©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amplification of a Circular Episome Carrying an Inverted Repeat of the DFR1 Locus and Adjacent Autonomously Replicating Sequence Element of Saccharomyces cerevisiae(*)

Tun Huang , Judith L. Campbell (§)

From the (1) From Braun Laboratories, MC147-75, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.() 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 Dfrat 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.


EXPERIMENTAL PROCEDURES

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).

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 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.




RESULTS

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. Dfrclones were plated on MC medium lacking uracil and a DfrUraclone, 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 Dfrtransformant, 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 Uraprototroph 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 Dfrtransformant, 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 Dfrtransformant, 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.

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 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.

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 .

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 10-10dTMP-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 10-10transformants 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.




DISCUSSION

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 segregants in derivatives of strain 25-D11 selected for high levels of MTX resistance. Further studies will show if the Uraclones 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.

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.


FOOTNOTES

*
This work was supported by Grants GM 25508 and GM 47281 from the U. S. Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 818-395-6053; Fax: 818-405-9452; E-mail, campbellj@starbase1.caltech.edu.

T. Huang, B. J. Barclay, R. C. von Borstel, P. J. Hastings, and S. M. Rosenberg, manuscript in preparation.

The abbreviations used are: MTX, methotrexate; ARS, autonomously replicating sequence; kb, kilobase(s); MOPS, 4-morpholinepropanesulfonic acid; ADA, [(carbamoylmethyl)imino]diacetic acid.


REFERENCES
  1. Tlsty, T. D., Margolin, B. H., and Lum, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9441-9445 [Abstract]
  2. Tlsty, T. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3132-3136 [Abstract]
  3. Wright, J. A., Smith, H. S., Watt, F. M., Hancock, M. C., Hudson, D. L., and Stark, G. R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1791-1795 [Abstract]
  4. Livingstone, L. R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T. D. (1992) Cell 70, 923-935 [Medline] [Order article via Infotrieve]
  5. Yin, Y., Tainsky, M. A., Bischoff, F. Z., Strong, L. C., and Wahl, G. M. (1992) Cell 70, 937-948 [Medline] [Order article via Infotrieve]
  6. Hartwell, L. (1992) Cell 71, 543-546 [Medline] [Order article via Infotrieve]
  7. Schimke, R. T. (1986) Cancer 57, 1912-1917 [Medline] [Order article via Infotrieve]
  8. Stark, G. R., Debatisse, M., Giulotto, E., and Wahl, G. M. (1989) Cell 57, 901-908 [Medline] [Order article via Infotrieve]
  9. Windle, B. E., and Wahl, G. M. (1992) Mutat. Res. 276, 199-224 [CrossRef][Medline] [Order article via Infotrieve]
  10. Lavi, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 6144-6148 [Abstract]
  11. Pellegrini, S., Dailey, L., and Basilico, C. (1984) Cell 36, 943-949 [Medline] [Order article via Infotrieve]
  12. Burhans, W. C., Selegue, J. E., and Heintz, N. H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7790-7794 [Abstract]
  13. Heintz, N. H., Milbrandt, J. D., Greisen, K. S., and Hamlin, J. L. (1983) Nature 302, 439-441 [Medline] [Order article via Infotrieve]
  14. Carroll, S. M., Derose, M. L., Kolman, J. L., Nonet, G. H., Kelly, R. E., and Wahl, G. M. (1993) Mol. Cell. Biol. 13, 2971-2981 [Abstract]
  15. Stark, G. R., and Wahl, G. M. (1984) Annu. Rev. Biochem. 53, 447-491 [CrossRef][Medline] [Order article via Infotrieve]
  16. Cowell, J. K. (1982) Annu. Rev. Genet. 16, 21-59 [Medline] [Order article via Infotrieve]
  17. Wahl, G. M. (1989) Cancer Res. 49, 21-59
  18. Fried, M., Feo, S., and Heard, E. (1991) Biochem. Biophys. Acta 1090, 143-155 [Medline] [Order article via Infotrieve]
  19. Aladjem, M. I., and Lavi, S. (1992) Mutat. Res. 276, 339-344 [Medline] [Order article via Infotrieve]
  20. Hyrien, O., Debatisse, M., Buttin, G., and De Saint Vincent, B. R. (1988) EMBO J. 7, 407-417 [Abstract]
  21. Passananti, C., Davies, B., Ford, M., and Fried, M. (1987) EMBO J. 6, 1697-1703 [Abstract]
  22. Huang, T. (1993) In Vivo Disruption and de Novo Amplification of the DFR1 Gene Encoding for Dihydrofolate Reductase in Saccharomyces cerevisiae. Ph. D. thesis, University of Alberta, Edmonton, Canada
  23. Sherman, F., Fink, G. R., and Hicks, J. (1986) Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Habor, NY
  24. Huang, T., Barclay, B. J., Kalman, T. I., von Borstel, R. C., and Hastings, P. J. (1992) Gene ( Amst.) 121, 167-171 [CrossRef][Medline] [Order article via Infotrieve]
  25. Maniatis, T., Frisch, E., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Morelle, G. (1989) Focus 11, 7
  27. Lee, F. S. (1992) BioTechniques 12, 677 [Medline] [Order article via Infotrieve]
  28. Nonet, G. H., Carroll, S. M., Derose, M. L., and Wahl, G. M. (1993) Genomics 15, 543-558 [CrossRef][Medline] [Order article via Infotrieve]
  29. Fakharzadeh, S. S., Rosenblumvos, L., Murphy, M., Hoffman, E. K., and George, D. L. (1993) Genomics 15, 283-290 [CrossRef][Medline] [Order article via Infotrieve]
  30. Kunes, S., Botstein, D., and Fox, M. S. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 617-628 [Medline] [Order article via Infotrieve]
  31. Kunes, S., Botstein, D., and Fox, M. S. (1985) J. Mol. Biol. 184, 375-387 [Medline] [Order article via Infotrieve]
  32. Kunes, S., Botstein, D., and Fox, M. S. (1990) Genetics 124, 67-80 [Abstract/Free Full Text]
  33. Fogel, S., and Welch, J. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5342-5346 [Abstract]
  34. Fogel, S., Welch, J. W., Cathala, G., and Karin, M. (1983) Curr. Genet. 7, 347-355
  35. Walton, J. D., Paquin, C. E., Kaneko, K., and Williamson, V. M. (1986) Cell 46, 857-863 [Medline] [Order article via Infotrieve]
  36. Dorsey, M., Peterson, C., Bray, K., and Paquin, C. E. (1992) Genetics 132, 943-950 [Abstract/Free Full Text]
  37. Dorsey, M. J., Hoeh, P., and Paquin, C. E. (1993) Curr. Genet. 23, 392-396 [Medline] [Order article via Infotrieve]
  38. Hamlin, J. L. (1992) Mutat. Res. 276, 179-187 [Medline] [Order article via Infotrieve]
  39. Siede, W., Friedberg, A. S., and Friedberg, E. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7985-7989 [Abstract/Free Full Text]
  40. Cross, F. R. (1988) Mol. Cell. Biol. 8, 4675-4684 [Medline] [Order article via Infotrieve]
  41. Barclay, B. J., Huang, T., Nagel, M. G., Misener, V. L., Game, J. C., and Wahl, G. M. (1988) Gene ( Amst.) 63, 175-185 [CrossRef][Medline] [Order article via Infotrieve]
  42. Fling, M. E., Kopf, J., and Richards, C. A. (1988) Gene ( Amst.) 63, 165-174 [Medline] [Order article via Infotrieve]
  43. Lagosky, P. A., Taylor, G. R., and Haynes, R. H. (1987) Nucleic Acids Res. 15, 10355-10371 [Abstract]
  44. Nath, K., and Baptist, E. W. (1984) Curr. Genet. 8, 265-270

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