currentGeneucs
Current Genetics 3, 83-89 (1981)
© Springer-Verlag 1981
Curing of Saccharomyces cerevisiae 2-pm DNA by Transformation Elke Erhart and Cornelis P. Hollenberg Institut f'tirMikrobiologie, Universit~itDiisseldorf, Universit~itsstraBe1, D-4000 Diisseldorf, Federal Republic of Germany
Summary. A general procedure for the curing o f 2-/~m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-~m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2-/am DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-/ma DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-gin DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the ceils completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir + and cir ° strains has been compared. Key words: 2-~-n DNA curing - Yeast transformation Incompatibility - Kanamycin resistance
Introduction Most laboratory strains of Saccharomyces cerevisiae contain 2-/am DNA (Hollenberg et al. 1970; Guerineau et al. 1971), a plasmid molecule present in 5 0 100 copies per cell. The few strains found to lack this DNA are mostly Saccharomyces carlsbergensis strains (Livingston 1977, Tabak 1977) or other wild brewery strains (Stewart, et al. 1980). Until now it has been impossible to inhibit specifically the replication or transmission of 2-/ma DNA in S. cerevisiae and generate 2-/~m DNA-less, so called cir ° strains. None of the methods applied for the curing of bacterial plasmids were proven to be successful for yeast. Cir ° strains of a defined phenotype can be used as a host for defective 2-/xm DNA molecules to detect es-
sential genes on the plasmid. Furthermore cir ° strains are useful hosts for recombinant plasmids carrying the 2-#m DNA repilcon, as such plasmids cannot be altered by recombination with endogenous 2-/xm DNA molecules. Finally, a cir ° strain in one case has been shown to give more stable transformants (Blanc et al. 1979). During the analysis of tile expression of antibiotic resistance genes on recombinant plasmids in yeast (Hollenberg 1979) we occasionally observed cells that had lost not only the recombinant plasmid, but also the endogenous 2-/Ira DNA. On the basis of this observation we developed a method to cure yeast 2-/~m DNA and to derive cirU strains. In this communication we describe the method and apply it to two yeast strains S. cerevisiae AH22 and GRF18 (LL20), which are frequently used as hosts in transformation systems. By using a kanamycin resistance carrying plasmid pMP81 also prototrophic strains can be cured. The application of the method has been mentioned in a previous report (Hollenberg et al. 1980).
Materials and Methods Strains andReeombinantPlasmid~ Three yeast strains were used as recipients for transformation: AH22, a double leu2 mutant (Itinnen et aL 1978) GRF18 (LL20) ~ leu2-3, leu2-12, his3-11, his3-15 and YT6-2-1L, a cir ° strain derived from AH22. The
strains were transformed with the recombinant plasmids pMP78 (Hollenberg 1979) and pJDB219 (Beggs 1978). Media. The complex medium used for the growth of yeast was
YEPD (1% yeast extract, 2% peptone, 2% glucose). The defined medium, YNB, contained 0,67% yeast nitrogen base without amino acids (Difco) and 2% glucose. Required amino acids were added to 20 ~g]ml. For the solid media 1.87~agar was supplemented. G418 was kindly donated by Schering-Plough Corp. Yeast transformation was performed according to the procedure
of Beggs (1978). O172-8083/81/0003/0083/$01.40
84
E. Erhart and C. P. Hollenberg: Curing of Yeast 2-/~m DNA
RA I ~~? IRB I EcoRI~ Fig. 1. pMP78-1 contains pBR325 (Bolivar 1978) and the HindIII fragment 3 including the lcu2 gene isolated from pJDB219 (Beggs 1978). pMP81 contains pCRI and a double EcoRI fragment including the leu2 gene and isolated from pJDB219
Yeast colony hybridization was performed as described by Grunstein and Hogness (1975) with modifications by Hinrlen et al. (1978).
~-Laetamase-Assay. The &laetamase-aetivity of yeast cells transformed with plasmid pMP78 was determined as described by Oaevallier and Aigle (1979).
DNA Analysis Yeast-DNA was isolated from 2 ml YEPD cultures grown overnight according to the method for rapid yeast DNA preparations of Struhl et al. (1979). Total yeast DNA was digested with the restriction endonueleases HindIII or EcoRI (Boehringer, Mannheim, FRG). Restriction digests were separated on 0.7% agarose gels and transferred to nitrocellulose filters by the Southern blotting procedure (Southern 1975). DNA was labetled with 32p by nick-translation using DNA-polymerase I from E. coli (Boehringer, Mannheim, FRG) according to the method of Maniafis et al. (1975). The specific activity of the labelled DNA was about 3 • 107 epm/~zg DNA.
Remits Detection o f Different Plasmids in Yeast Transformants In this study we used a combination o f analytical methods that have proven very useful for a quick determination o f plasmid DNA sequences in yeast transformants. Most o f the recombinant plasmids (Fig. 1) used here contain E. coli plasmid pBR 325 (Bolivar et al. 1978) and part o f pJDB219 (Beggs 1978). The presence o f plasmids that contain pBR325 is determined b y the /3-1actamase activity, the expression product o f the bacterial ampicillin resistance gene (Hollenberg 1979). The activity can be detected b y streaking colonies on an indicator plate (Chevallier and ?dgle 1979) and m a n y colonies can be tested in a single overnight assay (Fig. 2 0 . The presence o f recombinant plasmids containing pBR325 can be further demonstrated by colony hybridization with labelled pBR325 DNA. 2-/~m DNA is detected b y colony hybridization with the HindIII fragment 4 o f 2-pro DNA. This fragment does not occur in most o f the recombinant
Fig. 2 (a). Detection of fl-lactamase producing transformants of AH22 with the plasmid pMM8. Colonies were streaked out on the test-plates and incubated at 30 °C for 2 days. Then the penicillin containing I2/KI-mixture was carefully poured on the plates, The plates were kept for 1 h at 30 °C. After 12 h at 4 °C $1actamase producing strains showed a white halo against the deep blue background. (b) Detection of cir° and pMP780 yeast colonies. Colonies were grown on Miilipore HAWP filters overnight and treated in situ by zymolyase. Protoplasts were lysed, the DNA denatured and the filters were washed and finally baked at 80 °C for 1 - 2 h. The filters were hybridized with 32p-BTYP-2 plasmid DNA for about 14 h, washed 2 x in SSCP at 50 °C and exposed with Kodak X-Omat S film. The positive colonies contain either endogenous 2-gm DNA or pMP78 or both plasmids. The negative ones lost both plasmids. Two controls were present: positive AH22 with 2-pm DNA on the bottom of the filter and the cir0 strain YT6-2-1L on the top of the filters
plasmids used and therefore is a specific probe for endogenous 2-gin DNA. The colony hybridization is shown in Fig. 2b, where strains with and w i t h o u t 2-/Jxn DNA are used as a control. The presence o f the leu2 gene introduced with the recombinant plasmid into leuhost strains is determined b y growth on plates lacking leucine.
Curing o f S. eerevisiae GRF18 for 2-pro DNA by pMP78 In the first experiment to be reported here, we used strain G R F 1 8 and transformed it with pMP78-1. S. cerevisiae G R F 1 8 leu2 his2 was transformed with pMP78-1 b y the procedure of Beggs (1978) and analyzed for the presence of 2-/~m DNA and pMP78-1 after increasing numbers o f generations of growth b y the methods described above. In addition, the colonies were hybridized with radioactively labelled BTYP-2 DNA (Hollenberg 1978), a plasmid that carries the total 2-/am DNA in pBR322 inserted at the PstI site. Each transforrnant colony was grown in medium lacking leucine for about 15 generations after which the cells were tested for the presence of the leu2 gene b y
E. Erhart and C. P. Hollenberg: Curing of Yeast 2-#m DNA
85
Table 1. Properties of transformant YT32. GRF18, leu2, his3 was transformed with pMP78-1 following mainly the procedure described by Beggs (1978). Two transformants YT32-1 and YT32-2 were further analyzed. The results for both transformants were similar and the numbers in the table are the averages of both experiments. The transformants were grown on selective medium YNBH (YNB plus 20#g histidine per ml) for 15 generations. Culture samples were plated out on selectivemedium and on YEPD to determine the fraction of cells that are Leu +. For colony hybridization small amounts of cells were transferred with toothpicks to millipore filters and grown on YEPD medium. Hybridization probes: BTYP-2 DNA, a 2-#m DNApBR322 plasmid; and purified 2-;zm DNA HindlII fragment 4 (Hollenberg 1978) Growth conditions
15 generations in YNBH 36 generations in YEPD 72 generations in YEPD
Properties o f the transformants ¢~-Lactamase Production (%)
BTYP-2 Leu + Hybridization (%) (%)
85 15 10
85 20 10
90 15 10
cir 0
(%) 10 75 90
Table 2. Properties of cir ° transformants isolated after 15 generations. For detail see legend Table 1 Transformant &Lactamase BTYP-2 leu Production Hybridization 10, 81 15 87
-
+ -
+ -
cir
+ +
plating out equal amounts of cells on the same medium and on complete medium. About 240 of the colonies grown on the selective or on the non-selective plates were streaked on millipore filters and tested for the presence of pMP78-1 and 2-gm DNA by colony hybridization. In parallel the ~-lactamase activity of those colonies was tested. The results in Table 1 show that after 15 generations in selective medium 10% of the cells have lost the leu2 gene. On further testing about 15% of the colonies did not show/3-1actamase activity (Fig. 2a) or hybridization with BTYP-2 (Fig. 2b) and apparently had lost both pMP78 and 2-grn DNA. In a colony hybridization experiment with the specific 2-~m DNA probe no 2-/am DNA could be detected in about 10% of the colonies, which presumably had become cir °. A further analysis of individual transformants at this stage of the experiment showed different phenotypes of the c/r ° transformants (Table 2). The most abundant type (e.g. 10, 81) has lost both 2-/ma DNA and pMP78.
The loss of 2-/ma DNA can be explained by proposing a displacement of 2-/ira DNA by pMP78, which presumably uses the saff*e mode of replication and transmission. An analogy with the phenomenon of plasmid incompatibility in bacterial systems seems to be indicated. The loss of plasmid pMP78 in transformants 10 and 81 probably occurred on the non-selective plates following the loss of 2-/ira DNA (Table 1). More rare were the phenotypes of transformants 87 or 15. Transformant 87 is L e u ÷, but has lost both plasmids. We assume that at least the leu2 gene has integrated in the genome (Hinnen et al. 1978), a situation not detected by our colony hybridization. Transformant 15 seems to have lost only 2-pro DNA. These initial data indicate that cir ° strains, obtained by transformation with pMP78, can be isolated already after about 15 generations of growth o f the transformed colony. To calculate the actual number of generations of these transformants, we have to add the 17 generations leading from the transformed cell to the first colony, which brings it to a total of approx. 30 generations. After 15 generations on selective medium, the cells were transferred to complete medium (YEPD) and after 36 and 72 generations, they were tested for the same parameters. As shown in Table 1, the percentage o f L e u + cells declines under these conditions to 10%. The percentage of fi-lactamase+ cells decreases with about the same rate, a phenomenon that presumably has to be attributed to loss of pMP78 from the transformants. It is remarkable that we also observe in these cells a rapid loss of 2-/~m DNA. Already after 72 generations only 10% of the cells still have pMP78 and only 10% retain 2-/.tm DNA. The majority of the transformants has lost all plasmid DNA and is p M P 7 8 ° and cir °. A minority of the transformants still contains sequences of both plasmids although in some cases the ~-lactamase expression was very weak, suggesting that pMP78 is present in a low copy number or in a small number of cells. About half of the transformants that were stillLeu + and Cir + after 72 generations, had lost the /~.lactamase activity completely or nearly completely. These transformants will be discussed further below. Differences in stability between the initial transformant colonies were remarkable. About 5% were very stable and still had both plasmids after 60 generations, another 10% were very unstable and had already lost all plasmids after 20 generations. The majority of the transformants behaved as shown in Table 1 and 3.
Transformant Analysis o n S o u t h e r n Blots
To confirm the results of the colony hybridization and to analyze the arrangement of the plasmid DNA sequences, DNA of different transformants was isolated and
86
E. Erhart and C. P. Hollenberg: Curingof Yeast 2-tzmDNA
Table 3. Curing effect of pJDB219 and pMP78. Strain AH22, isogenicwith GRF18, was transformed with the plasmids pMP78-1 and pJDB219. 20 different transformant colonies were analyzed. Each transformant was grown selectively for 15 generations, followed by 60 generations in a non-selective medium. AIiquots of cells were plated on YEPD. Individual colonies were tested for ~qactamase activity and the presence of an active leu2 gene. The presence of 2-gin DNA and pMP78 was determined by colony hybridization. The data represent the averagevalues for the 20 transformants tested. Hasmid
~-Lactamase Production (%)
BTYP-2 Leu + Hybridization (%) (%)
pMP78 pJDB219
50 -
55 90
68 90
analyzed on Southern blots. The data in Fig. 3a show that indeed the DNA of transformant 81 does not give any hybridization in the Southern gels, which confirms the negative colony hybridization. The total absence of plasmid DNA sequences was also confirmed in Southern blots for transformants that were isolated after longer growth in complete medium. In all cases the hybridization conditions were such that DNA sequences present in a single copy per cell could be detected (Saunders et al. 1979). We can, therefore, conclude that most of the transformants that were scored as cir ° a n d p M P 7 8 ° by colony hybridization had lost both plasmids completely. Transformant 15, representing a minor class of transformants, after 15 generations of selective growth displayed only the presence of plasmid pMP78 in the Southern gel (Fig. 3b). Apparently 2-gm DNA is lost, while pMP78 is still present, a situation that might precede the
complete loss of plasmids and will be discussed further below. The loss o f both plasmids in the leu + transformant 87 is confirmed by the hybridization data in Fig. 3a. We assume that the L e u + phenotype is caused by an exchange of leu2 gene sequences between plasmid pMP78 and the chromosome.
The Stability o f D i f f e r e n t R e c o m b i n a n t Plasmids in a cir ° Strain
In our selection procedure transformants were isolated that are l e u - and then, in most cases, have lost both pMP78 and 2-pm DNA. It cannot yet be decided with certainty whether 2-/~m DNA is lost first, followed by a loss of the recombinant plasmid under non-selective conditions or that both plasmids are lost concomitantly. If 2-grn DNA is lost first, the transformants would thereafter contain only the recombinant plasmid. The appearance of l e u - cells would thus reflect the stability of the recombinant plasmid in a cir ° strain. We therefore tested the stability of different plasmids under nonselective conditions in strain YT6-2-1 L, a cir ° derivative of AH22. We found that in comparison to pJDB219, pMP78 is extremely unstable in YT6-2-1L After 60 generations 100% of the pMP78 transformant colonies had lost their plasmids, whereas pJDB219 was still present in over 80% of the colonies. This finding is in agreement with the data of Broach and Hicks (1980), who showed that in naturally isolated eir ° strains, the region of 2-gm DNA designated Baker (Hartley and Donelson 1980), is required for stable replication. The data here show that absence of this gene also leads to instability in an isogenic cir ° strain and therefore, the instability cannot be due to the requirement of additional genes absent from non-isogenic natural cir ° strains.
Fig. 3a. Autoradiograph of a Southern blot of undigested (right lanes) and HindIII digested DNA (left lanes) isolated from different transformant strains. A, DNA of transformant 32; B, transformant 81; C, transformant 57 and D, transformant 87. Hybridizationprobe 32p-labelled BTYP-2 DNA. b Autoradiograph of a Southern blot of Hind III digested DNAof some transformants. A and B, DNA of transformant 15. Only the two HindIII fragments of plasmid pMP78, pBR325 and the 3.3 kb band (3-1eu2 fragment) are present. C-G, HindIII fragments of DNA of different transformants. HindIII fragments I and 3 are ab sent in all transformants, D - F in addition lack pBR325. Hybridizationprobe 32P-labelled BTYP-2DNA
E. Erhart and C. L Hollenberg: Curing of Yeast 2-~zmDNA
87
Table 4. Different phenotypes of C/r+ transformants Strain
83/7 40/84 82 118/121/39 92/16/32/57
Phenotypes of Or + Transformants f~-Lactamase BTYP leu Hybridization
eir
(+)s (+)s +
+ + -* + +
+ + + +
+ + + +
Fig. 4. Autoradiograph of a Southern blot of HindlII digested (left lanes) and undigested DNA (right lanes) of yeast transformants. A and C, DNA of strains which lack pBR325, HindlII fragments 1 and 3 of 2-~zm DNA, but still are Leu + and show a new HindlIl fragment (transformants 83 and 7); B and D, transformant s 92 and 16 which still co ntain pBR325; E, transformant 57 containing pMP78, 2-pro DNA and the new fragment which presumable is Hind lII 1-1eu2;F, transformant 118 contains 2-pro DNA only; G, control mixture of 2-#m DNA from AH22 and pMP78 DNA. In all blots with digested DNA HindlII fragment 5 of 2-pro DNA is not visible. Bands a, a', b, b' and e, c' of lanes with undigested DNA indicate the supercoiled and open circular forms of respectively 2-~m DNA, pMP78 and 2-~zm DNA-leu2. Bands of higher molecular weights presumably represent multimerle form~ Hfcbridizationprobe 32p-labeUed BTYP-2 DNA
The Curing E f f e c t o f Plasmid pJDB219, Carrying the Whole 2-1gn D N A
In Table 3 the appearance o f l e u - cells among transformants that carry pMP78 or pJDB219 can be compared. The presence o f the recombinant plasmid pJDB219 is not easy to detect, because it does not carry the ~-lactamase gene. However, it is clear that after 60 generations only about 10% o f the cells are l e u - , and up to 90%
still contain pJDB219 and possibly also 2-pro DNA. The BTYP-2 colony hybridization shows that at least 5 - 1 0 % o f the cells lack the endogenous 2-/.tm DNA. In this case, however, these cells do not become l e u - , since pJDB219 as shown above is maintained more stably in a eir ° strain. The colony hybridization with the 2-pm DNA HindIII fragment 4 cannot be used for a direct screening o f endogenous 2-pro DNA. Only a Southern analysis can show the constitution o f the plasmid DNA in such transformants. From different transformants that were Leu + and positive in colony hybridization with labelled BTYP-2, the DNA was analyzed on Southern blots. Out of 5 clones examined, 4 still contained both plasmids, 2-/xm DNA and the recombinant plasmid pJDB219. One clone had lost 2-pro DNA and only carried pJDB219. In another experiment AH22 transformed with pJDB219 was first grown on plates under selective conditions for about 60 generations and then kept under non-selective conditions for 60 generations. After this time more than 90% o f the colonies were still Leu + and hybridized with labelled BTYP-2 DNA in colony hybridization experiments. The DNA from 8 strains was isolated and analyzed. All o f them had lost their 2-/1m DNA and only retained pJDB219. In summary these results show a similar curing effect of pJDB219 when the transformants are grown for a longer period under selective conditions. After the endogenous 2-pm DNA has disappeared, pJDB219 is rather stably replicated and the cir ° cells can be detected only by Southern hybridization.
Cir+ Transformants with Altered Plasmids
Among the transformantstested in the curing experiments, many colonies were observed that apparently had undergone changes other than just a plasmid loss. In Table 4 some representative strains are listed. The/3-1actamase activity was often low or completely absent in transformants that still were Leu + and contained 2-pro DNA sequences. The Southern hybridization of such transformants showed different plasmid arrangements. Mostly the pBR325 sequences were lost and the Ieu2 gene had recombined into the endogenous 2-pm DNA (Fig. 4). In Some cases the normal 2-pro DNA had been lost completely, detectable by the absence o f HindIII fragments 1 and 3. In other cases a small amount o f r~ormal 2-pm DNA was still present. In some weakly ~-lactamasepositive strains the pBR325 fragment was still present, but in reduced amounts. Tranformant 92 contains as smaller supercoil only the presumptive 2-pm DNA.leu2 plasmid. The HindIII digest shows the absence o f 2-pm DNA fragments 1 and 3, but the presence of pBR325, fragment 1-leu2, and 3-leu2. pBR325 probably is present
88
E. Erhart and C. P. HoUenberg: Curing of Yeast 2-#m DNA
in a larger supercoll, presumably a pMP78/2-/~m DNAleu2 cointegrate. The absence of fragment 3 points to the fact that the region of DNA carrying the leu2 fragment had recombined with a 2-/~m DNA molecule of type 14 leading to a fragment of 3.3 kb also pre sent in pMP78. The same event can take place with a type 23 2-/am DNA molecule and results then in the enlargement of HindIII fragment 1 from 4 kb to about 5 kb. The initial recombination could have taken place with only one type of molecule followed by an intramolecular recombination leading to the other from (Beggs 1978).
Application Strains
o f this Curing M e t h o d
to Prototrophic
The described curing method depends on the availability of auxotrophic markers, in general not present in industrial yeast strains. To extend the method for prototrophic strains we made use of plasmid pMP81 (Fig. 1) which contains approximately the same yeast DNA sequences as pMP78-1, but integrated at the EcoRI site of pCRI. pCRI carries the transposable resistance element Tn601 which contains a gene conferring resistance to kanamycin (Hollenberg 1979) and gentamycin 418 (Jimenez and Davis 1980) to yeast transformants. We transformed strain AH22 with plasmid pMP81 and plated the transformation mixture on complete medium (YEP - 2% glucose) containing 600 #g of CA18 per ml. After 5 days of incubation, the colonies were tested for the presence of the leu2 gene. It was found that 20% of the colonies were L e u 2 + and thus represented transformants that contain plasmid pMP81. In control experiments L e u 2 + revertants were not observed. The leu2- colonies presumably result from spontaneous resistant cells (Jimenez and Davis 1980). pMP81 gives the same curing effect as pMP78-1 and it should therefore be possible to cure any wild type strain for 2-/~m DNA. Gentamycin resistant colonies are grown according to the scheme used for pMP78, i.e. 15 generations selectively and 60 generations non-selectively. Then aliquots of each culture are plated out and screened for gentamycin resistance. Those transformant cultures that give over 50% of gentamycin sensitive cells should contain a high percentage of cir ° cells, which can be detected by colony hybridization or Southern hybridization of mini-lysates.
Discussion In this communication we have presented a procedure for the curing of 2-/~m DNA from S. cerevisiae strains by transformation. We have developed the method using strains AH22 and GRF18, auxotrophic mutants allowing
direct selection of a leu2 gene on the recombinant plasmid pMP78. This enables the curing of strains that are l e u 2 - and presumably other auxotrophic mutants for which the corresponding wild-type genes are available and can be integrated in pMP78 or a similar vector. For prototrophic strains and special industrial strains we propose the use of pMP81, which carries a kanamycin resistance gene and confers resistance against kanamycin (Hollenberg 1979) and G418 (Jimenez and Davis 1980). This plasmid is able to cure in a similar manner as pMP78 and can be selected in the transformants by direct growth on CA18. Not all colonies growing on G418 plates are transformants due to the fact that many spontaneous resistant cells arise (Jimenez and Davis 1980). In our experiment, however, 20% of the tested resistant colonies were real transformants, a percentage already high enough to allow for their efficient and fast isolation. Cir ° strains can offer an advantage for transformation with 2-pro DNA vectors. The most promising transformants with regard to stability are formed by a cir ° host strain and a recombinant plasmid containing most of 2-gin DNA. The curing data suggest that the replication of yeast plasmids is subjected to thephenomenon of incompatibility, so extensively studied in bacterial systems. Incompatibility in yeast seems to be a logical consequence of the fact that the endogenous2-~tmDNA ismaintained at a constant number of molecules per cell. Recombinant DNA molecules that use the 2-/~m DNA replication and transmission system can only be maintained in the cell at the expense of endogenous 2-/.tin DNA molecules. The observation of Gerbaud and Guerineau (1980) that the total number of recombinant and endogenous plasmid molecules in yeast transformants is identical to the number of endogenous 2-pro DNA in the host cell, supports this notion. The replication and distribution efficiency of both types of plasmids will decide which of the two plasmids will be segregated out. The combination of pMP78 and 2-/.tm DNA in one cell presents a peculiar case. The presence of pMP78 causes the loss of endogenous 2-~ma DNA, but pMP78 can only be stably maintained if 2-/Jxn DNA is present. This ist most probably due to a gene that has been shown to be required for stable replication (Broach and Hicks 1980) and that is not present on pMP78. As soon as pMP78 has displaced the endogenous 2-/1m DNA, it cannot be stablymaintained and pMP78 ° cells are segregated. In this way the cured cells can be identified directly by their l e u - phenotype. In a previous communication (Hollenberg et al. 1980) we reported that, under non-selective conditions, pMP78 was in a cir ° host almost as stable as in a cir+ host cell. The non-selective growth medium was yeast nitrogen base - 2% glucose plus histidine and leucine. Apparently this medium, although containing leucine, is selective and gives AH22 cells with a leu2 gene an advantage.
E. Erhart and C. P. Hollenberg: Curing of Yeast 2-~m DNA The data obtained with pJDB219 show that conditions selective for the recombinant plasmid in a transformant are favorable for the displacement o f endogenous 2-/.ma DNA. We do not k n o w whether this selection directly induces a higher c o p y number o f the recombinant plasmid. In the case o f the leu2 gene one would assume that a single copy could meet the cellular requirements unless its functioning on the plasmid is not as efficient as in the genome and multiple copies are needed for growth. On the other hand it is conceivable that the 2-grn DNA fragment carrying the leu2 gene could contain a DNA sequence that favors its replication and transmission over that o f endogenous 2-/2m DNA. New DNA sequences created b y the integration o f the leu2 gene fragment in 2-pan DNA or the AT sequence used to integrate the DNA fragment in the original PstI site (Beggs 1978) could be responsible. When this manuscript was in preparation Dobson et al. (1980) reported the observation o f a loss o f 2-/~m DNA in some yeast transformants carrying pJDB219.
Aclcnowledgernent~ We thank Helga Derichsweiler for excellent technical assistance and Dr. Barbara Sears for reading the manuscript. This work was supported by the Deutsche Forschnngsgemeinschaft.
References Beggs JD (1978) Nature 275:104-109 Blanc H, Gerbaud C, Slonimski PP, Guerineau M (1979) Mol Gen Genet 176:335-342 Bolivar F (1978) C,ene 4:121-136 Broach JR, Hicks JB (in press) In: Von Wettstein D, Friis J, Kiel-
89 land-Brandt M, Stenderup A (eds) Molecular genetics in yeast. Alfred Benzon Symp 16 Munksgaard Copenhagen Chevallier MR, Aigle M (1979) FEBS Lett 108:179-180 Dobson MJ, Fntcher AB, Cox BC (1980) Curt Genet 2: 201-205 Gerbaud C, Guerinean M (1980) Curr Genet 1:219-228 Grunstein M, Hogness DS (1975) Proe Natl Acad Sci USA 72: 3961-3965 Guerineau M, Grandchamp C, Paoletti J, Slonimski P (1971) Biochem Biophys Res Commun 42:550-557 Hartley JL, Donelson JE (1980) Nature 286:860-865 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933 HoUenberg CP (1978) Mol Gen Genet 162:23-34 HoUenberg CP (1979) In: Cummings DJ, Borst P, Dawid IB, Weissman SM, Fox CF (eds) Extrachromosomal DNA. ICN-UCLA Symp 15:325-338 Academic Press New York Hollenberg CP, Borst P, van Bruggen EFJ (1970) Biochim Biophys Acta 209:1-15 Hollenberg CP, Kustermann-Kuhn B, Mackedonski V, Erhart E (in press) In: Von Wettstein D, Friis J, Kielland-Brandt M, Stenderup A (eds) Molecular genetics in yeast. Alfred Benzon Syrup 16 Munksgaard Copenhagen Jimenez A, Davis J (1980) Nature 287:869-871 Livingston DM (1977) Genetics 86:73-84 Maniatis T, Jeffrey A, Kleid DG (1975) Proc Natl Acad Sci USA 72:1184-1188 Saunders GW, Rank GH, Kustermann-Kuhn B, HoUenberg CP (1979) Mol Gen Genet 175:45-52 Southern EM (1975) J Mol Biol 98:503-517 Stewart GG, Russell I, Panchal CJ (1980) Abstracts VI Int Syrup on Yeasts London (Ontario), Canada, p 212 Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035-1039 Tabak HF (1977) FEBS Letters 84:67-70 Communicated b y F. Kaudewitz Received February 6, 1981
Current Genetics 3, 83-89 (1981)
© Springer-Verlag 1981
Curing of Saccharomyces cerevisiae 2-pm DNA by Transformation Elke Erhart and Cornelis P. Hollenberg Institut f'tirMikrobiologie, Universit~itDiisseldorf, Universit~itsstraBe1, D-4000 Diisseldorf, Federal Republic of Germany
Summary. A general procedure for the curing o f 2-/~m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-~m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2-/am DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-/ma DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-gin DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the ceils completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir + and cir ° strains has been compared. Key words: 2-~-n DNA curing - Yeast transformation Incompatibility - Kanamycin resistance
Introduction Most laboratory strains of Saccharomyces cerevisiae contain 2-/am DNA (Hollenberg et al. 1970; Guerineau et al. 1971), a plasmid molecule present in 5 0 100 copies per cell. The few strains found to lack this DNA are mostly Saccharomyces carlsbergensis strains (Livingston 1977, Tabak 1977) or other wild brewery strains (Stewart, et al. 1980). Until now it has been impossible to inhibit specifically the replication or transmission of 2-/ma DNA in S. cerevisiae and generate 2-/~m DNA-less, so called cir ° strains. None of the methods applied for the curing of bacterial plasmids were proven to be successful for yeast. Cir ° strains of a defined phenotype can be used as a host for defective 2-/xm DNA molecules to detect es-
sential genes on the plasmid. Furthermore cir ° strains are useful hosts for recombinant plasmids carrying the 2-#m DNA repilcon, as such plasmids cannot be altered by recombination with endogenous 2-/xm DNA molecules. Finally, a cir ° strain in one case has been shown to give more stable transformants (Blanc et al. 1979). During the analysis of tile expression of antibiotic resistance genes on recombinant plasmids in yeast (Hollenberg 1979) we occasionally observed cells that had lost not only the recombinant plasmid, but also the endogenous 2-/Ira DNA. On the basis of this observation we developed a method to cure yeast 2-/~m DNA and to derive cirU strains. In this communication we describe the method and apply it to two yeast strains S. cerevisiae AH22 and GRF18 (LL20), which are frequently used as hosts in transformation systems. By using a kanamycin resistance carrying plasmid pMP81 also prototrophic strains can be cured. The application of the method has been mentioned in a previous report (Hollenberg et al. 1980).
Materials and Methods Strains andReeombinantPlasmid~ Three yeast strains were used as recipients for transformation: AH22, a double leu2 mutant (Itinnen et aL 1978) GRF18 (LL20) ~ leu2-3, leu2-12, his3-11, his3-15 and YT6-2-1L, a cir ° strain derived from AH22. The
strains were transformed with the recombinant plasmids pMP78 (Hollenberg 1979) and pJDB219 (Beggs 1978). Media. The complex medium used for the growth of yeast was
YEPD (1% yeast extract, 2% peptone, 2% glucose). The defined medium, YNB, contained 0,67% yeast nitrogen base without amino acids (Difco) and 2% glucose. Required amino acids were added to 20 ~g]ml. For the solid media 1.87~agar was supplemented. G418 was kindly donated by Schering-Plough Corp. Yeast transformation was performed according to the procedure
of Beggs (1978). O172-8083/81/0003/0083/$01.40
84
E. Erhart and C. P. Hollenberg: Curing of Yeast 2-/~m DNA
RA I ~~? IRB I EcoRI~ Fig. 1. pMP78-1 contains pBR325 (Bolivar 1978) and the HindIII fragment 3 including the lcu2 gene isolated from pJDB219 (Beggs 1978). pMP81 contains pCRI and a double EcoRI fragment including the leu2 gene and isolated from pJDB219
Yeast colony hybridization was performed as described by Grunstein and Hogness (1975) with modifications by Hinrlen et al. (1978).
~-Laetamase-Assay. The &laetamase-aetivity of yeast cells transformed with plasmid pMP78 was determined as described by Oaevallier and Aigle (1979).
DNA Analysis Yeast-DNA was isolated from 2 ml YEPD cultures grown overnight according to the method for rapid yeast DNA preparations of Struhl et al. (1979). Total yeast DNA was digested with the restriction endonueleases HindIII or EcoRI (Boehringer, Mannheim, FRG). Restriction digests were separated on 0.7% agarose gels and transferred to nitrocellulose filters by the Southern blotting procedure (Southern 1975). DNA was labetled with 32p by nick-translation using DNA-polymerase I from E. coli (Boehringer, Mannheim, FRG) according to the method of Maniafis et al. (1975). The specific activity of the labelled DNA was about 3 • 107 epm/~zg DNA.
Remits Detection o f Different Plasmids in Yeast Transformants In this study we used a combination o f analytical methods that have proven very useful for a quick determination o f plasmid DNA sequences in yeast transformants. Most o f the recombinant plasmids (Fig. 1) used here contain E. coli plasmid pBR 325 (Bolivar et al. 1978) and part o f pJDB219 (Beggs 1978). The presence o f plasmids that contain pBR325 is determined b y the /3-1actamase activity, the expression product o f the bacterial ampicillin resistance gene (Hollenberg 1979). The activity can be detected b y streaking colonies on an indicator plate (Chevallier and ?dgle 1979) and m a n y colonies can be tested in a single overnight assay (Fig. 2 0 . The presence o f recombinant plasmids containing pBR325 can be further demonstrated by colony hybridization with labelled pBR325 DNA. 2-/~m DNA is detected b y colony hybridization with the HindIII fragment 4 o f 2-pro DNA. This fragment does not occur in most o f the recombinant
Fig. 2 (a). Detection of fl-lactamase producing transformants of AH22 with the plasmid pMM8. Colonies were streaked out on the test-plates and incubated at 30 °C for 2 days. Then the penicillin containing I2/KI-mixture was carefully poured on the plates, The plates were kept for 1 h at 30 °C. After 12 h at 4 °C $1actamase producing strains showed a white halo against the deep blue background. (b) Detection of cir° and pMP780 yeast colonies. Colonies were grown on Miilipore HAWP filters overnight and treated in situ by zymolyase. Protoplasts were lysed, the DNA denatured and the filters were washed and finally baked at 80 °C for 1 - 2 h. The filters were hybridized with 32p-BTYP-2 plasmid DNA for about 14 h, washed 2 x in SSCP at 50 °C and exposed with Kodak X-Omat S film. The positive colonies contain either endogenous 2-gm DNA or pMP78 or both plasmids. The negative ones lost both plasmids. Two controls were present: positive AH22 with 2-pm DNA on the bottom of the filter and the cir0 strain YT6-2-1L on the top of the filters
plasmids used and therefore is a specific probe for endogenous 2-gin DNA. The colony hybridization is shown in Fig. 2b, where strains with and w i t h o u t 2-/Jxn DNA are used as a control. The presence o f the leu2 gene introduced with the recombinant plasmid into leuhost strains is determined b y growth on plates lacking leucine.
Curing o f S. eerevisiae GRF18 for 2-pro DNA by pMP78 In the first experiment to be reported here, we used strain G R F 1 8 and transformed it with pMP78-1. S. cerevisiae G R F 1 8 leu2 his2 was transformed with pMP78-1 b y the procedure of Beggs (1978) and analyzed for the presence of 2-/~m DNA and pMP78-1 after increasing numbers o f generations of growth b y the methods described above. In addition, the colonies were hybridized with radioactively labelled BTYP-2 DNA (Hollenberg 1978), a plasmid that carries the total 2-/am DNA in pBR322 inserted at the PstI site. Each transforrnant colony was grown in medium lacking leucine for about 15 generations after which the cells were tested for the presence of the leu2 gene b y
E. Erhart and C. P. Hollenberg: Curing of Yeast 2-#m DNA
85
Table 1. Properties of transformant YT32. GRF18, leu2, his3 was transformed with pMP78-1 following mainly the procedure described by Beggs (1978). Two transformants YT32-1 and YT32-2 were further analyzed. The results for both transformants were similar and the numbers in the table are the averages of both experiments. The transformants were grown on selective medium YNBH (YNB plus 20#g histidine per ml) for 15 generations. Culture samples were plated out on selectivemedium and on YEPD to determine the fraction of cells that are Leu +. For colony hybridization small amounts of cells were transferred with toothpicks to millipore filters and grown on YEPD medium. Hybridization probes: BTYP-2 DNA, a 2-#m DNApBR322 plasmid; and purified 2-;zm DNA HindlII fragment 4 (Hollenberg 1978) Growth conditions
15 generations in YNBH 36 generations in YEPD 72 generations in YEPD
Properties o f the transformants ¢~-Lactamase Production (%)
BTYP-2 Leu + Hybridization (%) (%)
85 15 10
85 20 10
90 15 10
cir 0
(%) 10 75 90
Table 2. Properties of cir ° transformants isolated after 15 generations. For detail see legend Table 1 Transformant &Lactamase BTYP-2 leu Production Hybridization 10, 81 15 87
-
+ -
+ -
cir
+ +
plating out equal amounts of cells on the same medium and on complete medium. About 240 of the colonies grown on the selective or on the non-selective plates were streaked on millipore filters and tested for the presence of pMP78-1 and 2-gm DNA by colony hybridization. In parallel the ~-lactamase activity of those colonies was tested. The results in Table 1 show that after 15 generations in selective medium 10% of the cells have lost the leu2 gene. On further testing about 15% of the colonies did not show/3-1actamase activity (Fig. 2a) or hybridization with BTYP-2 (Fig. 2b) and apparently had lost both pMP78 and 2-grn DNA. In a colony hybridization experiment with the specific 2-~m DNA probe no 2-/am DNA could be detected in about 10% of the colonies, which presumably had become cir °. A further analysis of individual transformants at this stage of the experiment showed different phenotypes of the c/r ° transformants (Table 2). The most abundant type (e.g. 10, 81) has lost both 2-/ma DNA and pMP78.
The loss of 2-/ma DNA can be explained by proposing a displacement of 2-/ira DNA by pMP78, which presumably uses the saff*e mode of replication and transmission. An analogy with the phenomenon of plasmid incompatibility in bacterial systems seems to be indicated. The loss of plasmid pMP78 in transformants 10 and 81 probably occurred on the non-selective plates following the loss of 2-/ira DNA (Table 1). More rare were the phenotypes of transformants 87 or 15. Transformant 87 is L e u ÷, but has lost both plasmids. We assume that at least the leu2 gene has integrated in the genome (Hinnen et al. 1978), a situation not detected by our colony hybridization. Transformant 15 seems to have lost only 2-pro DNA. These initial data indicate that cir ° strains, obtained by transformation with pMP78, can be isolated already after about 15 generations of growth o f the transformed colony. To calculate the actual number of generations of these transformants, we have to add the 17 generations leading from the transformed cell to the first colony, which brings it to a total of approx. 30 generations. After 15 generations on selective medium, the cells were transferred to complete medium (YEPD) and after 36 and 72 generations, they were tested for the same parameters. As shown in Table 1, the percentage o f L e u + cells declines under these conditions to 10%. The percentage of fi-lactamase+ cells decreases with about the same rate, a phenomenon that presumably has to be attributed to loss of pMP78 from the transformants. It is remarkable that we also observe in these cells a rapid loss of 2-/~m DNA. Already after 72 generations only 10% of the cells still have pMP78 and only 10% retain 2-/.tm DNA. The majority of the transformants has lost all plasmid DNA and is p M P 7 8 ° and cir °. A minority of the transformants still contains sequences of both plasmids although in some cases the ~-lactamase expression was very weak, suggesting that pMP78 is present in a low copy number or in a small number of cells. About half of the transformants that were stillLeu + and Cir + after 72 generations, had lost the /~.lactamase activity completely or nearly completely. These transformants will be discussed further below. Differences in stability between the initial transformant colonies were remarkable. About 5% were very stable and still had both plasmids after 60 generations, another 10% were very unstable and had already lost all plasmids after 20 generations. The majority of the transformants behaved as shown in Table 1 and 3.
Transformant Analysis o n S o u t h e r n Blots
To confirm the results of the colony hybridization and to analyze the arrangement of the plasmid DNA sequences, DNA of different transformants was isolated and
86
E. Erhart and C. P. Hollenberg: Curingof Yeast 2-tzmDNA
Table 3. Curing effect of pJDB219 and pMP78. Strain AH22, isogenicwith GRF18, was transformed with the plasmids pMP78-1 and pJDB219. 20 different transformant colonies were analyzed. Each transformant was grown selectively for 15 generations, followed by 60 generations in a non-selective medium. AIiquots of cells were plated on YEPD. Individual colonies were tested for ~qactamase activity and the presence of an active leu2 gene. The presence of 2-gin DNA and pMP78 was determined by colony hybridization. The data represent the averagevalues for the 20 transformants tested. Hasmid
~-Lactamase Production (%)
BTYP-2 Leu + Hybridization (%) (%)
pMP78 pJDB219
50 -
55 90
68 90
analyzed on Southern blots. The data in Fig. 3a show that indeed the DNA of transformant 81 does not give any hybridization in the Southern gels, which confirms the negative colony hybridization. The total absence of plasmid DNA sequences was also confirmed in Southern blots for transformants that were isolated after longer growth in complete medium. In all cases the hybridization conditions were such that DNA sequences present in a single copy per cell could be detected (Saunders et al. 1979). We can, therefore, conclude that most of the transformants that were scored as cir ° a n d p M P 7 8 ° by colony hybridization had lost both plasmids completely. Transformant 15, representing a minor class of transformants, after 15 generations of selective growth displayed only the presence of plasmid pMP78 in the Southern gel (Fig. 3b). Apparently 2-gm DNA is lost, while pMP78 is still present, a situation that might precede the
complete loss of plasmids and will be discussed further below. The loss o f both plasmids in the leu + transformant 87 is confirmed by the hybridization data in Fig. 3a. We assume that the L e u + phenotype is caused by an exchange of leu2 gene sequences between plasmid pMP78 and the chromosome.
The Stability o f D i f f e r e n t R e c o m b i n a n t Plasmids in a cir ° Strain
In our selection procedure transformants were isolated that are l e u - and then, in most cases, have lost both pMP78 and 2-pm DNA. It cannot yet be decided with certainty whether 2-/~m DNA is lost first, followed by a loss of the recombinant plasmid under non-selective conditions or that both plasmids are lost concomitantly. If 2-grn DNA is lost first, the transformants would thereafter contain only the recombinant plasmid. The appearance of l e u - cells would thus reflect the stability of the recombinant plasmid in a cir ° strain. We therefore tested the stability of different plasmids under nonselective conditions in strain YT6-2-1 L, a cir ° derivative of AH22. We found that in comparison to pJDB219, pMP78 is extremely unstable in YT6-2-1L After 60 generations 100% of the pMP78 transformant colonies had lost their plasmids, whereas pJDB219 was still present in over 80% of the colonies. This finding is in agreement with the data of Broach and Hicks (1980), who showed that in naturally isolated eir ° strains, the region of 2-gm DNA designated Baker (Hartley and Donelson 1980), is required for stable replication. The data here show that absence of this gene also leads to instability in an isogenic cir ° strain and therefore, the instability cannot be due to the requirement of additional genes absent from non-isogenic natural cir ° strains.
Fig. 3a. Autoradiograph of a Southern blot of undigested (right lanes) and HindIII digested DNA (left lanes) isolated from different transformant strains. A, DNA of transformant 32; B, transformant 81; C, transformant 57 and D, transformant 87. Hybridizationprobe 32p-labelled BTYP-2 DNA. b Autoradiograph of a Southern blot of Hind III digested DNAof some transformants. A and B, DNA of transformant 15. Only the two HindIII fragments of plasmid pMP78, pBR325 and the 3.3 kb band (3-1eu2 fragment) are present. C-G, HindIII fragments of DNA of different transformants. HindIII fragments I and 3 are ab sent in all transformants, D - F in addition lack pBR325. Hybridizationprobe 32P-labelled BTYP-2DNA
E. Erhart and C. L Hollenberg: Curing of Yeast 2-~zmDNA
87
Table 4. Different phenotypes of C/r+ transformants Strain
83/7 40/84 82 118/121/39 92/16/32/57
Phenotypes of Or + Transformants f~-Lactamase BTYP leu Hybridization
eir
(+)s (+)s +
+ + -* + +
+ + + +
+ + + +
Fig. 4. Autoradiograph of a Southern blot of HindlII digested (left lanes) and undigested DNA (right lanes) of yeast transformants. A and C, DNA of strains which lack pBR325, HindlII fragments 1 and 3 of 2-~zm DNA, but still are Leu + and show a new HindlIl fragment (transformants 83 and 7); B and D, transformant s 92 and 16 which still co ntain pBR325; E, transformant 57 containing pMP78, 2-pro DNA and the new fragment which presumable is Hind lII 1-1eu2;F, transformant 118 contains 2-pro DNA only; G, control mixture of 2-#m DNA from AH22 and pMP78 DNA. In all blots with digested DNA HindlII fragment 5 of 2-pro DNA is not visible. Bands a, a', b, b' and e, c' of lanes with undigested DNA indicate the supercoiled and open circular forms of respectively 2-~m DNA, pMP78 and 2-~zm DNA-leu2. Bands of higher molecular weights presumably represent multimerle form~ Hfcbridizationprobe 32p-labeUed BTYP-2 DNA
The Curing E f f e c t o f Plasmid pJDB219, Carrying the Whole 2-1gn D N A
In Table 3 the appearance o f l e u - cells among transformants that carry pMP78 or pJDB219 can be compared. The presence o f the recombinant plasmid pJDB219 is not easy to detect, because it does not carry the ~-lactamase gene. However, it is clear that after 60 generations only about 10% o f the cells are l e u - , and up to 90%
still contain pJDB219 and possibly also 2-pro DNA. The BTYP-2 colony hybridization shows that at least 5 - 1 0 % o f the cells lack the endogenous 2-/.tm DNA. In this case, however, these cells do not become l e u - , since pJDB219 as shown above is maintained more stably in a eir ° strain. The colony hybridization with the 2-pm DNA HindIII fragment 4 cannot be used for a direct screening o f endogenous 2-pro DNA. Only a Southern analysis can show the constitution o f the plasmid DNA in such transformants. From different transformants that were Leu + and positive in colony hybridization with labelled BTYP-2, the DNA was analyzed on Southern blots. Out of 5 clones examined, 4 still contained both plasmids, 2-/xm DNA and the recombinant plasmid pJDB219. One clone had lost 2-pro DNA and only carried pJDB219. In another experiment AH22 transformed with pJDB219 was first grown on plates under selective conditions for about 60 generations and then kept under non-selective conditions for 60 generations. After this time more than 90% o f the colonies were still Leu + and hybridized with labelled BTYP-2 DNA in colony hybridization experiments. The DNA from 8 strains was isolated and analyzed. All o f them had lost their 2-/1m DNA and only retained pJDB219. In summary these results show a similar curing effect of pJDB219 when the transformants are grown for a longer period under selective conditions. After the endogenous 2-pm DNA has disappeared, pJDB219 is rather stably replicated and the cir ° cells can be detected only by Southern hybridization.
Cir+ Transformants with Altered Plasmids
Among the transformantstested in the curing experiments, many colonies were observed that apparently had undergone changes other than just a plasmid loss. In Table 4 some representative strains are listed. The/3-1actamase activity was often low or completely absent in transformants that still were Leu + and contained 2-pro DNA sequences. The Southern hybridization of such transformants showed different plasmid arrangements. Mostly the pBR325 sequences were lost and the Ieu2 gene had recombined into the endogenous 2-pm DNA (Fig. 4). In Some cases the normal 2-pro DNA had been lost completely, detectable by the absence o f HindIII fragments 1 and 3. In other cases a small amount o f r~ormal 2-pm DNA was still present. In some weakly ~-lactamasepositive strains the pBR325 fragment was still present, but in reduced amounts. Tranformant 92 contains as smaller supercoil only the presumptive 2-pm DNA.leu2 plasmid. The HindIII digest shows the absence o f 2-pm DNA fragments 1 and 3, but the presence of pBR325, fragment 1-leu2, and 3-leu2. pBR325 probably is present
88
E. Erhart and C. P. HoUenberg: Curing of Yeast 2-#m DNA
in a larger supercoll, presumably a pMP78/2-/~m DNAleu2 cointegrate. The absence of fragment 3 points to the fact that the region of DNA carrying the leu2 fragment had recombined with a 2-/~m DNA molecule of type 14 leading to a fragment of 3.3 kb also pre sent in pMP78. The same event can take place with a type 23 2-/am DNA molecule and results then in the enlargement of HindIII fragment 1 from 4 kb to about 5 kb. The initial recombination could have taken place with only one type of molecule followed by an intramolecular recombination leading to the other from (Beggs 1978).
Application Strains
o f this Curing M e t h o d
to Prototrophic
The described curing method depends on the availability of auxotrophic markers, in general not present in industrial yeast strains. To extend the method for prototrophic strains we made use of plasmid pMP81 (Fig. 1) which contains approximately the same yeast DNA sequences as pMP78-1, but integrated at the EcoRI site of pCRI. pCRI carries the transposable resistance element Tn601 which contains a gene conferring resistance to kanamycin (Hollenberg 1979) and gentamycin 418 (Jimenez and Davis 1980) to yeast transformants. We transformed strain AH22 with plasmid pMP81 and plated the transformation mixture on complete medium (YEP - 2% glucose) containing 600 #g of CA18 per ml. After 5 days of incubation, the colonies were tested for the presence of the leu2 gene. It was found that 20% of the colonies were L e u 2 + and thus represented transformants that contain plasmid pMP81. In control experiments L e u 2 + revertants were not observed. The leu2- colonies presumably result from spontaneous resistant cells (Jimenez and Davis 1980). pMP81 gives the same curing effect as pMP78-1 and it should therefore be possible to cure any wild type strain for 2-/~m DNA. Gentamycin resistant colonies are grown according to the scheme used for pMP78, i.e. 15 generations selectively and 60 generations non-selectively. Then aliquots of each culture are plated out and screened for gentamycin resistance. Those transformant cultures that give over 50% of gentamycin sensitive cells should contain a high percentage of cir ° cells, which can be detected by colony hybridization or Southern hybridization of mini-lysates.
Discussion In this communication we have presented a procedure for the curing of 2-/~m DNA from S. cerevisiae strains by transformation. We have developed the method using strains AH22 and GRF18, auxotrophic mutants allowing
direct selection of a leu2 gene on the recombinant plasmid pMP78. This enables the curing of strains that are l e u 2 - and presumably other auxotrophic mutants for which the corresponding wild-type genes are available and can be integrated in pMP78 or a similar vector. For prototrophic strains and special industrial strains we propose the use of pMP81, which carries a kanamycin resistance gene and confers resistance against kanamycin (Hollenberg 1979) and G418 (Jimenez and Davis 1980). This plasmid is able to cure in a similar manner as pMP78 and can be selected in the transformants by direct growth on CA18. Not all colonies growing on G418 plates are transformants due to the fact that many spontaneous resistant cells arise (Jimenez and Davis 1980). In our experiment, however, 20% of the tested resistant colonies were real transformants, a percentage already high enough to allow for their efficient and fast isolation. Cir ° strains can offer an advantage for transformation with 2-pro DNA vectors. The most promising transformants with regard to stability are formed by a cir ° host strain and a recombinant plasmid containing most of 2-gin DNA. The curing data suggest that the replication of yeast plasmids is subjected to thephenomenon of incompatibility, so extensively studied in bacterial systems. Incompatibility in yeast seems to be a logical consequence of the fact that the endogenous2-~tmDNA ismaintained at a constant number of molecules per cell. Recombinant DNA molecules that use the 2-/~m DNA replication and transmission system can only be maintained in the cell at the expense of endogenous 2-/.tin DNA molecules. The observation of Gerbaud and Guerineau (1980) that the total number of recombinant and endogenous plasmid molecules in yeast transformants is identical to the number of endogenous 2-pro DNA in the host cell, supports this notion. The replication and distribution efficiency of both types of plasmids will decide which of the two plasmids will be segregated out. The combination of pMP78 and 2-/.tm DNA in one cell presents a peculiar case. The presence of pMP78 causes the loss of endogenous 2-~ma DNA, but pMP78 can only be stably maintained if 2-/Jxn DNA is present. This ist most probably due to a gene that has been shown to be required for stable replication (Broach and Hicks 1980) and that is not present on pMP78. As soon as pMP78 has displaced the endogenous 2-/1m DNA, it cannot be stablymaintained and pMP78 ° cells are segregated. In this way the cured cells can be identified directly by their l e u - phenotype. In a previous communication (Hollenberg et al. 1980) we reported that, under non-selective conditions, pMP78 was in a cir ° host almost as stable as in a cir+ host cell. The non-selective growth medium was yeast nitrogen base - 2% glucose plus histidine and leucine. Apparently this medium, although containing leucine, is selective and gives AH22 cells with a leu2 gene an advantage.
E. Erhart and C. P. Hollenberg: Curing of Yeast 2-~m DNA The data obtained with pJDB219 show that conditions selective for the recombinant plasmid in a transformant are favorable for the displacement o f endogenous 2-/.ma DNA. We do not k n o w whether this selection directly induces a higher c o p y number o f the recombinant plasmid. In the case o f the leu2 gene one would assume that a single copy could meet the cellular requirements unless its functioning on the plasmid is not as efficient as in the genome and multiple copies are needed for growth. On the other hand it is conceivable that the 2-grn DNA fragment carrying the leu2 gene could contain a DNA sequence that favors its replication and transmission over that o f endogenous 2-/2m DNA. New DNA sequences created b y the integration o f the leu2 gene fragment in 2-pan DNA or the AT sequence used to integrate the DNA fragment in the original PstI site (Beggs 1978) could be responsible. When this manuscript was in preparation Dobson et al. (1980) reported the observation o f a loss o f 2-/~m DNA in some yeast transformants carrying pJDB219.
Aclcnowledgernent~ We thank Helga Derichsweiler for excellent technical assistance and Dr. Barbara Sears for reading the manuscript. This work was supported by the Deutsche Forschnngsgemeinschaft.
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89 land-Brandt M, Stenderup A (eds) Molecular genetics in yeast. Alfred Benzon Symp 16 Munksgaard Copenhagen Chevallier MR, Aigle M (1979) FEBS Lett 108:179-180 Dobson MJ, Fntcher AB, Cox BC (1980) Curt Genet 2: 201-205 Gerbaud C, Guerinean M (1980) Curr Genet 1:219-228 Grunstein M, Hogness DS (1975) Proe Natl Acad Sci USA 72: 3961-3965 Guerineau M, Grandchamp C, Paoletti J, Slonimski P (1971) Biochem Biophys Res Commun 42:550-557 Hartley JL, Donelson JE (1980) Nature 286:860-865 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933 HoUenberg CP (1978) Mol Gen Genet 162:23-34 HoUenberg CP (1979) In: Cummings DJ, Borst P, Dawid IB, Weissman SM, Fox CF (eds) Extrachromosomal DNA. ICN-UCLA Symp 15:325-338 Academic Press New York Hollenberg CP, Borst P, van Bruggen EFJ (1970) Biochim Biophys Acta 209:1-15 Hollenberg CP, Kustermann-Kuhn B, Mackedonski V, Erhart E (in press) In: Von Wettstein D, Friis J, Kielland-Brandt M, Stenderup A (eds) Molecular genetics in yeast. Alfred Benzon Syrup 16 Munksgaard Copenhagen Jimenez A, Davis J (1980) Nature 287:869-871 Livingston DM (1977) Genetics 86:73-84 Maniatis T, Jeffrey A, Kleid DG (1975) Proc Natl Acad Sci USA 72:1184-1188 Saunders GW, Rank GH, Kustermann-Kuhn B, HoUenberg CP (1979) Mol Gen Genet 175:45-52 Southern EM (1975) J Mol Biol 98:503-517 Stewart GG, Russell I, Panchal CJ (1980) Abstracts VI Int Syrup on Yeasts London (Ontario), Canada, p 212 Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035-1039 Tabak HF (1977) FEBS Letters 84:67-70 Communicated b y F. Kaudewitz Received February 6, 1981
