGCGGTAGTTTATCACAGTT, and YAC SPE1-LR1 5'-CTTACTAGTGGTGTGGTCGCCATGATCGCG generating
a 329 bp product. The RA sequences were made by amplification with the primer
pairs YAC XBA-RP 5'-CATTCTAGAATCATCGTCGCGCTCCAGCGA, and YAC SPE1-RR3 5'-CTAACTAGTCTCGCCACTTCGGGCTCATGA, generating
a 354 bp product. PCR was performed in buffer containing 1.5 mM MgCl
2
for 30 cycles of 93oC for 1 min, 55oC for 1 min, 70oC for 1 min and 70oC for 5 min final extension. LA and RA arm sequences were gel purified in 2% low melting point agarose, melted at
68oC and incubated with 6 U [beta]-agarase (NEB) at 37oC for 16 h. DNA was then extracted twice with phenol, precipitated in
ethanol and resuspended in a final volume of 20 [mu]l of 1* TE. The LA and RA sequences were digested in separate reactions with
Spe
I, before ligating the two fragments at the
Spe
I site. The ligated product (~700 bp) was gel purified and digested with
Xba
I in manufacturer's buffer and ligated into the
Spe
I site in the polylinker sequences of pRS303 (
8
) to create pH901 and pRS305 (
8
) to create pL983. The [beta]-galactosidase gene (pCMV[beta] expression vector, Clontech) regulated by the cytomegalovirus
(CMV) promoter was also incorporated into the
Eco
RI-
Sal
I polylinker site of pH901 to form pHG901, and the
Pst
I site of pL983 to form pLG983 (Fig.
1
). pLG901 and pLG983 were linearised with
Spe
I prior to transformation.
Two non-overlapping human Y chromosome YACs were transferred from AB1380 (
MAT
a
ade2-1
,
can1-100
,
lys2-1
,
ura3
,
trp1
,
his5
,
[psi+]
;
9
) to the same haploid cell in CGY2570 (
MATa
Gal
+
ura3-52
,
trp1-63
,
leu2-1
,
lys2-202
,
his3-200
,
ade2-1
;
10
) via an
intermediary
kar1
strain. The left arm of the YAC clone 758G1 (1.7 Mb;
11
) was first modified in AB1380 (
MAT
a) by retrofitting with pLGTEL1 (which introduces the LYS2 gene;
12
), and the YAC was then transferred to the recipient
kar1
strain EJL434-3D (
MAT
[alpha]
ade2-101
,
lys2
,
trp1
,
leu2
,
ura3-52
,
cyh
R
kar1-del 13
;
13
) as described (
14
). Large colonies were selected on standard agar plates lacking lysine,
tryptophan and uracil but containing cycloheximide, and cells were analysed by
PCR for the
MAT
[alpha] genotype (
15
). High molecular weight DNA was prepared from positive cells, digested with
Sfi
I and analysed by PFGE, and hybridised to a total human DNA probe to detect any
rearrangements. 758G1 was then transferred from EJL434-3D to CGY2570 (
MAT
a) which was previously modified to be canavanine sensitive (
14
). Large colonies were selected on agar plates lacking lysine, tryptophan and
uracil, but containing canavanine. The PCR identified positive cells with
MAT
a genotype.
A second YAC 62C1212 (370 kb;
11
) was initially modified in the right arm with pRAN4 (containing the ADE2 gene;
16
) in AB1380, and transferred to EJL434-3D. Large colonies were selected on agar plates containing cycloheximide,
but lacking tryptophan and adenine. Following identification of cells with
mating type
a, YAC DNA was prepared, digested with
Eco
RI and hybridised to RA and LA probes. The RA detected a 1.7 kb band from pRAN4,
and the LA detected a 10.2 kb band due to a rearrangement of the pYAC4 arm following transfer to the
kar1
strain. DNA was also digested with
Sfi
I, analysed by PFGE and hybridised to a total human DNA probe, to detect any rearrangement of the insert DNA. 62C1212 was then transferred to the recipient strain CGY2570
carrying the 758G1 YAC, and the two YACs were selected on agar plates lacking
tryptophan, lysine, uracil and adenine, but containing canavanine. Large
colonies were checked for mating type a by PCR, and YAC DNA was prepared from
positive cells.
Spheroplasts of haploid cells (4 * 10
8
/ml;
17
) containing both YACs were then transformed with either 1 [mu]g pHG901 or pLG983 linearised with
Spe
I. Transformants were selected on agar plates lacking lysine, tryptophan,
histidine and adenine or lysine, tryptophan, leucine and adenine, respectively.
Trp
+
Lys
+
Ade
+
His
+
Ura
-
Leu
-
or Trp
+
Lys
+
Ade
+
Leu
+
Ura
-
His
-
transformants, respectively, were isolated from appropriate selective media plates lacking sorbitol, and grown in liquid media at 30oC for 24-36 h. YAC DNA was prepared in agarose plugs as described (
2
). Undigested YAC DNA was analysed by PFGE, and hybridised to a total human DNA
probe which was radiolabelled as described (
18
). YAC DNA was also digested with
Eco
RI and fractionated on a 0.8% agarose gel in 1* TBE, and hybridised to radiolabeled probes prepared from the RA and LA
of pYAC4.
YAC HC32 was transferred to mouse LA9 cells by yeast spheroplast fusion (
19
). Yeast cells (1 * 10
8
) were fused with 3 * 10
6
LA9 cells. Positive colonies, selected for neomycin resistance with geneticin
(G418), appeared in 14 days. Cells were monitored for [beta]-galactosidase activity 14 days after the fusion by staining with X-gal, and high molecular weight genomic DNA was prepared from positive clones as described (
2
). DNA was digested with
Eco
RI and hybridised to radiolabelled RA and LA YAC probes, total human and yeast
DNA.
The method used to link YACs is shown schematically in Figure
2
. The chosen YACs, in the vector pYAC4 and the host strain AB1380, were first
genetically modified to allow selection of recombinants. As described in
Materials and Methods, YAC 758G1 and YAC 62C1212 were then transferred into the
same cell of the host strain CGY2570 by kar transfer. The procedure was done in
two stages. Initially, YAC 758G1 was transferred from AB1380 to CGY2570 via the
intermediary kar strain; then YAC 62C1212 was transferred from AB1380 to the
same cell in CGY2570 containing YAC 758G1 via the intermediary kar strain as
depicted in Figure
2
. Spheroplasts prepared from haploid cells containing both 62C1212 and 758G1
were then transformed in separate experiments with either pHG901 or pLG983.
DNA from the six recombinant clones was digested with
Eco
RI and analysed by conventional electrophoresis. Figure
4
A shows the size of fragments detected by the RA and LA probes, and Figure
4
B indicates the expected fragment sizes of clones linked with either pH901 or
pL983. Lanes 4-6 and 13-15 contain clones linked by pHG901 (HC6, HA45 and HC32) and lanes
7-9 and 16-18 contain clones linked by pLG983 (L23, LC31 and LC39). The RA
probe detects a 1.7 kb band from pRAN4 in 758G1/62C1212 and 62C1212 in lanes 1
and 2 and in 4-9, respectively, and a 3.6 kb band in lanes 1 and 3 from the right pYAC4
arm of 758G1. The RA detects a 9.4 kb fragment in lanes 4 and 6 from pHG901,
and a 5.4 kb band in lanes 7-9 from pLG983 as depicted in Figure
4
B. Lanes 5 and 8 contain additional bands of 17 and 9 kb resulting from a
rearrangement of pHG901 and pLG983.
To show that the [beta]-galactosidase and neo genes remained functional in mammalian cells,
one linked clone HC32 was transferred to mouse LA9 cells by yeast spheroplast
fusion. Three G418 positive fusion clones (F1, F4 and F5) were expanded after
14 days and the [beta]-galactosidase gene was found to be expressed in at least 50% of the
cells (data not shown). High molecular weight DNA, prepared from the positive
clones, was digested with
Eco
RI and analysed by conventional electrophoresis. DNA was hybridised to the RA
and LA (Fig.
5
), total human and yeast DNA probes (data not shown). The RA probe detected a
1.7 kb fragment common to all lanes except LA9 DNA (lane 5), which is
diagnostic for the right arm vector pRAN4. The probe also detected a 9.4 kb
fragment in HC32 which was present in F1, F4 and F5 (a slight difference in
migration of the bands is a result of quantitative loading discrepancies of
yeast and genomic DNA), and comes from the linking vector pHG901. The LA
detected a 13 kb band in HC32 (lane 6) derived from the left arm of pLGTEL1,
which is a telomeric
Eco
RI fragment. A larger band was present in F5 (lane 7) as a result of integration
and joining to mouse DNA, and the region has been deleted from F4 and F5. The LA also detected a 0.6 kb fragment in all lanes except LA9 (lane 10), which was the 0.6 kb
Eco
RI fragment from pHG901. The total human DNA probe detected a similar
fingerprint in all lanes except LA9, indicating that no major rearrangement of
the insert DNA in HC32 occurred following transfer to LA9 cells. However, F1 and F4 were missing several bands (data not shown), consistent with the YAC being truncated in these two clones, since they did not
contain the left arm from pLGTEL1. The total yeast DNA probe detected positive
bands in HC32 only.
We have designed linking vectors which allow any two YACs in the most widely
used vector pYAC4 to be recombined via unique sequences on the left and right
YAC arms. The overlap region between the vector sequences and the corresponding
homologous region on the left or right YAC arm is ~320 bp which is sufficient for mitotic recombination, with an overall
frequency of ~2% of initial transformants. The vectors contain the LEU2 and HIS3 markers
and we have constructed a similar vector containing LYS2. In addition, we
incorporated the [beta]-galactosidase gene under control of the CMV promoter as a reporter
for gene expression in mammalian cells. This method of recombination is
relatively simple to perform and requires a genetic selection rather than a microscopic dissection of haploid spores, and a major advantage is that the entire recombination procedure can be performed in a
single strain.
A series of YACs can be linked sequentially. For example, as described above, if
ADE2 is introduced on the right arm of one YAC and LYS2 on the left arm of the
second YAC, then this will ensure selection of the correct transformant following recombination with the pHG901 vector containing HIS3 (Fig.
6
a). A further recombination between another YAC containing the TRP1 and URA3
markers on the left and right arm respectively is now possible with the pLG983
vector containing LEU2, and this will result in the loss of ADE2 (Fig.
6
b). Lastly, recombination could occur via a linking vector containing LYS2 if
the left arm of the recombined YAC was modified to contain TRP1, and the right arm of the YAC to be linked contained the ADE2 marker (Fig.
6
c). This will result in a single construct from the recombination of four YACs
(Fig.
6
d). It might even be possible to link an additional YAC carrying the URA3 marker
by selecting against URA3 after transformation with any linking vector. The efficiency of linking in this way may be low, but if successful, the procedure could be
continued indefinitely.
The method described here will be useful for linking YACs from different parts
of the genome, or even from different species. For example, the genes for the
subunits of a multi-subunit protein such as haemoglobin or the immunoglobulins are often
located on different chromosomes and could be recombined into a single YAC
before use in expression studies, or sets of genes that interact in a
biochemical or developmental pathway could be linked together. In more
ambitious experiments, it may be possible to link genes to a centromere and any
other necessary sequences to construct an MAC. Extensive manipulation of
complex genomes will be increasingly important in the future, and YAC linking vectors provide a useful addition to the techniques available for such work.
We thank Ed Louis and Rhona Borts for providing the kar strain, EJL434-3D, and advice on the Kar1
-
matings, and Diana Wylie for help with tissue culture. Z.L. was supported by
the CRC and the Wellcome Trust, S.S.T. by the MRC and C.T.S. by the CRC.
*To whom correspondence should be addressed. Tel: +44 1865 222677; Fax: +44 1865
222500; Email: zlarin@molbiol.ox.ac.uk
+
Present address: Department of Cell Biology, Harvard Medical School, 240
Longwood Avenue, Boston, MA 02215, USA
{
1996 Oxford University Press
REFERENCES
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