Current Genetics 2, 201-205 (1980)
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© b y Springer-Verlag 1980
Loss of 2 um DNA from Saccharomyces cerevisiae Transformed with the Chimaeric Plasmid pJDB219 Melanie J. Dobson, A. Bruce Futcher, and Brian S. Cox Botany School, South Parks Road, Oxford 0X1 3RA England
Summary. Two plasmids containing Saccharomyces cerevisiae 2 # m DNA sequences and the S. cerevisiae L E U 2 gene have been found to display incompatibility with 2 #m DNA; in the presence o f the L E U 2 plasmids, 2/~m DNA can be lost. The L E U 2 plasmids can be lost spontaneously after (and before) 2 #m DNA loss has occurred, so that strains completely lacking 2/ira DNA sequences can be obtained routinely.
yeast transformed with the chimaeric plasmid pJDB219 (Beggs 1978), that a given strain could produce b o t h relatively stable and unstable transformants, and that at least some o f these stable transformants lacked 2/2m DNA. This paper reports an investigation o f this 2 #m DNA toss.
Materials and Key words: Chimaeric plasmid instability - 2 #m DNA loss.
Introduction
Virtually all strains of Saccharomyces cerevisiae contain 20 to 100 copies per cell of a circular DNA plasmid called 2/am DNA (Sinclair et al. 1967; Gibbins et al. 1977; Clark-Walker and Miklos 1974; Guerineau et al. 1976; Gerbaud and Guerineau 1980). This plasmid is not required b y the cell, since strains lacking 2 #m DNA do occur (Tabak 1977; Guerineau et al. 1974; Livingston 1977; Saunders et al. 1979). Attempts have been made to 'cure' yeast of their 2 #m DNA b y treatments which cure other extra-nuclear determinants found in yeast. So far, these attempts have been unsuccessful (Clark-Walker 1972; Griffiths et al. 1975; C. Mundy, M. Tuite, P. Lund and B. Cox, unpublished results). It has often been noted that chimaeric yeast - E. coli plasmids replicating from a 2 # m DNA origin are unstable during mitotic growth in yeast (Blanc et al. 1979; Struhl et al. 1979; Broach et al. 1979). This phenomenon may be related to the presence o f endogenous 2 #m DNA (Blanc et al. 1979). We found, during an investigation o f Offprint requests to: B. S. Cox
Methods
DNA preparation, plasmid purification, gel electrophoresis, nicktranslation, Southern transfers, hybridization, transformation of yeast, and restriction digestions were by standard methods, and were exactly as described in Dobson et al. 1980. Yeast Strains. MC16 (aade2-1 his4-712 leu2 SUF2 [2/~m+]) was obtained from G. Fink. LL20 (a leu2-3 leu2-112 his3-11 his3.15 [2/~m+]) was obtained from G. Fink via R. Storms. MD38 (a/a ade2-1/ade2-1 leu2/leu2 ura3-1/URA SQ5/SQ5 his? [2t~m+]) and MD35/4d (a leu2 ade2-1 SQ5 his? [2#m+]) were constructed during the course of this work. Media. YEPD was 2% glucose, 2% peptone (Difco), and 1% yeast extract (Difco), supplemented with 1.5% agar when solid medium was required. YNB + leu was 1% glucose, 0.67% yeast nitrogen base without amino acids (Difco), supplemented with 20~zg/ml adenine, histidine, and uracil, and 30 #g/ml leucine. YNB - leu was the same without leucine. 2% agar was added when solid medium was required. Experimental Procedures. Selective plate transfer: clones were streaked thinly onto YNB - leu agar plates, grown at 28 ° C to stationary phase, and then restreaked onto a fresh YNB - leu plate. Selective liquid transfer: 25 ml of YNB - leu in a 50 ml flask was innoeulated with 4 x 103 ceUs/ml and shaken at 28 °C for 24 h, at which time a sample was titred using a haemocytometer. The suspension was then diluted into 25 ml of fresh YNB - leu, so that the cell titre was once again 4 x 103 ceils/ml, and the process repeated. Non-selective liquid transfer was exactly as selective liquid transfer, except that YEPD was used instead of YNB - leu. Plamicls. The plasmids pJDB219 (Beggs 1978), pJDB248 (Beggs 1978) and YpR141 were the gifts of Dr. J. D. Beggs. 0172-8083/80/0002/0201/$01.00
202
M.J. Dobson et al.: Loss of 2 t~m DNA from Saccharomyces cerevisiae
Fig. 1. Electrophoretic separation on a 0.7% agarose gel of unrestricted DNA. 1, MC16 L+I [pYX+]; 2, MC16 L+2/5 [pJDB219+]; 3, MC16 L+8 [2 #m +] [pJDB219+]; 4, MC16 [2~m+]; 5, pJDB219, r X and sX = relaxed and supereoiled forms of pYX. rJ, lJ and sd = relaxed, linear and supercoiled forms of pJDB219, r2# and s2~ = relaxed and supereoiled 2 tzm DNA. m = multimeric forms of the plasmids
pJDBzlg-B
pYX- B
0-7 L4 ~ 7 ~
2.4
many generations by selective plate transfer. DNA preparations were made at regular intervals, and examined for the types of plasmid present by gel eleetrophoresis. A typical gel is shown in Fig. 1, and complete results are given in Table 1. These results show that: i) in transformed clones, a new plasmid appeared. We have designated it pYX and give its structure in Fig. 2. As shown by Dobson et al. (1980), pYX is composed of the entire 2 pm DNA plus the LEU2 fragment carried by pJDB219. It does not carry any bacterial sequences, and cannot be used to transform E. coli. It confers the LEU + phenotype when present in yeast, and, like the plasmid pSLel of Toh-e et al. (1980) is only rarely lost during mitotic growth, pYX is probably generated by a recombinational event or events that result in the transfer of the LEU2 fragment carried by pJDB219 to the endogenous 2 pm DNA plasmid. ii) In all but one of the transformed clones, 2 pm DNA is lost in favour of pJDB219, or of pYX, or of both. To prove that 2/.tm had been completely lost, and not merely reduced in copy number, Southern transfers were made, and 32P-labelled nick-translated pJDB219 was used as a hybridization probe. Results for MC16 L + 1 are shown in Fig. 3. Various forms of pYX have been detected, but 2 pm DNA has not. Single copy sensitivity has been achieved, since the LEU2 gene present in a single copy per celt in the high molecular weight DNA has been detected, both in the MC16 L+I track (where this hybridization is partially obscured by hybridization to a multimeric form of pYX) and in a control track, where DNA from a [2 pm ° ] [pYX° ] strain has been run. Since no 2 pm DNA circles have been detected even though single copy sensitivity has been achieved, we conelude that 2 pm DNA circles are completely absent in most, probably all, of the-cells extracted. Applicability to Other Strains. To extent these results
5-3 Fig. 2. Structure of the B form o f p Y X and pJDB219. (v) EcoRI sites, ()BamHI sites, (I---l) 2 pm DNA, (rv~-~) inverted repeat sequences of 2 gm DNA, (t----q) yeast chromosomal DNA carrying the LEU2 + gene, ( ~ ) pMB9 DNA. EcoRI fragment sizes are given in kilobases
to other strains, clones of LL20 and MD38 transformed to LEU + with pJDB219 were put through 100 generations of growth by the selective liquid transfer technique, and DNA preparations made. The results were similar to those Obtained with MC16; by gel electrophoresis, the LL20 contained pJDB219 and pYX, while MD38 contained only pYX. No 2 #m DNA was evident (data not shown). Both pJDB219 and p Y X can Eliminate 2 lim DNA. It is
Results Elimination o f the 2 tzm DNA o f MC16. MC 16 was transformed with pJDB219, and LEU + transformants were
selected. Four transformed clones,.designated MC 16 L+ 1, MC16 L+2, MC16 L+3, and MC16 L+8, were grown for
clear from the behaviour of MC16 L+3 and MC16 L÷2/5 as given in Table 1 that pJDB219 can compete with 2/~m DNA to its exclusion in the presence or absence of pYX. To find out if pYX can compete with 2/~m DNA in the absence of pJDB219, a clone of MC16 L+I ([pYX + ] [pJDB219 ° ] [2/lm ° ]) was crossed to MD35/4d,
M. J. Dobson et al.: Loss of 2/~m DNA from Saccharomyees cerevisiae
203
Table 1. Types of DNA plasmids present in pJDB219 - transformed clones of MC16 in successive samples from cultures on YNB - leu medium
DN•AP•sType of
LEU+ transformant
mid prep- \ arationa ~
MC16L+I
MC16 L+2
MC16 L+2/5
MC16 L+3
MC16 L+8
1
b
+
0
+
+
0
n.t.
n.t.
n.t.
+
+
0
n.t.
n.t.
n.t.
2 3 4 5 6
0 0 0 0 n.t.
+ 0 0 0 n.t.
+ + + + n.t.
n.t. 0 n.t. 0 n.t.
n.t. + n.t. + n.t.
n.t. + n.t. + n.t.
0 0 n.t. n.t. 0
+ + n.t. n.t. +
0 0 n.t. n.t. 0
n.t. 0 n.t. n.t. 0
n.t. + n.t. n.t. +
n.t. 0 n.t. n.t. 0
+ b + + n.t.
+ + + + n.t.
+ 0 0 0 n.t.
2 #m pJDB219 pYX 2 #m pJDB219 pYX 2/~m pJDB219 pYX 2 #m pJDB219 pYX 2 ~m pJDB219 pYX
a approximately 75 generations between DNA preparations b data insufficient + plasmid present 0 plasmid absent n.t. = not tested
the resulting diploid sporulated, and M D 3 7 / l a ([pYX ÷ ] [2/am+]) obtained. After 0, 7, 14, 21, 28, 35 and 42 generations o f growth in YNB - leu, clones were chosen and DNA preparations made. Gel electrophoresis showed that the clones chosen after 0, 7, and 14 generations each contained 2/am DNA and pYX, while four o f the five clones chosen after 21 generations contained pYX but not 2/am DNA. The fifth clone, from generation 35, contained both plasmids. We conclude that pYX competes with 2/am DNA, as does pJDB219.
Selection is not Required for 2 prn DNA Elimination. M D 3 7 / l a was grown by non-selective liquid transfer for 85 generations, and DNA extracted from one LEU + clone. Gel electrophoresis showed that pYX was present, and that 2/am DNA was not.
Fig. 3. Autoradiogram of a Southern transfer (from a 0.7% agarose gel) probed with 32p-pJDB219. 1, 5 #g of DNA extracted from MC16 L+I [2#m °] [pYX+]; 2, 0.0025 /~g of pJDB219 DNA (the equivalent of 1 copy per cell with reference to track 1); 3, 5/~g of DNA from a strain 'cured' of 2/~m DNA; 4, 2 #m -DNA. rX, lX and sX = relaxed, linear and supercoiledpYX. ~1= supercoiled pJDB219, r2/~ and s2# = relaxed and supercoiled 2 ~m DNA. e = high molecular weight chromosomal DNA. mX= multimeric forms ofpYX, rn2~ = multimeric forms of 2 tzm DNA
Generation o f 2 p~n DNA Sequence-less Strains. In order to obtain a strain free o f 2/am DNA sequences, whether on a 2/am DNA circle, a chimaeric plasmid, or integrated into the chromosomal DNA, a clone of MC 16 L + 1 which had been shown by hybridization to contain pYX b u t not 2/am DNA circles or pJDB219 was grown for 500 generations b y the non-selective liquid transfer technique, after which tfme 32% o f the ceils in the population were leucine auxotrophs. A DNA preparation was made from an a u x o t r o p h designated MC16 L+1-39-8. No plasmid band o f any sort could be seen after gel electrophoresis (Fig. 4). A Southern transfer was made, and 32P-labelled nick-translated pJDB248 (a plasmid similar to pJDB219, b u t with a larger LEU2 fragment - Beggs, 1978) showed the expected hybridization to the high molecular weight DNA (due to the presence, in a single copy per cell, o f the chromosomal leu2 gene), b u t to no other bands
204
M.J. Dobson et al.: Loss of 2 tzm DNA from Saeeharomyces cerevisiae
Fig. 4. Electrophoretic separation of unrestricted DNA on 0.7% agarose gels (1, 3, 5) and matching autoradiograms (2, 4, 6) where either 32p-YpR141 or 32p-pJDB248 has been hybridized to a Southern transfer of the gel. 1, DNA extracted from MC16 [2 t~m+]; 2, 32p-YpR141 hybridized to track 1 ; 3, 5 ug of DNA extracted from MC16 L+1-39-8; 4, 32p-pJDB248 hybridized to track 3; 5, 5 tzg of DNA extracted from MC16 L+1-39-8; 6, 32p-YpR141 hybridized to track 5. C = high molecular weight chromosomal DNA. r21~ and s2tz = relaxed and supercoiled 2 tzm DNA. rn2~ = multimeric forms of 2 ~m DNA. The 'exposed' area at the top of track 6 is due to an imperfection in the film, not to hybridization
Fig. 5. Electrophoretic separation of unrestricted DNA on a 0.7% agarose gel (1, 6) and matching autoradiograms (2, 5) where either 32p-pJDB248 or 32p-YpR141 has been hybridized to a Southern transfer of the gel. 1, 5/zg of DNA extracted from LL20-11-2; 2, 32p-pJDB248 hybridized to track 1; 3, a control showing hybridization to 0.0005 tzg of pJDB219 (the equivalent of 1/5 copy per cell with respect to tracks 1 and 2); 4, a control showing hybridization of 32p-YpR141 to 0.0005 ~g ofpJDB219 (the equivalent of 1/5 copy per cell with respect to tracks 5 and 6); 5, 32p-YpR141 hybridized to- track 6 ;, 6, 5 ~g of DNA extracted from LL20-11-2. O = origin. C = high molecular weight chromosomal DNA. rJ and sJ = relaxed and supercoiled pJDB219. r2t~ and s21~ indicate the positions at which relaxed and supercoiled 2 #m DNA marker ran in an adjacent slot
(Fig. 4). 32P-labelled YpR141 (a plasmid consisting o f pMB9 and a complete 2/zm DNA circle, but no other yeast DNA - J. Beggs, personal communication), however, did not hybridize to any portion of the gel track, though a separate single copy control showed that single copy sensitivity had been achieved. Therefore we conclude that no 2/~m DNA sequences are present in MC16 L+1-39-8, since any 2 # m DNA sequences on plasmids would have been detected in b o t h hybridization experiments described above, while any integrated sequences present in the high molecular weight DNA would have been detected when 32P-labelled YpR141 was used as a probe.
To prove that pJDB248 hybridizes to the high molecular weight DNA solely because of its homology to the chromosomal leu2 gene, we digested DNA from MC16 L+1-39-8 with BamHI, and found that pJDB248 hybridizes to a single fragment, 7 kb in size, the size expected for the leu2 BamHI fragment (Storms et al. 1979). YpR141 shows no hybridization to BamHI digested DNA from this strain. Similar.experiments have been done with an isolate o f strain LL20 designated LL20-11-2 (which arose as a leu- clone in a population of LEU +, pJDB219 transformed cells) and it, too, appears to be free of any 2/~m DNA sequences (Fig. 5).
M. J. Dobson et al.: Loss of 2 #m DNA from Saccharomycescerevisiae
Discussion While it is clear that 2/am DNA is mitotically unstable in the presence of either pYX or pJDB219, the reason for this instability is not known. The obvious speculation, which is supported by the recent work of Gerbaud and Guerineau (1980), is that the total number of 2/am DNA origins (whether on 2/am DNA circles or chimaeric plasmids) is subject to copy number control. Random segregation would then generate some cells in which the entire 'allowed' complement of 2/am DNA origins is located on chimaeric plasmids. Preferential replication of the chimaeric plasmid would increase the rate of generation of such cells, while preferential replication of 2/am would decrease it. The difficulty with this speculation is that it predicts that the chimaeric plasmids will be mitotically stable in [2/am ° ] strains, whereas in fact some instability is observed. This difficulty would be resolved if 2/am DNA circles themselves are slightly unstable, as has been suggested by Guerineau et al. (1974). However, the near ubiquity of 2/am DNA in laboratory strains argues against this, unless [2/am ° ] strains are at a selective disadvantage when compared with their [2/am + ] counterparts. There are several genetic determinants, including [psi] (Cox 1965), [ URE3] (Lacroute 1971, [20sRNA ] (Garvik and Haber 1978), and [NEX], [HOK] and [EXL] (Wickner 1980), which are inherited in a 4 : 0 , non-Mendelian pattern as are 2/am DNA circle (Livingston 1977). [2/am °] strains generated after transformation of strains carrying determinants such as those mentioned above can be used to test whether there is a strict correlation between the loss of 2/am DNA and the loss of any such non-Mendelian phenotype. [2 /am °] strains have at least one advantage over isogenic [2/~m + ] strains as chimaeric plasmid hosts in that 2/am DNA based chimaeric plasmids recombine with endogenous 2/am DNA after transformation (Storms et al. 1979), generating new plasmids which can complicate genetic analysis considerably. No new plasmids can be generated in this way in [2/am ° ] strains.
205
Acknowledgements.We would like to thank Mr. Stephen Kearsey for introducing us to nick-translation, Drs. I. W. Craig and A. J. Kingsman for gifts of radioactive nucleotide, Dr. J. D. Beggs for her plasmids, and Drs. J. Friesen and R. Storms for encouragement and helpful discussions.
References Beggs JD (1978)Nature 275:104-109 Blanc H, Gerbaud C, Slonimski PP, Guerineau M (1979) Mol Gen Genet 176:335-342 Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121-133 Clark-Walker GD (1972) Proc Natl Acad Sci USA 69:388-392 Clark-Walker GD, MiNos GLG (1974) Eur J Biochem 41:359365 Cox BS (1965) Heredity 20:505-521 Dobson M, Futcher AB, Cox BS (1980) Curt Gen 2:193-200 Garvik B, Haber JE (1978) J Bacteriol 134:261-269 Gerbaud C, Guerineau M (1980) Curr Gen 1 : 219-228 Griffiths DE, Lancashire WE, Zanders ED (1975) FEBS Lett 53: 126-130 Gubbins E J, Newlon CS, Kann MD, Donelson JE (1977) Gene 1: 185-207 Guerineau M, Grandchamp C, Slonimski PP (1976) Proc Natl Acad Sci USA 73:3030-3034 Guerineau M, Slonimski PP, Avner PR (1974)Biochem Biophys Res Commun 61:462-469 Lacroute F (1971) J Bacteriol 106:5.19-522 Livingston DM (1977) Genetics 86: 73-84 Saunders GW, Rank GH, Kustermann-Kuhn B, HoUenberg CP (1979) Mol Gen Genet 175:45-52 Sinclair JH, Stevens RJ, Sanghavi P, Rabinowitz M (1967) Science 156:1234-1237 Storms RK, McNeil JB, Khandekar PS, Parker J, An GJ, Friesen JD (1979) J Bacteriol 140:73-82 Strum K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035-1039 Tabak HF (1977) FEBS Letters 84:67-70 Toh-e A, Guerry-Kopecko P, Wickner RB (1980) J Bacteriol 141:413-416 Wickner RB (1980) Cell 21:217-226
Communicated by F. Kaudewitz Received June 30, 1980
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© b y Springer-Verlag 1980
Loss of 2 um DNA from Saccharomyces cerevisiae Transformed with the Chimaeric Plasmid pJDB219 Melanie J. Dobson, A. Bruce Futcher, and Brian S. Cox Botany School, South Parks Road, Oxford 0X1 3RA England
Summary. Two plasmids containing Saccharomyces cerevisiae 2 # m DNA sequences and the S. cerevisiae L E U 2 gene have been found to display incompatibility with 2 #m DNA; in the presence o f the L E U 2 plasmids, 2/~m DNA can be lost. The L E U 2 plasmids can be lost spontaneously after (and before) 2 #m DNA loss has occurred, so that strains completely lacking 2/ira DNA sequences can be obtained routinely.
yeast transformed with the chimaeric plasmid pJDB219 (Beggs 1978), that a given strain could produce b o t h relatively stable and unstable transformants, and that at least some o f these stable transformants lacked 2/2m DNA. This paper reports an investigation o f this 2 #m DNA toss.
Materials and Key words: Chimaeric plasmid instability - 2 #m DNA loss.
Introduction
Virtually all strains of Saccharomyces cerevisiae contain 20 to 100 copies per cell of a circular DNA plasmid called 2/am DNA (Sinclair et al. 1967; Gibbins et al. 1977; Clark-Walker and Miklos 1974; Guerineau et al. 1976; Gerbaud and Guerineau 1980). This plasmid is not required b y the cell, since strains lacking 2 #m DNA do occur (Tabak 1977; Guerineau et al. 1974; Livingston 1977; Saunders et al. 1979). Attempts have been made to 'cure' yeast of their 2 #m DNA b y treatments which cure other extra-nuclear determinants found in yeast. So far, these attempts have been unsuccessful (Clark-Walker 1972; Griffiths et al. 1975; C. Mundy, M. Tuite, P. Lund and B. Cox, unpublished results). It has often been noted that chimaeric yeast - E. coli plasmids replicating from a 2 # m DNA origin are unstable during mitotic growth in yeast (Blanc et al. 1979; Struhl et al. 1979; Broach et al. 1979). This phenomenon may be related to the presence o f endogenous 2 #m DNA (Blanc et al. 1979). We found, during an investigation o f Offprint requests to: B. S. Cox
Methods
DNA preparation, plasmid purification, gel electrophoresis, nicktranslation, Southern transfers, hybridization, transformation of yeast, and restriction digestions were by standard methods, and were exactly as described in Dobson et al. 1980. Yeast Strains. MC16 (aade2-1 his4-712 leu2 SUF2 [2/~m+]) was obtained from G. Fink. LL20 (a leu2-3 leu2-112 his3-11 his3.15 [2/~m+]) was obtained from G. Fink via R. Storms. MD38 (a/a ade2-1/ade2-1 leu2/leu2 ura3-1/URA SQ5/SQ5 his? [2t~m+]) and MD35/4d (a leu2 ade2-1 SQ5 his? [2#m+]) were constructed during the course of this work. Media. YEPD was 2% glucose, 2% peptone (Difco), and 1% yeast extract (Difco), supplemented with 1.5% agar when solid medium was required. YNB + leu was 1% glucose, 0.67% yeast nitrogen base without amino acids (Difco), supplemented with 20~zg/ml adenine, histidine, and uracil, and 30 #g/ml leucine. YNB - leu was the same without leucine. 2% agar was added when solid medium was required. Experimental Procedures. Selective plate transfer: clones were streaked thinly onto YNB - leu agar plates, grown at 28 ° C to stationary phase, and then restreaked onto a fresh YNB - leu plate. Selective liquid transfer: 25 ml of YNB - leu in a 50 ml flask was innoeulated with 4 x 103 ceUs/ml and shaken at 28 °C for 24 h, at which time a sample was titred using a haemocytometer. The suspension was then diluted into 25 ml of fresh YNB - leu, so that the cell titre was once again 4 x 103 ceils/ml, and the process repeated. Non-selective liquid transfer was exactly as selective liquid transfer, except that YEPD was used instead of YNB - leu. Plamicls. The plasmids pJDB219 (Beggs 1978), pJDB248 (Beggs 1978) and YpR141 were the gifts of Dr. J. D. Beggs. 0172-8083/80/0002/0201/$01.00
202
M.J. Dobson et al.: Loss of 2 t~m DNA from Saccharomyces cerevisiae
Fig. 1. Electrophoretic separation on a 0.7% agarose gel of unrestricted DNA. 1, MC16 L+I [pYX+]; 2, MC16 L+2/5 [pJDB219+]; 3, MC16 L+8 [2 #m +] [pJDB219+]; 4, MC16 [2~m+]; 5, pJDB219, r X and sX = relaxed and supereoiled forms of pYX. rJ, lJ and sd = relaxed, linear and supercoiled forms of pJDB219, r2# and s2~ = relaxed and supereoiled 2 tzm DNA. m = multimeric forms of the plasmids
pJDBzlg-B
pYX- B
0-7 L4 ~ 7 ~
2.4
many generations by selective plate transfer. DNA preparations were made at regular intervals, and examined for the types of plasmid present by gel eleetrophoresis. A typical gel is shown in Fig. 1, and complete results are given in Table 1. These results show that: i) in transformed clones, a new plasmid appeared. We have designated it pYX and give its structure in Fig. 2. As shown by Dobson et al. (1980), pYX is composed of the entire 2 pm DNA plus the LEU2 fragment carried by pJDB219. It does not carry any bacterial sequences, and cannot be used to transform E. coli. It confers the LEU + phenotype when present in yeast, and, like the plasmid pSLel of Toh-e et al. (1980) is only rarely lost during mitotic growth, pYX is probably generated by a recombinational event or events that result in the transfer of the LEU2 fragment carried by pJDB219 to the endogenous 2 pm DNA plasmid. ii) In all but one of the transformed clones, 2 pm DNA is lost in favour of pJDB219, or of pYX, or of both. To prove that 2/.tm had been completely lost, and not merely reduced in copy number, Southern transfers were made, and 32P-labelled nick-translated pJDB219 was used as a hybridization probe. Results for MC16 L + 1 are shown in Fig. 3. Various forms of pYX have been detected, but 2 pm DNA has not. Single copy sensitivity has been achieved, since the LEU2 gene present in a single copy per celt in the high molecular weight DNA has been detected, both in the MC16 L+I track (where this hybridization is partially obscured by hybridization to a multimeric form of pYX) and in a control track, where DNA from a [2 pm ° ] [pYX° ] strain has been run. Since no 2 pm DNA circles have been detected even though single copy sensitivity has been achieved, we conelude that 2 pm DNA circles are completely absent in most, probably all, of the-cells extracted. Applicability to Other Strains. To extent these results
5-3 Fig. 2. Structure of the B form o f p Y X and pJDB219. (v) EcoRI sites, ()BamHI sites, (I---l) 2 pm DNA, (rv~-~) inverted repeat sequences of 2 gm DNA, (t----q) yeast chromosomal DNA carrying the LEU2 + gene, ( ~ ) pMB9 DNA. EcoRI fragment sizes are given in kilobases
to other strains, clones of LL20 and MD38 transformed to LEU + with pJDB219 were put through 100 generations of growth by the selective liquid transfer technique, and DNA preparations made. The results were similar to those Obtained with MC16; by gel electrophoresis, the LL20 contained pJDB219 and pYX, while MD38 contained only pYX. No 2 #m DNA was evident (data not shown). Both pJDB219 and p Y X can Eliminate 2 lim DNA. It is
Results Elimination o f the 2 tzm DNA o f MC16. MC 16 was transformed with pJDB219, and LEU + transformants were
selected. Four transformed clones,.designated MC 16 L+ 1, MC16 L+2, MC16 L+3, and MC16 L+8, were grown for
clear from the behaviour of MC16 L+3 and MC16 L÷2/5 as given in Table 1 that pJDB219 can compete with 2/~m DNA to its exclusion in the presence or absence of pYX. To find out if pYX can compete with 2/~m DNA in the absence of pJDB219, a clone of MC16 L+I ([pYX + ] [pJDB219 ° ] [2/lm ° ]) was crossed to MD35/4d,
M. J. Dobson et al.: Loss of 2/~m DNA from Saccharomyees cerevisiae
203
Table 1. Types of DNA plasmids present in pJDB219 - transformed clones of MC16 in successive samples from cultures on YNB - leu medium
DN•AP•sType of
LEU+ transformant
mid prep- \ arationa ~
MC16L+I
MC16 L+2
MC16 L+2/5
MC16 L+3
MC16 L+8
1
b
+
0
+
+
0
n.t.
n.t.
n.t.
+
+
0
n.t.
n.t.
n.t.
2 3 4 5 6
0 0 0 0 n.t.
+ 0 0 0 n.t.
+ + + + n.t.
n.t. 0 n.t. 0 n.t.
n.t. + n.t. + n.t.
n.t. + n.t. + n.t.
0 0 n.t. n.t. 0
+ + n.t. n.t. +
0 0 n.t. n.t. 0
n.t. 0 n.t. n.t. 0
n.t. + n.t. n.t. +
n.t. 0 n.t. n.t. 0
+ b + + n.t.
+ + + + n.t.
+ 0 0 0 n.t.
2 #m pJDB219 pYX 2 #m pJDB219 pYX 2/~m pJDB219 pYX 2 #m pJDB219 pYX 2 ~m pJDB219 pYX
a approximately 75 generations between DNA preparations b data insufficient + plasmid present 0 plasmid absent n.t. = not tested
the resulting diploid sporulated, and M D 3 7 / l a ([pYX ÷ ] [2/am+]) obtained. After 0, 7, 14, 21, 28, 35 and 42 generations o f growth in YNB - leu, clones were chosen and DNA preparations made. Gel electrophoresis showed that the clones chosen after 0, 7, and 14 generations each contained 2/am DNA and pYX, while four o f the five clones chosen after 21 generations contained pYX but not 2/am DNA. The fifth clone, from generation 35, contained both plasmids. We conclude that pYX competes with 2/am DNA, as does pJDB219.
Selection is not Required for 2 prn DNA Elimination. M D 3 7 / l a was grown by non-selective liquid transfer for 85 generations, and DNA extracted from one LEU + clone. Gel electrophoresis showed that pYX was present, and that 2/am DNA was not.
Fig. 3. Autoradiogram of a Southern transfer (from a 0.7% agarose gel) probed with 32p-pJDB219. 1, 5 #g of DNA extracted from MC16 L+I [2#m °] [pYX+]; 2, 0.0025 /~g of pJDB219 DNA (the equivalent of 1 copy per cell with reference to track 1); 3, 5/~g of DNA from a strain 'cured' of 2/~m DNA; 4, 2 #m -DNA. rX, lX and sX = relaxed, linear and supercoiledpYX. ~1= supercoiled pJDB219, r2/~ and s2# = relaxed and supercoiled 2 ~m DNA. e = high molecular weight chromosomal DNA. mX= multimeric forms ofpYX, rn2~ = multimeric forms of 2 tzm DNA
Generation o f 2 p~n DNA Sequence-less Strains. In order to obtain a strain free o f 2/am DNA sequences, whether on a 2/am DNA circle, a chimaeric plasmid, or integrated into the chromosomal DNA, a clone of MC 16 L + 1 which had been shown by hybridization to contain pYX b u t not 2/am DNA circles or pJDB219 was grown for 500 generations b y the non-selective liquid transfer technique, after which tfme 32% o f the ceils in the population were leucine auxotrophs. A DNA preparation was made from an a u x o t r o p h designated MC16 L+1-39-8. No plasmid band o f any sort could be seen after gel electrophoresis (Fig. 4). A Southern transfer was made, and 32P-labelled nick-translated pJDB248 (a plasmid similar to pJDB219, b u t with a larger LEU2 fragment - Beggs, 1978) showed the expected hybridization to the high molecular weight DNA (due to the presence, in a single copy per cell, o f the chromosomal leu2 gene), b u t to no other bands
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M.J. Dobson et al.: Loss of 2 tzm DNA from Saeeharomyces cerevisiae
Fig. 4. Electrophoretic separation of unrestricted DNA on 0.7% agarose gels (1, 3, 5) and matching autoradiograms (2, 4, 6) where either 32p-YpR141 or 32p-pJDB248 has been hybridized to a Southern transfer of the gel. 1, DNA extracted from MC16 [2 t~m+]; 2, 32p-YpR141 hybridized to track 1 ; 3, 5 ug of DNA extracted from MC16 L+1-39-8; 4, 32p-pJDB248 hybridized to track 3; 5, 5 tzg of DNA extracted from MC16 L+1-39-8; 6, 32p-YpR141 hybridized to track 5. C = high molecular weight chromosomal DNA. r21~ and s2tz = relaxed and supercoiled 2 tzm DNA. rn2~ = multimeric forms of 2 ~m DNA. The 'exposed' area at the top of track 6 is due to an imperfection in the film, not to hybridization
Fig. 5. Electrophoretic separation of unrestricted DNA on a 0.7% agarose gel (1, 6) and matching autoradiograms (2, 5) where either 32p-pJDB248 or 32p-YpR141 has been hybridized to a Southern transfer of the gel. 1, 5/zg of DNA extracted from LL20-11-2; 2, 32p-pJDB248 hybridized to track 1; 3, a control showing hybridization to 0.0005 tzg of pJDB219 (the equivalent of 1/5 copy per cell with respect to tracks 1 and 2); 4, a control showing hybridization of 32p-YpR141 to 0.0005 ~g ofpJDB219 (the equivalent of 1/5 copy per cell with respect to tracks 5 and 6); 5, 32p-YpR141 hybridized to- track 6 ;, 6, 5 ~g of DNA extracted from LL20-11-2. O = origin. C = high molecular weight chromosomal DNA. rJ and sJ = relaxed and supercoiled pJDB219. r2t~ and s21~ indicate the positions at which relaxed and supercoiled 2 #m DNA marker ran in an adjacent slot
(Fig. 4). 32P-labelled YpR141 (a plasmid consisting o f pMB9 and a complete 2/zm DNA circle, but no other yeast DNA - J. Beggs, personal communication), however, did not hybridize to any portion of the gel track, though a separate single copy control showed that single copy sensitivity had been achieved. Therefore we conclude that no 2/~m DNA sequences are present in MC16 L+1-39-8, since any 2 # m DNA sequences on plasmids would have been detected in b o t h hybridization experiments described above, while any integrated sequences present in the high molecular weight DNA would have been detected when 32P-labelled YpR141 was used as a probe.
To prove that pJDB248 hybridizes to the high molecular weight DNA solely because of its homology to the chromosomal leu2 gene, we digested DNA from MC16 L+1-39-8 with BamHI, and found that pJDB248 hybridizes to a single fragment, 7 kb in size, the size expected for the leu2 BamHI fragment (Storms et al. 1979). YpR141 shows no hybridization to BamHI digested DNA from this strain. Similar.experiments have been done with an isolate o f strain LL20 designated LL20-11-2 (which arose as a leu- clone in a population of LEU +, pJDB219 transformed cells) and it, too, appears to be free of any 2/~m DNA sequences (Fig. 5).
M. J. Dobson et al.: Loss of 2 #m DNA from Saccharomycescerevisiae
Discussion While it is clear that 2/am DNA is mitotically unstable in the presence of either pYX or pJDB219, the reason for this instability is not known. The obvious speculation, which is supported by the recent work of Gerbaud and Guerineau (1980), is that the total number of 2/am DNA origins (whether on 2/am DNA circles or chimaeric plasmids) is subject to copy number control. Random segregation would then generate some cells in which the entire 'allowed' complement of 2/am DNA origins is located on chimaeric plasmids. Preferential replication of the chimaeric plasmid would increase the rate of generation of such cells, while preferential replication of 2/am would decrease it. The difficulty with this speculation is that it predicts that the chimaeric plasmids will be mitotically stable in [2/am ° ] strains, whereas in fact some instability is observed. This difficulty would be resolved if 2/am DNA circles themselves are slightly unstable, as has been suggested by Guerineau et al. (1974). However, the near ubiquity of 2/am DNA in laboratory strains argues against this, unless [2/am ° ] strains are at a selective disadvantage when compared with their [2/am + ] counterparts. There are several genetic determinants, including [psi] (Cox 1965), [ URE3] (Lacroute 1971, [20sRNA ] (Garvik and Haber 1978), and [NEX], [HOK] and [EXL] (Wickner 1980), which are inherited in a 4 : 0 , non-Mendelian pattern as are 2/am DNA circle (Livingston 1977). [2/am °] strains generated after transformation of strains carrying determinants such as those mentioned above can be used to test whether there is a strict correlation between the loss of 2/am DNA and the loss of any such non-Mendelian phenotype. [2 /am °] strains have at least one advantage over isogenic [2/~m + ] strains as chimaeric plasmid hosts in that 2/am DNA based chimaeric plasmids recombine with endogenous 2/am DNA after transformation (Storms et al. 1979), generating new plasmids which can complicate genetic analysis considerably. No new plasmids can be generated in this way in [2/am ° ] strains.
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Acknowledgements.We would like to thank Mr. Stephen Kearsey for introducing us to nick-translation, Drs. I. W. Craig and A. J. Kingsman for gifts of radioactive nucleotide, Dr. J. D. Beggs for her plasmids, and Drs. J. Friesen and R. Storms for encouragement and helpful discussions.
References Beggs JD (1978)Nature 275:104-109 Blanc H, Gerbaud C, Slonimski PP, Guerineau M (1979) Mol Gen Genet 176:335-342 Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121-133 Clark-Walker GD (1972) Proc Natl Acad Sci USA 69:388-392 Clark-Walker GD, MiNos GLG (1974) Eur J Biochem 41:359365 Cox BS (1965) Heredity 20:505-521 Dobson M, Futcher AB, Cox BS (1980) Curt Gen 2:193-200 Garvik B, Haber JE (1978) J Bacteriol 134:261-269 Gerbaud C, Guerineau M (1980) Curr Gen 1 : 219-228 Griffiths DE, Lancashire WE, Zanders ED (1975) FEBS Lett 53: 126-130 Gubbins E J, Newlon CS, Kann MD, Donelson JE (1977) Gene 1: 185-207 Guerineau M, Grandchamp C, Slonimski PP (1976) Proc Natl Acad Sci USA 73:3030-3034 Guerineau M, Slonimski PP, Avner PR (1974)Biochem Biophys Res Commun 61:462-469 Lacroute F (1971) J Bacteriol 106:5.19-522 Livingston DM (1977) Genetics 86: 73-84 Saunders GW, Rank GH, Kustermann-Kuhn B, HoUenberg CP (1979) Mol Gen Genet 175:45-52 Sinclair JH, Stevens RJ, Sanghavi P, Rabinowitz M (1967) Science 156:1234-1237 Storms RK, McNeil JB, Khandekar PS, Parker J, An GJ, Friesen JD (1979) J Bacteriol 140:73-82 Strum K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035-1039 Tabak HF (1977) FEBS Letters 84:67-70 Toh-e A, Guerry-Kopecko P, Wickner RB (1980) J Bacteriol 141:413-416 Wickner RB (1980) Cell 21:217-226
Communicated by F. Kaudewitz Received June 30, 1980
