From the
Lack of suitable amplification markers has hindered the use of
the yeast system for investigating the mechanism of gene amplification
in a eukaryote with a simple genome and well defined genetic system.
Recently, methotrexate has been used to select for Saccharomyces cerevisiae mutants with de novo amplification of
the dihydrofolate reductase gene ( DFR1) (Huang, T. (1993)
In Vivo Disruption and de Novo Amplification of the DFR1 Gene
Encoding Dihydrofolate Reductase in Saccharomyces cerevisiae. Ph.
D. thesis, University of Alberta, Edmonton, Canada). We report here the
detailed structure of a DFR1 episome amplified in
methotrexate-resistant strain 25-1. The extrachromosomal DNA is
found predominantly as a single 11-kilobase circular molecule. It
consists of a 5.5-kilobase inverted duplication that contains the
DFR1 locus and adjacent ARS (autonomously replicating
sequence) element. This molecular configuration mimics the inferred
structure of double minute chromosomes observed in a number of
mammalian amplification systems and suggests that mechanisms that
generate amplified DNAs are conserved from yeast to mammals.
Gene amplification occurs frequently in tumor cells or in cells
resistant to cytotoxic agents. It has only recently become appreciated
that amplification is a rare event in normal mammalian cells
(1, 2, 3) . Similarly, amplification is rarely
seen in normal yeast cells. This suggests that mechanisms that prevent
amplification in normal cells are conserved from yeast to mammals.
Recent studies
(4, 5) reveal that mutations in the
tumor suppressor gene p53 lead to an increase in gene amplification
rate. One can envision that in the p53 mutants, cells carrying damaged
DNA, which normally arrest in the cell cycle to allow repair, proceed
into S phase with damaged chromosomes
(6) . This can perturb
replication and lead to gene amplification in a number of ways. Stalled
replication forks, reinitiation from a single origin, and DNA strand
breakages, possible outcomes of direct or indirect inhibition of DNA
replication, have been proposed to be the early events of gene
amplification
(7, 8, 9) . One assumption
underlying the above hypothesis is that a replication origin or a
cis-acting element involved in replication is present within amplicons.
Carcinogen-induced amplification of polyomavirus and SV40 DNA does
require a functional viral origin of replication
(10, 11) . In addition, cis-acting elements putatively
involved in mammalian replication have been found within the sequences
co-amplified with the Chinese hamster dihydrofolate reductase gene
(12, 13) and murine adenosine deaminase gene
(14) . Thus, definition of the role of cell cycle control and
DNA replication in gene amplification is crucial for understanding the
mechanisms that control gene amplification.
Structural
characterization of amplified DNA has been an important experimental
approach in dissecting the mechanism of gene amplification. Such
characterization is very challenging in mammalian cells, however,
because of their highly complex genomes and the large amount of DNA
involved in an amplification event. Mammalian amplified DNAs are found
either as multiple repeats within chromosomes or as multiple copies of
extrachromosomal elements
(8, 15) . Extrachromosomal
amplified DNAs often appear as double minute chromosomes or episomes,
which are acentric, self-replicating circular elements
(16, 17) . Inverted repeats are a common, though not
invariant, feature of both episomal and chromosomal amplified DNAs
(18) . Models to explain the mechanism of amplification are
therefore often predicated on the known properties of inverted
sequences
(9, 19, 20, 21) .
It is
desirable to be able to investigate the mechanisms of gene
amplification in an organism that has both tractable genetics and low
genome complexity. The yeast system would be ideal, but studies of gene
amplification have been hampered by the fact that conditions for
increasing the basal level of these rare events are not known and by
the paucity of appropriate markers to select out the rare amplification
events. Recently, Huang et al.
A similar
protocol for circular DNA purification from yeast cells was used when
QIAGEN was employed to purify the DNA. Briefly, the yeast spheroplasts
were resuspended in 4 ml of buffer (100 µg/ml RNase A, 50
m
M Tris-HCl, pH 8.0, 10 m
M EDTA). An equal volume of
lysis solution (200 m
M NaOH, 1% SDS) was added and mixed.
After 5 min at room temperature, 4 ml of chilled neutralization buffer
(750 m
M NaCl, 50 m
M MOPS, 15% ethanol, pH 7.0, 0.15%
Triton X-100) was added. The suspension was centrifuged at 4 °C for
30 min at 15,000 rpm, and the supernatant was applied to a QIAGEN-tip
100 equilibrated with 4 ml of buffer (1.0
M NaCl, 50
m
M MOPS, 15% ethanol, pH 7.0). The tip was washed and DNA
eluted as described by the manufacturer. The relatively high copy
numbers of the DFR1 circle in the MTX-resistant cells appear
to facilitate the efficient purification of the yeast circular DNAs.
Total yeast DNA was prepared from a 5-ml culture by the small scale
purification procedure of Sherman et al. (23) with
modifications suggested by Lee
(27) . Yeast spheroplasts were
resuspended in 0.5 ml of 3
If the SalI
fragment is present as an inverted repeat, then one junction (J1) of
the inverted repeat would lie, based on the foregoing, less than 0.2 kb
upstream of the 5`- SalI site. If this is true, a restriction
map would be expected such that the size of any specific fragment would
be the sum of the 0.2-kb SalI fragment and twice the length
from the 5`- SalI site to the first downstream restriction site
in question (Fig. 4 A). For example, DNA sequence data
reveal that 1.04 kb is the length from the 5`- SalI site to its
first downstream HindIII site (Fig. 3). Thus, there
would be a HindIII fragment of about 2.2 kb (0.20 + 1.04
+ 1.04) from the 11-kb DFR1 circle and a HindIII
fragment of about 1.14 kb (0.20/2 + 1.04) from the 5.5-kb linear
DFR1 fragment (Fig. 4 A). In fact, two bands
with these expected sizes are detected by probe HH1.38 in the
HindIII digests of the yeast circular DNA sample (Fig.
4 B, lane 2). We also observed the 3.02-kb
EcoRI, 3.76-kb BamHI, 4.10-kb BglII, and
4.56-kb XbaI fragments predicted from the 11-kb DFR1 circle and the 1.51-kb EcoRI, 1.88-kb BamHI,
2.05-kb BglII, and 2.28-kb XbaI fragments of the
5.5-kb linear DFR1 fragment (Fig. 4 B). The
consistently lower intensity of the smaller band in each digest
probably reflects that there are more 11-kb DFR1 circles than
5.5-kb linear DFR1 molecules in this particular circular
DFR1 sample.
To characterize the junction (J2) that is the
junction at the SalI site 3` to DFR1, we used the
probe BS1.57 (Fig. 3). As shown in Fig. 4 C, this
probe detects the 1.82-kb HindIII, 1.88-kb EcoRI,
2.5-kb BglII, and 3.34-kb BamHI fragments expected
from the J2 junction of the 11-kb DFR1 circle and the 0.91-kb
HindIII, 0.94-kb EcoRI, 2.25-kb BglII, and
1.67-kb BamHI fragments of the 5.5-kb linear DFR1 molecule (Fig. 4 C). These observations are
consistent with the J2 junction lying within a region encompassing
about 0.2 kb downstream of the 3`- SalI site. The detection of
the additional 3.05-kb EcoRI and HindIII fragments by
probe BS1.57 is also expected (Fig. 4 C).
Most, if not
all, restriction fragments from both episomal DFR1 molecules
should be detected by probing with the entire 5.3-kb SalI
fragment. Following are the sizes of restriction fragments predicted
from the proposed structure of the episomal DFR1 molecules and
the restriction map of the DFR1 locus (Fig. 3):
SalI (2
Analysis with double restriction digests further
supports the proposed structure of the DFR1 molecules
(Fig. 4, C and D). One example of this analysis
is the BamHI- EcoRI digestion shown in lane 3 of Fig. 4 D. Both the 3.76- and 3.34-kb
BamHI junction bands are absent in the double digest (compare
lanes 1 and 3 of Fig. 4 D). Bands of
3.02, 1.95, 1.88, 1.51, 0.94, and 0.73 kb are observed. The 3.02-kb
band is the expected product of EcoRI digestion of the 3.76-kb
BamHI fragment proposed to span the J1 junction (Figs. 3 and
4 A). On the other hand, the 1.88- and 0.73-kb bands are the
expected products of EcoRI digestion of the 3.34-kb
BamHI fragment at the other junction (J2). Two identical
0.73-kb bands are generated due to the inverted duplication. The
1.95-kb band is consistent with BamHI digestion of the 3.05-kb
EcoRI band. Similarly, in the BglII/ HindIII
double digest (Fig. 4 D, lane 7), the
4.1-kb BglII fragment at the J1 junction disappears (Fig.
4 D, compare lanes 6 and 7), and the
two new 2.28- and 0.91-kb bands are the expected products of a
HindIII digestion of the 4.1-kb BglII fragment. Two
identical 0.91-kb bands are generated due to the inverted duplication.
In addition, the 2.14- and 0.91-kb bands are the expected products of a
BglII digestion of the 3.05-kb HindIII fragment
located in the middle of the 5.3-kb SalI fragment (
Fig. 3
and Fig. 4 D, lane 7).
Stronger hybridization is seen in all of these bands as they are
present twice in the 11-kb DFR1 circle and once in the 5.5-kb
linear DFR1 molecule. Taken together, the restriction mapping
data support the map of the yeast DFR1 episome shown in
Fig. 3
.
We have determined the molecular structure of a
yeast-amplified DFR1 episome and identified a cis-acting
element required for its autonomous replication. The overall sequence
organization of the yeast DFR1 episome resembles some
mammalian amplified DNAs and suggests some possible mechanisms
underlying its formation. In particular, the inverted repeat structure
and the association of a putative origin of replication is similar to a
recently characterized mouse ADA amplisome
(14, 28) ,
although the extent of the amplified region is much smaller in yeast.
Extrachromosomal circular elements are common products or intermediates
of mammalian gene amplification
(17) . There are, nonetheless,
only a couple of episomes or double minute chromosomes whose entire
sequence organization is known. As mentioned above, Nonet et al. (28) found that a mouse 500-kb ADA episome is circular and
is composed of a simple inverted duplication, similar to that of the
yeast DFR1 episome. A more complex structure was found in a
mouse 4000-kb double minute chromosome containing the mdm2 oncogene, which apparently consists of two large inverted repeats
that are inverted duplications
(29) . The yeast DFR1 episome is remarkably stable during repeated selections since no
rearrangement was found between the episome purified from cells derived
from the original MTX-resistant isolate 25-1 and the episome
purified from a MTX-resistant transformant of a dfr1 mutant
with purified DFR1 episome. Interestingly, the mouse ADA
episome that has a simple inverted repeat structure also constitutes
the predominant species in the amplified cell lines. Even the mouse
mdm2 episome that has a more complex duplication is found to
be more or less homogeneous.
The DFR1 episome could arise
in a number of ways. Possible mechanisms fall into two general classes:
1) fragmentation and extrachromosomal formation of an inverted repeat
or 2) intrachromosomal formation of the inverted repeat followed by
fragmentation. For instance, the DFR1 episome may be derived
from one or two acentric fragments deleted from a replicating
chromosome. The yeast mutant described in this report was derived from
a haploid strain, and the primary resistant cells still have a copy of
the DFR1 gene in its native chromosome, suggesting that a
conservative mechanism was used during the formation of the DFR1 episome. We therefore feel that it is unlikely that both fragments
are independently deleted from a replicating chromosome and joined to
form the dimeric DFR1 episome. This conclusion rests on the
assumption that over-replication in the DFR1 region within a
single cell cycle does not occur. A dimeric circular molecule could
also be derived through generation from a chromosome of a single
acentric fragment that is capable of autonomously replicating to
produce a second fragment. We have demonstrated in this report that the
yeast DFR1 episome does contain a cis-acting ARS element.
Joining two extrachromosomal fragments to form a dimeric molecule may
be the consequence of repairing the broken ends of the acentric
fragments. Kunes et al. (30, 31, 32) have demonstrated that a dimeric plasmid can be formed
after transformation of yeast with ARS-containing linear fragments
whose free ends have no homology with the yeast genome. One condition
required for the formation of the dimeric plasmid is that
single-stranded carrier DNAs must be used during the yeast
transformation. It has been suggested that the carrier DNAs may mimic
the damaged chromosomal DNAs and may induce a DNA repair system. Thus,
the initiation of gene amplification may also be influenced by certain
specific physiological conditions. Once the first dimeric episome is
generated, its further amplification may then result from
over-replication and/or unequal segregation of the episome during
subsequent cell division. For example, in the case of episomes with
inverted repeats, the amplification process can be accounted for by the
``double rolling circle model'' of replication proposed by
Passananti et al. (21) , which allows amplification
without reinitiation within a single cell cycle. This method of
replication is well documented in yeast for the endogenous plasmid
2-µm circle. On the other hand, yeast ARS plasmids that lack
centromeres have been shown to replicate once per cell cycle and to be
present in high copy number by virtue of unequal segregation. The
second class of models, that is, intrachromosomal formation of the
inverted duplication, is embodied by the chromosomal spiral model of
gene amplification
(20) . We are currently characterizing the
chromosomal DFR1 locus of the yeast-amplified mutant and the
sequences at the novel joints of the amplified DNAs to determine
whether there is any initiating event within the chromosomal DFR1 locus that may lead to the formation of the circular DFR1 episome.
Integration of submicroscopic episomes into
chromosomes has been proposed to be a possible mechanism for the
generation of homogeneously staining regions, the cytological hallmark
of chromosomal amplified DNAs, in mammalian cells. This pathway has
been difficult to establish because of the complex structure of
eukaryotic amplisomes. Studying integration of the yeast DFR1 episome described here should be much easier because of the
tendency toward homologous recombination observed in yeast plasmid
integration events and because the structure of both the episome and
chromosomal locus is well understood. We have already observed
Ura
Amplified DNA with the structure of the DFR1 episome
reported here has not been observed in cases of gene amplification
studied previously in yeast, although a preliminary description of a
circular episomal amplification has appeared (see below). The first
description of gene amplification was at the CUP1 locus. As
many as 15 copies of direct tandem repeats of a 2-kb fragment are seen
at the CUP1 locus, which confers copper resistance in many
yeast strains
(33) . However, de novo CUP1 amplification from a single copy to two or more at its native
chromosomal location has not been observed yet. Change in the copy
number of the amplified CUP1 repeats has been shown to be
mediated by unequal sister chromatid exchange between the pre-existing
repeats
(33, 34) . Amplification of the ADH4 gene was observed in a subset of yeast antimycin A-resistant
mutants. In a few cases, ADH4 amplicons were found with as
many as 50 copies of an extrachromosomal linear palindromic element
flanked by telomeres
(35, 36) . This linear 42-kb
ADH4 amplicon is produced by a fusion of two copies of the
terminal 21-kb sequence of chromosome VII where the ADH4 locus
is located. A recent report reveals that when a different and more
sensitive method than that used in the original study is used to detect
amplification events at the ADH4 locus, the extra copies of
the ADH4 DNA can be observed both within the chromosome and
apparently as circular episomes, although the extrachromosomal location
and circular structure have not been proved
(37) . It will be
interesting if the ADH4 amplicons also involve inverted
repeats as does the DFR1 episome. The different forms of
amplified DNA observed so far in the yeast system argue that more than
one mechanism can underlie yeast gene amplification events. Similar
categories of amplified DNAs are observed in mammalian systems
(9, 38) , making it likely that at least some aspects of
the underlying mechanisms may be conserved.
We have described a
convenient yeast amplification marker and suggest that its use, in
combination with other markers such as ADH4 and CUP1,
might allow one to take advantage of the yeast genetic system and the
small genome size to shed light on the mechanisms that control the
frequency of gene amplification events and, perhaps more generally,
that control genetic integrity of normal cells. Importantly, yeast
mutants should expedite testing the idea that the mechanisms of gene
amplification control may be intimately linked to the controls of cell
cycle progression and DNA replication
(6) . For instance,
synchronized yeast cells elongate the G1 phase of the cell cycle in
response to UV irradiation, presumably allowing repair of the genome
before entry into S phase where lesions might give rise to lethal or
mutagenic damage
(39) . rad9 mutants fail to elongate
the G1 period in response to UV irradiation
(39) . One might
envision, by analogy with the effect of the p53 mutation on gene
amplification rate, that either the frequency or the nature of
amplification of DFR1 might be altered in rad9 mutations after UV irradiation. Other mutations in yeast that
accelerate progress through G1, such as dominant CLN mutations
(40) , may also lead to an impaired ability to repair damage and
thus to an increased frequency of gene amplification, and amplification
can now be studied in such mutants. It is important to pursue studies
with mutants because until genetic or physiological conditions are
found to increase gene amplification rates in yeast, even with powerful
selective markers, the rate occurrence of amplification events such as
the one described here will continue to limit the usefulness of the
yeast system.
(
)
have
been able to use methotrexate to select Saccharomyces cerevisiae mutants with de novo amplification of the DFR1 locus encoding dihydrofolate reductase
(22) . Briefly, an
initial selection was carried out using the minimal lethal dose of
MTX
(
)
(25 µg/ml in rich medium). Of 32
MTX-resistant mutants, 7 were shown to have an elevated copy number of
the DFR1 gene. In three isolates, the amplified DNA seemed to
be chromosomal. However, in one isolate, strain 25-1, amplified
DNA was found primarily, if not exclusively, as multiple copies of an
extrachromosomal element(s) that appeared to be circular based on its
behavior on pulsed field gels
(22) .
The association
of an autonomously replicating sequence (ARS) with the DFR1 episomes was inferred from their extrachromosomal location and by
the ability of the DNAs purified from MTX-resistant cultures to
transform Dfr-yeast to Dfr
at high frequency
(22) .
We report here the detailed molecular
structure of the major species of the DFR1 episome/amplisome
found in the 25-1 mutant strain. We present molecular evidence
that the episome is circular. Furthermore, we show that the episome is
similar to extrachromosomal amplified DNA in mammalian cells in that it
is composed of an inverted repeat of sequences at the DFR1 locus, although the extent of the duplication is more limited.
Finally, we show that the episome is associated with an ARS that is
located at a position 3` to the DFR1 gene within the episome.
Strains, Media, and Selective
Conditions
Yeast complex (YPD) and synthetic (MC) media
used were previously described
(23) . M1-2B ( MATa
ura3-52 leu2-3, 112 trp1) is a haploid strain of
S. cerevisiae. Strain 25-1 was isolated as a mutant
strain from M1-2B by selecting for resistance to 25 µg/ml
methotrexate in YPD medium supplemented with 5 mg/ml sulfanilamide
(22) . This selective condition inhibits the growth of
M1-2B, which carries a single copy of the DFR1 gene, but
not strain M1-2B/pIUD-1, which was constructed by integration of
plasmid pIUD-1 carrying a cloned DFR1 gene, and therefore has
two copies of the DFR1 gene at the DFR1 locus. The
yeast dfr1::URA3 mutant YH5 ( MATa ura3-52
leu2-3, 112 trp1 tup dfr1::URA3) and its parental strain YH1
have been previously described
(24) . Strain 25-D11 is strain
YH5 transformed with DNA from strain 25-1 as described in Fig.
1 B.
Purification of DNA
The yeast circular
DNA samples used in Figs. 2 and 4 were prepared according to the
alkaline lysis-based procedure commonly used for plasmid purification
from Escherichia coli. Prior to the application of these
protocols, yeast spheroplasts were first generated with zymolyase from
20 to 50 ml of MTX-resistant cultures. As described by Maniatis et
al. (25) and modified by Morelle
(26) , the yeast
spheroplasts were resuspended in 200 ml of lysis buffer (50 m
M
glucose, 25 m
M Tris-HCl, pH 8, 10 m
M EDTA). After 5
min at room temperature, 400 ml of a freshly prepared alkaline solution
(0.2
N NaOH, 1% SDS) was added and mixed by inverting the
tubes 3-6 times. After 5 min on ice, 300 ml of ammonium acetate
(7.5
M, pH 7.8, without adjustment) was added, and the
contents of the tube were mixed gently for a few seconds. After 10 min
at 0 °C, the suspension was centrifuged for 3 min at 10,000 rpm.
The clear supernatant was transferred to a second tube, and 0.6 volumes
of isopropanol was added. The pellet was collected by centrifugation at
12,000 rpm for 10 min, washed with 70% ethanol, and dissolved in TE (10
m
M Tris-HCl, pH 8.0, 1 m
M EDTA).
TE buffer, and 25 ml of 20% SDS was
added. The tube was inverted 10 times to mix and then incubated at 65
°C for 20 min. DNA was collected by isopropanol precipitation,
washed, and resuspended in 300 ml of TE buffer. To ensure the recovery
of all small circular DNAs, the samples were spun for 5 min instead of
only 1 s as suggested in the original protocol.
Miscellaneous Methods
DNA probes used for
Southern hybridization are indicated in Fig. 3.
Figure 3:
Restriction map of the DFR1 region and proposed structure of the DFR1 episome. The
restriction map is compiled from the data of our studies and those of
others (41-44). Shown on the top are the cloned DFR1 fragments available to us. Below this map are the
sequences used as probes in Southern hybridization, which are
designated SS5.3 for the 5.3-kb SalI fragment, HH 1.38 for the
1.38-kb HindIII fragment, etc. In the middle is the
detailed restriction map of the 5.3-kb SalI fragment. On the
bottom is the proposed structure of the amplified DFR1 molecules. J1 and J2 indicate the upstream and downstream
junctions, respectively, with reference to the orientation of the
DFR1 gene.
Demonstration of Amplification of the DFR1
Gene
Amplification of the DFR1 gene in strain
25-1, a MTX-resistant mutant, was proposed in a previous study to
be due to the formation of episomes carrying the DFR1 gene.The initial observation underlying the proposal
that the amplified DNA is episomal is that the amplified DNA does not
comigrate in pulsed field gels with yeast chromosomes XV.
When the DNA of a control strain (Fig. 1 A, lane 1) and of the MTX-resistant isolate 25-1
(Fig. 1 A, lane 2) was analyzed by
pulsed field gel and Southern hybridization, signals detected with a
DFR1 probe were much stronger in the MTX-resistant isolate,
even though a smaller amount of DNA was loaded (Fig. 1 A,
lanes 3 and 4). The major hybridization
signal is detected in a position below the chromosome XV band and is
much stronger than the hybridization signal to chromosome XV itself
(Fig. 1 A, lane 4). These observations
suggest that most, and probably all, of the amplified DFR1 genes are not located on a native chromosome XV but possibly in an
episomal element.
Figure 1:
Amplification of the DFR1 gene. A, pulsed field gel and hybridization analysis of
DNA from MTX-resistant strain 25-1. DNA samples were prepared in
agarose plugs. Lanes 1 and 3, wild-type
control strain M1-2B; lanes 2 and 4, a
culture of strain 25-1 grown in MC medium supplemented with 50
µg/ml MTX and 5 mg/ml sulfanilamide. This is an approximately
25-fold increase in the MTX resistance level compared with the
wild-type strain, which is sensitive to about 2 µg/ml MTX and 5
mg/ml sulfanilamide in MC medium. Lanes 1 and 2 are the ethidium staining of a 1% pulsed field gel run at 200
volts at 14 °C in 0.5 TBE with the CHEF-DR II system from
Bio-Rad at 70-s switch time for 15 h and 120-s switch time for a
further 11 h. Lanes 3 and 4 are Southern
hybridization of blots from the pulsed field gel in lanes 1 and 2, probing with the BH0.74 DFR1 sequence (The probe is described in Fig. 3). B, pulsed
field gel and hybridization analysis of DNA from a strain transformed
with purified amplified DNA. Circular DNA was purified from strain
25-1 as described under ``Experimental Procedures'' and
used to transform the dfr1 strain YH5 to dTMP independence.
YH5 does not grow on YPD medium lacking dTMP due to insertion of the
URA3 gene into the DFR1 locus (24). Selection for
transforming DFR1 DNA was therefore carried out on YPD medium.
The frequency of transformation was
10
-10
/mg of DNA. Dfr
clones were plated on MC medium lacking uracil and a
Dfr
Ura
clone, 25-D11, was selected
for further study. Strain 25-D11 is MTX resistant. Lanes 1 and 2, wild-type and strain 25-D11, respectively, are the
ethidium bromide staining of a 1% pulsed field gel run at 200 volts at
14 °C in 0.5
TBE with the CHEF-DR II system from Bio-Rad at
70-s switch time for 16 h and 120-s switch time for a further 16 h.
Lanes 3 and 4 are hybridization of blots of
lanes 1 and 2, probing with the BH0.74
DFR1 sequence. The arrows indicate the position of
chromosome XV ( Chr. XV) carrying the DFR1 locus.
A second observation suggesting an episomal
location of the amplified DFR1 genes is that when DNA is
isolated by an alkaline lysis method from the original MTX-resistant
mutant, the purified DNA can transform at high frequency a
dfr1::URA3 mutant to a Dfrphenotype (the
ability to grow in the absence of dTMP in rich medium)
(Fig. 1 B, Ref. 16).
We now demonstrate that
the transforming DNA in the Dfr
transformant, 25-D11,
has a similar migration in pulsed field gels to the amplified DNA in
the original strain 25-1 (compare Fig. 1 B with
Fig. 1A). It is also worth noting that the chromosomal
DFR1 locus of the transformant had been disrupted by insertion
of the URA3 gene and that the transformant carrying the high
copy number DFR1 episome remains a Ura
prototroph even when subsequently selected for high level MTX
resistance. Thus, insertion by gene conversion does not appear to have
occurred. The pulsed field gel analysis further supports the idea that
the amplified DNA might be episomal in both the original isolate,
25-1, and in the Dfr
transformant, 25-D11.
Furthermore, the size and structure of the amplified DFR1 episomes in the transformant are to a first order the same as in
the original isolate. The experiments reported below characterize in
further detail the structure of the DFR1 genes both in strain
25-1 and in the Dfr
transformant, 25-D11.
The Amplified DNA Is on a Circular
Molecule
The slow migration of the amplified DNA in the
pulsed field gel, its lack of comigration with any linear chromosome,
and its ability to transform are consistent with a circular structure.
To test the idea of circularity further, DNA was purified from the
MTX-resistant mutant 25-1 by the alkaline lysis method, which
yields circular DNAs essentially free of chromosomal DNA. Three major
DNA species and some minor ones that hybridize to a DFR1 probe
were observed (Fig. 2, lane 1). To determine which,
if any, of the DNAs was circular, the DNA preparation was treated with
topoisomerase I, which relaxes the supercoiled form of circular
molecules, resulting in reduced mobility in agarose gels. Disappearance
of the middle major band and simultaneous increased hybridization in
the upper band after topoisomerase I treatment suggests that the upper
two bands correspond to relaxed and supercoiled topoisomers of the same
circular DNA (Fig. 2, lane 2). Circular
molecular weight standards on the gel suggest a molecular size of
approximately 11 kb for the circular species. Accordingly, digestion of
a mixture of the open and closed circular species with XbaI
gives two fragments of 6.4 and 4.6 kb (see below). The 2-µm
endogenous plasmid in these preparations was present at about the
level of the amplified DFR1 DNAs, as revealed by both ethidium
bromide staining and hydridization with a 2-µm DNA probe (data not
shown).
Figure 2:
Circularity of the DFR1 episome.
Lane 1, undigested yeast circular DNA purified by an
alkaline lysis method from a culture of strain 25-1 grown in MC
medium supplemented with 400 µg/ml MTX and 5 mg/ml sulfanilamide.
Lane 2, the same yeast circular DNA used in lane 1 was treated with topoisomerase I (Life Technologies,
Inc.) as recommended by the manufacturer. Lane 3,
undigested total yeast DNA purified from an unamplified control strain
YH5. Lane 4, undigested total yeast DNA purified from
a culture of strain 25-D11, a Dfrtransformant of a
dfr1 mutant with purified DFR1 episome, grown in MC
medium supplemented with 400 µg/ml MTX and 5 mg/ml sulfanilamide.
Lane 5, DNA from the same sample used in lane 4 was heated at 100 °C for 10 min for denaturing and
then annealed by slowly cooling to room temperature. Lane 6, DNA from the same sample used in lane 4 was denatured in 0.2
M NaOH and then neutralized with 0.2
volume of 3
M sodium acetate. Lane 7, DNA
from the same sample used in lane 4 was subjected to
the procedure of an alkaline plasmid purification. The size markers are
HindIII-digested
DNA fragments and 1-kb ladder (Life
Technologies, Inc.). The gels were blotted and probed with the BH0.74
DFR1 sequence. The arrow indicates the 5.5-kb linear
DFR1 fragment.
On the other hand, the smallest DFR1 hybridizing
molecule (Fig. 2) does not appear to be sensitive to
topoisomerase I. Its mobility corresponds to a 5.5-kb linear molecule
since it migrates with a 5.5-kb linear marker DNA under many different
electrophoresis conditions (Fig. 2; also, see Fig. 4and
data not shown). A linear species is unexpected, however, since linear
DNAs are thought to be excluded by the alkaline lysis purification
procedure. Several facts support the notion that the 5.5-kb species
might be a linear hairpin molecule arising from the circular species.
The ratio of the 5.5- to the 11-kb DFR1 species varies widely
between alkaline preparations of DNA. However, a species corresponding
to the 5.5-kb DFR fragment was not seen in total yeast DNA
samples prepared by non-denaturing procedures, even when they were
prepared from the same MTX-resistant cultures used for selective
purification of the yeast circular DNAs (Fig. 2, lane 4). To test whether the 5.5-kb species could be generated
by denaturing conditions, total yeast DNA was prepared under native
conditions and then denatured (either by heating or by alkaline
treatment) and renatured. The 5.5-kb DFR1 molecule was now
visible (Fig. 2, lanes 5 and 6). This
novel fragment was also seen if the total yeast DNA preparation was
submitted to the entire alkaline plasmid purification ( lane 7). We propose that the 11-kb DFR1 molecule may
consist of an inverted duplication and that the 5.5-kb band is derived
from the 11-kb circle by nicking, strand separation, and intrastrand
pairing during the circular DNA preparation. We also tentatively
conclude that the 11-kb species is the primary if not the only
amplified species in the cells and that the 5.5-kb species arises
during in vitro manipulation.
Figure 4:
Restriction enzyme mapping of the DFR1 episome. Yeast DNAs were isolated by the alkaline plasmid
purification method from MTX-resistant cultures of a dTMP-independent
transformant 25-D11 grown in MC medium supplemented with 400 µg/ml
MTX and 5 µg/ml sulfanilamide. Restriction enzymes used to digest
the DNA samples are indicated. Panels B-E are
Southern hybridizations using the probes defined in Fig. 3. B,
probed with HH1.38; C, probed with BS1.57; D, the
same blot shown in C probed with SS5.3; E, probed
with SS5.3. Restriction enzymes KpnI and XhoI do not
cut both DFR1 molecules ( lanes 2 and 5 of E). B, BamHI; Bg,
BglII; E, EcoRI; H,
HindIII; X, XbaI.
Attempts to Clone the Amplified DNAs
To
determine the precise structure of the amplified DNA and verify the
existence of an inverted repeat, we wanted to clone the amplisomes and
determine the DNA sequence. In the course of attempting to clone the
DFR1 episomes into an E. coli plasmid or a M13 phage
cloning vector, however, we have found that while some restriction
fragments were readily cloned, no full-length clones could be obtained.
We reasoned that this might be due to the presence of a large inverted
duplication in the amplified DNA since it is generally impossible to
clone large palindromic DNA sequences in a plasmid in E. coli.
We failed, however, with the use of not only the common E. coli cloning hosts such as DH5a (Life Technologies, Inc.) and XL1
(Stratagene) but also special cloning strains such as Sure (Stratagene)
and E. coli sbcBC that facilitate the cloning of DNAs with
secondary structure including inverted duplications in phage.
Since we were not able to clone the whole fragment in E. coli,
we have deduced the structure of the amplisome cloned in yeast (strain
25-D11, Fig. 1 B) using a refined restriction enzyme map
of amplified DNAs (Fig. 3 and 4).
Restriction Enzyme Mapping of the Amplified
Species
Amplified DNAs were purified from strain 25-D11 by
the alkaline lysis method and submitted to restriction enzyme
digestion. Restriction fragments were visualized by hybridization to
probes available to us from the previously cloned and characterized
DFR1 region. The location of some restriction sites around the
DFR1 locus and probe nomenclature are defined in Fig. 3.
SalI digestion of the purified circular DFR1 DNAs at
first appeared to produce a single fragment of about 5.3 kb, suggesting
a linear map one-half the size of the circular DNA (Fig. 4, B ( lane 1) and E ( lane 3)). This result would be consistent with our proposal of
a circular, dimeric structure consisting of the two SalI
fragments joined by two smaller SalI fragments at the
junctions for a total size of 11 kb. In keeping with this, in addition
to the 5.3-kb SalI fragment, a 0.2-kb SalI fragment
was also detected by probes such as HH1.38 that contain sequence
upstream of the 5`- SalI site (see Fig. 3 and
Fig. 4B, lane 1). The 5.3-kb
SalI fragment was readily cloned in a plasmid in E.
coli. A number of observations indicate that it is a continuous
segment derived from the DFR1 locus. All probes indicated in
Fig. 3
hybridize to the fragment. Conversely, when the
SalI fragment from the episome is isolated and labeled, it
hybridizes only to the same SalI fragment in a wild-type
genomic blot. Digestion of the fragment with additional enzymes reveals
that it has the same restriction map as the DFR1 locus shown
in Fig. 3. For convenience of discussion, we designate the
orientation of this SalI fragment by referring to the
SalI site upstream of the DFR1 gene, with respect to
the coding sequence, as the 5` -SalI site and the downstream
SalI site as 3`- SalI site.
5.30 + 2
0.20 from the 11-kb
DFR1 circle and 5.30 + 2
0.10 from the 5.5-kb
linear DFR1 molecule; HindIII (2
3.05 +
2.28 + 1.82 + 2
0.40 and 3.05 + 1.14 + 0.91
+ 0.4); EcoRI (2
3.05 + 3.02 + 1.88 and
3.05 + 1.51 + 0.94); BamHI (3.76 + 3.34 +
2
1.95 and 1.95 + 1.88 + 1.67); BglII (4.10
+ 2.50 + 2
2.20 and 2.20 + 2.05 + 1.25);
XbaI (6.44 + 4.56 and 3.22 + 2.28). In some digests,
the sum of all bands detected by the SS5.3 probe is equal to the
expected size of both 11- and 5.5-kb DFR1 molecules. For
example, a total of four XbaI fragments are detected
(Fig. 4 E, lane 4). The 6.4- and 4.6-kb
XbaI fragments are derived from the 11-kb circle, as they are
more intensely hybridized (Fig. 4 E) and are only seen in
the XbaI digest of either the gel-purified 11-kb DFR1 molecule or the total DNA sample of the yeast-amplified strain
(data not shown). The 3.2- and 2.3-kb XbaI fragments are
derived from the 5.5-kb linear fragment, as they are less intensely
hybridized (Fig. 4 E) and are only seen in the
XbaI digest of a gel-purified 5.5-kb DFR1 molecule
(data not shown). In other cases, the sum of all bands detected by
probe SS5.3 in each digest is smaller than the total size of both 11-
and 5.5-kb DFR1 molecules, as some of the restriction
fragments are present in both DFR1 molecules and/or present
more than once within the DFR1 episome. For example, there are
a total of five BamHI bands of 3.76, 3.34, 1.95, 1.88, and
1.67 kb in the BamHI digests of a circular DFR1 DNA
sample (Fig. 4 D, lane 1). The most
intensely hybridized fragment is the 1.95-kb band. This is expected as
it is present twice in the 11-kb circle and once in the 5.5-kb linear
molecule. Again, the intensity of the 3.76- and 3.34-kb BamHI
fragments is greater than that of the 1.88- and 1.67-kb fragments,
suggesting that there are relatively more 11 kb than 5.5-kb molecules
in this DNA sample.
Mapping of a Functional ARS Element in the Amplified
DNA
The episomal location and high copy number of the
DFR1 circle suggest that it replicates autonomously.
Furthermore, circular DFR1 DNAs purified from MTX-resistant
cultures are capable of transforming a yeast dfr1 mutant,
which cannot grow on complete medium lacking dTMP, to dTMP
independence. The frequency of transformation is equivalent to that
produced by an ARS-carrying plasmid (at least
10-10
dTMP-independent transformants per
milligram of DFR1 DNA).
In addition, the
transformants become resistant to 25 µg/ml MTX, just like the
original isolate. As shown above, the transforming DFR1 DNA
remains as multicopies of a circular episomal element that can be
purified by an alkaline lysis plasmid purification method. A
transformation assay was then used to directly map the cis-acting
elements responsible for the autonomous replication of the DFR1 episome. A number of restriction fragments derived from the
DFR1 circle and other available cloned sequences around the
DFR1 gene were subcloned into the E. coli vector
pGEM4Z and/or a yeast-integrating vector YIp5 to test their ability to
transform yeast at high frequency (Fig. 5). DNA fragments such as the
5.3-kb SalI fragment containing the DFR1 gene were
subcloned in pGEM4Z and used to transform a dfr1 mutant. DNA
fragments that do not contain the DFR1 gene were subcloned
into YIp5 and then used to transform a ura3 mutant strain such
as YH1. The results summarized in Fig. 5reveal that over a
region of about 12.52 kb around the DFR1 locus, sequences that
supported transformation at 10
-10
transformants per mg of DNA were detected only within the 0.7-kb
PstI- BamHI fragment that lies just downstream of the
DFR1 gene (also see Fig. 3). By definition, then, this
small fragment must contain an ARS element. Since this fragment is
contained within the 5.3-kb SalI fragment that is present on
the episome, we propose that the PstI- BamHI fragment
contains the ARS responsible for the autonomous replication of the
episome.
Figure 5:
Localization of an ARS activity in the
DFR1 locus. Restriction fragments tested for ARS activity by
transformation assay are indicated beneath the map of the
DFR1 region. The E. coli cloning vector pGEM4Z
carries the 9.0-kb BamHI, the 5.3-kb SalI, and the
1.78-kb BamHI- SalI fragments. These resultant
plasmids all have the DFR1 gene and therefore are used to
transform a dfr1 mutant. Transformants were selected for by
dTMP-independent growth in complete YPD medium. The remaining fragments
including the 1.78-kb BamHI- SalI fragment were
subcloned into yeast integration vector YIp5, which has the selectable
URA3 gene. Before inserting into the BamHI and
NheI sites of YIp5, the two BamHI- PstI
fragments were first converted into the BamHI- XbaI
fragments by inserting into the multiple cloning site of pGEM4Z, and
the BamHI- XbaI fragments, including the
BamHI- PstI sequences, were then cut out again from
multiple cloning sites. The resulting plasmids were used to transform a
ura3 mutant strain YH1 ( MATa ura3-52 leu2-3,
112 trp1 tup), and the transformants were selected for
Uraprototrophy. + indicates high frequency yeast
transformation. B, BamHI; S, SalI;
P, PstI.
segregants in derivatives of strain 25-D11
selected for high levels of MTX resistance. Further studies will show
if the Ura
clones result from gene conversion between
the episome and the chromosome, whether the putatively integrated DNA
is amplified, and what proportion of the DNA remains episomal.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.