pDblet, a stable autonomously replicating shuttle vector for Schizosaccharomyces pombe

Gene, 164 (1995) 173-177 ©1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

173

GENE 09213

pDblet, a stable autonomously replicating shuttle vector for

Schizosaccharomyces pombe (ARS; replication origin; plasmid; cloning; fission yeast)

Christine Brun*, Dharani D. Dubey* and Joel A. Huberman Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY14263, USA Received by A.D. Riggs: 6 March 1995; Revised/Accepted: 23 May/25 May 1995; Received at publishers: 17 July 1995

SUMMARY

We have constructed a new multipurpose stable shuttle vector for the fission yeast Schizosaccharomyces pombe (Sp). Plasmid pDblet was designed to provide convenient features for molecular work and to overcome the inconveniences of previously designed Sp vectors. It contains the Sp ura4 gene as selectable marker and a new highly efficient ARS (autonomously replicating sequence) element, allowing the vector to remain stable as a monomer in Sp. In addition, pDblet transforms Sp with high efficiency and has high mitotic stability and low copy number.

INTRODUCTION

Autonomously replicating sequences (ARS) from the fission yeast Schizosaccharomyces pombe (Sp) are poorly understood. This led to the initial use of cloning vectors that had been designed for the budding yeast Saccharomyces cerevisiae (Sc) for cloning studies in Sp. These vectors (reviewed in Russel, 1989) contain an Sc selectable marker able to complement an Sp mutation and the 2gm plasmid ARS-containing fragment, which functions as a ARS element in Sp (Beach and Nurse, 1981; Gaillardin et al., 1983). Other vectors especially Correspondence to: Dr. J.A. Huberman, Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. Tel. (1-716) 845-3047; Fax (1-716) 845-8126; e-mail: [email protected] * Present addresses: (C.B.) Ecole Normale Superieure de Lyon, CNRSUMR 49, 69364 Lyon Cedex 07, France. Tel. (33-72) 728-453; (D.D.D.) Department of Zoology, Kutir Postgraduate College, Chakkey, Jaunpur, U.P. 222146, India. Tel. (91-5452) 8725. Abbreviations: Ap, ampicillin; ARS (ars), autonomously replicating sequence; bp, base pair(s); E., Escherichia; kb, kilobase or 1000 bp; MCS, multiple cloning site; nt, nucleotide(s); ori, origin of DNA replication; R, resistant/resistance; Sc, Saccharomyces cerevisiae; Sp, Schizosaceharomyces pombe; ura4, gene encoding orotidine-5'phosphate decarboxylase.

SSDI 0378-1119(95)00497-1

constructed for Sp contain one of the first characterized Sp ARS elements, arsl (Losson and Lacroute, 1983; Maundrell et al., 1988). Although both types of vector are widely used due to their availability, they present inconveniences due to the weakness of their ARS elements; they are poorly transmitted through mitosis and meiosis, they multimerize (Sakaguchi and Yamamoto, 1982) and often rearrange. The propensity of Sp vectors to multimerize usually leads to low efficiency of plasmid recovery in E. coli (Sakaguchi and Yamamoto, 1982; Barbet et al., 1992). In order to minimize this negative consequence of vector multimerization, vectors containing additional rare cutter enzyme sites were engineered to permit plasmid monomerization prior to E. coli transformation (Barbet et al., 1992). Interestingly, the major flaws of Sp vectors containing arsl, low mitotic stability and multimerization, are corrected when a 1.3-kb Sp fragment called stb (for 'stability'; Heyer et al., 1986) is introduced into the vector along with arsl. However, pFL20, the only plasmid available with this combination of fragments (Losson and Lacroute, 1983), contains only a few cloning sites. Taking advantage of the first characterization of an Sp chromosomal replication origin region and its associated ARS elements in our laboratory (Zhu et al., 1992; 1994;

174 Dubey et al., 1994), we have constructed a new multipurpose shuttle vector, pDblet. This vector contains all the features of pBluescript II (Stratagene), transforms Sp with high efficiency, has high mitotic stability and low copy number and remains stable as a monomer in Sp. It can also be employed as a shuttle vector in Sc.

Aatll 6169 BamHl

615911

Ndel 163

BamHI5571

IRS doublet BamHI4983

ura4

Aatl14971

pDblet 6238 bp

EXPERIMENTALAND DISCUSSION

Ap R

(a) Cloning of the ARS doublet During our analysis of the ura4 origin region on chromosome III of Sp (Zhu et al., 1992; 1994; Dubey et al., 1994), a 581-bp subfragment of ars3002 (nt 3289-3870; Zhu et al., 1994) was cloned by PCR using two primers containing built-in BamHI sites. This fragment was then ligated into the BamHI site of pBluescript II S K + (Stratagene). Fortuitously, the plasmid recovered after bacterial transformation contained a duplication of the insert. This plasmid transformed Sp with high efficiency (7 × 104 transformants/~tg of DNA), even higher than that of a plasmid containing the complete ura4 origin region (3.4× 104 transformants/pg of DNA), which includes three independent A R S elements (Dubey et al., 1994). Furthermore, this plasmid was maintained in monomeric form throughout many generations, whereas a plasmid containing the single insert was recovered as a multimer (data not shown). These two particular features of the cloned doublet, high transformation efficiency and stability in monomeric form, suggested to us that the A R S doublet fragment might represent a good A R S element for the construction of a new Sp autonomously replicating vector.

(b) Construction of pDblet The vector pDblet (Fig. 1) is based on pBluescribe (Stratagene) and contains the 1.8-kb Sp ura4-containing restriction fragment (Grimm et al., 1988) inserted as an NdeI cassette at the NdeI site, and the A R S doublet inserted as an AatII cassette at the AatII site. Note that this cloning strategy was designed in order to permit easy recovery of the A R S doublet for further vector construction. In addition, the pBluescribe MCS (multiple cloning site) was replaced by the corresponding pBluescript II SK + (Stratagene) MCS because of its greater utility. All the sites of this polylinker are available for cloning in pDblet except for BamHI, EcoOl09I, EcoRV and AccI, which are present in the A R S doublet sequence, the pBluescribe sequence and the ura4 gene, respectively. As designed, this vector enables (i) blue/white color screen of bacterial recombinants, (ii) production of singlestranded DNA in vivo using the fl phage replication origin, (iii) in vitro synthesis of RNA using the T3/T7

fl(+)

1953

on •

LacZPr

MCS T3 promoter ---D~

Sac1. Sact I. BetX I, Eagl. Nott. Xbal. SpeL.Xmal. Sinai. P$ tl. EcoR t, Hind III. Clal. Sa II. Hinc ll. Xhol.A pal, Kp nl4F - T7 promoter

Fig. 1. Construction of pDblet. The original MCS of pBluescribe+ (Stratagene) was removed after PvulI digestion and replaced by the 448-bp PvulI MCS-containing fragment of pBluescript II SK+ (Stratagene). This intermediateplasmid was called pBSS.The Sp 1.8 kb HindlII fragment containing the ura4 gene was blunted by end filling, ligated to NdeI linkers and cloned into the NdeI site of pBSS in order to obtain pBSSura. The ARS doublet was releasedfrom pBluescript,in which it was initially cloned,by SpeI-SmaI digestion.The SpeI site was blunted by end filling, AatlI linkers were ligated to the blunted ends, and the fragmentwas ligated into the AatlI site of pBSSura. The resulting plasmid was called pDblet. The unique restriction sites located in the polylinker are shown below the diagram of pDblet. ApR, the [~-lactamase-encodinggene conferringAp resistance;ARS doublet, the duplicated region from ars3002 (see section a); ura4, the Sp orotidine5'-phosphate decarboxylasegene;fl (+), the fl phage origin of replication; LacZ, a portion of the lacZ gene; LacZPr, the lac gene promoter; ColE1 ori, the ColE1 origin of replication.

phage promoters, (iv) unidirectional ExoIII deletions (Henikoff, 1984) and (v) direct rescue into E. coll.

(c) Transformation efficiency, mitotic stability and copy number of pDblet, pFL20 and pSP2 in Sp The transformation efficiency obtained with pDblet was compared with the efficiencies observed with two available Sp vectors, pFL20 (Losson and Lacroute, 1983) and pSP2 (Cottarel et al., 1993). pFL20 and pSP2 both contain the Sc URA3 gene as selectable marker and Sp arsl as replication origin, pFL20 also bears the stb fragment conferring stability upon transformation (Heyer et al., 1986). The data obtained are summarized in Table I. pDblet reproducibly transforms Sp with an average efficiency twice as high as the other plasmids.

175 TABLE I Comparative transformation efficiency, mitotic stability and copy number for pDblet, pFL20 and pSP2 Plasmids

Number of transformants/~tg of DNA a

Mitotic stabilityb

Copy number c

(%)

pDblet pFL20 pSP2

Experiment 1

Experiment 2

Experiment 3

4.8 × 104 2.6 x 104 1.1 × 104

7.2 × 104 N.D. 4.4 × 104

1.3 × 106 0.5 × 106 N.D.

73 62 30

6 (8.3) 5 (8.1) 0.5 (1.7)

The Sp strain ura4-D18 (Grimm et al., 1988) was used for all experiments. a Experiments 1 and 2 were performed according to Gietz et al. (1992). Experiment 3 was performed using the frozen-yeast transformation kit (Zymo Research). b Percentage of plasmid-containing ceils after 13 generations of growth in selective medium. Average of 3 independent transformants. c Average copy number per cell. The values in parentheses represent the calculated copy number per plasmid-containing cell.

pDblet is also able to transform the budding yeast Sc at high frequency (data not shown). Mitotic stability was determined for three independent transformants. After a period of growth in selective medium, the same numbers of cells were plated in the presence or absence of selection to determine the percentages of plasmid-bearing cells. These percentages are summarized in Table I; pDblet is more stably transmitted through mitosis than pFL20 or pSP2. However, pFL20 has an average mitotic stability of 62% in our hands, but Heyer et al. (1986) reported a value of 95 +__5% for the same plasmid. This difference may be due to the use of different Sp strains. In order to determine the average number of copies of each vector per cell, a Southern blot bearing restricted total DNA from transformants containing each of the vectors (Fig. 2) was sequentially hybridized with (i) an ARS doublet probe and (ii) a probe revealing all the tested plasmids (the 0.69-kb DraI fragment contained in the Ap a gene). The ARS doublet probe reveals both pDblet (in the DNA from cells transformed by pDblet) and the appropriate chromosome III restriction fragment containing the 581-bp stretch of ars3002 in the DNAs from all the transformed cells. After quantifying the signals with a Phosphorlmager (Molecular Dynamics), the copy numbers were estimated for each plasmid, pDblet has a copy number of 8.3 per plasmid-containing cell (Table I). The copy number obtained for pFL20 (8.1 per plasmid-containing cell) is different from the 74-80 copies per plasmid-containing cell reported by Heyer et al. (1986). As for the difference in mitotic stability reported in the previous paragraph, this difference may be due to the difference in strains used.

(d) Maintenance status of pDblet, pFL20 and pSP2 When Sp cells are transformed with weak ARS-containing plasmids, the plasmids frequently multimerize. We therefore monitored the structures of the tested vectors

Cells transformedwith: pnblet pFL20 pSP2 pDblet pFL20 pSP2 DNA digestedwith: EcoRI BamHI EcoRI EcoRI BamHI EcoRI

7.9 kb

O

6.2 kb

4.0 kb

5.6 kb

i

Dblet probe

ApR probe

Fig. 2. Determination of vector copy numbers. DNA was isolated (Hoffman and Winston, 1987) from ura4-D18 cells (Grimm et al., 1988) transformed with each of the indicated vectors. The DNAs from pDbletand pSP2-containing cells were digested with EcoRI, and the DNA from pFL20-containing cells was digested with BamHI. Digestion with EcoRI was incomplete in the case of DNA from pSP2-containing cells. After electrophoresis through a 0.8% agarose gel, the DNA was transferred to a Hybond N membrane (Amersham) and hybridized sequentially with the following 32p-labeled probes: (i) the ARS doublet, which detects pDblet and the genomic restriction fragment containing ars3002 and (ii) the 0.69-kb DraI fragment from the Ap R gene, which detects vector sequences. Probes were labelled by random priming using a Multiprime kit (Amersham). The membrane was then exposed to a Phosphorlmager screen (Molecular Dynamics). The signals from individual bands were quantitated using ImageQuant software (Molecular Dynamics). The copy number for pDblet was calculated from the ratio of the total signal in plasmid bands (linear at 6.2 kb, supercoiled monomer, barely visible at this exposure, just below the 4.0-kb genomic fragment, and nicked circle at about 9 kb) divided by 2 (because there are 2 copies of ARS information in each pDblet molecule) to the signal in the genomic 4.0-kb restriction fragment. The Ap R probe signals for the other two vectors were then multiplied by the ratio of one half the doublet probe signal to the Ap rt signal for pDblet, to obtain the signals these vectors would have provided if they had each contained a single copy of ars3002. Finally, these values were divided by the signal from the corresponding ars3OO2-containing genomic fragment (detected with the ARS doublet probe) to yield copy numbers for pFL20 and pSP2. The calculated copy number values are listed in Table I.

for > 30 generations, in three independent transformants per vector. For this purpose, total DNA was prepared from each transformant after 12 to 19 generations, 23 to 26 generations and 30 to 35 generations, respectively. The

176

U D U D

U D U D

UD

U D

U D U D

U D

U D U D U D

SG, d

t~,rn 62kb

SC, m Fig. 3. Multimerization status of pDblet, pFL20 and pSP2. DNA was prepared (Hoffman and Winston, 1987) from Sp cells 12 to 19 generations, 23 to 26 generations and 30 to 35 generations after transformation with each of the indicated vectors. DNA was then digested with a restriction enzyme linearizing the plasmid: HindlII for pDblet (6.2 kb), BamHI for pFL20 (7.9 kb) and EcoRI for pSP2 (5.6 kb). For each DNA sample, 200 ng of undigested DNA and 200 ng of digested DNA were loaded on a 0.8% agarose gel. In the case of the pSP2-containing cells, only a small amount of DNA was recovered after 19 generations of growth because of the high mortality of the ceils before the multimerization of the plasmid. Consequently, only 25 ng of DNA were loaded in the 19 generations lanes. The two first lanes of each gel, indicated by the plasmid name, contained undigested and digested plasmid recovered from E. coli. In the case of pFL20, there is a difference in migration due to difference in extent of supercoiling between the undigested plasmid from E. coil and the undigested plasmid from yeast, All the gels were processed for transfer, hybridization, and exposure as described in the legend to Fig. 2. The 0.69-kb DraI fragment located in the Apg gene was used as probe. U, undigested DNA; D, digested DNA; SC, supercoiled form; NC, nicked circular form; d, plasmid dimer; m, plasmid monomer; multi, plasmid multimer; (~r), fragment generated by the EcoRI star activity. Additional bands in the pSP2 panel are presumably due to rearrangements. The relative positions of nicked circular and supercoiled dimer forms were inferred based on the following considerations. First, we assumed that nicked circles were less likely than supercoiled dimers to appear in the plasmid preparations from E. coli, since the alkaline lysis procedure employed would have selected against nicked circles. Second, we assumed that, when linear molecules were detected in the undigested DNA, nicked circles must also be present. Because these assumptions may be incorrect, it is possible that the indicated positions of supercoiled dimers and nicked circular monomers should be reversed. results obtained for one of the three tested transformants per plasmid are presented in Fig. 3. The results for the other transformants were similar, pDblet remained mainly m o n o m e r i c for at least 34 generations. However, a small p r o p o r t i o n of supercoiled dimers was also detected at all time points, p F L 2 0 did not reveal any multimer formation up to at least 35 generations. After 19 generations, a significant p r o p o r t i o n of p S P 2 was still monomeric. Some rearranged forms were also present. Later, the m a j o r species of pSP2 was the supercoiled dimer. Larger multimers and rearranged forms were also detectable. N o t e that cells transformed with pSP2 grew very poorly for the first 19 generations. Their growth rate increased significantly during the following 10 generations. There appeared to be a correlation between the g r o w t h rate of the transformants and the extent of multimerization of the plasmid.

mitosis and (iii) is stably maintained as a m o n o m e r , Although p F L 2 0 is also maintained as a m o n o m e r , it does not provide convenient features for molecular work. Also, pDblet can efficiently transform and replicate in Sc. An additional advantage of pDblet is that the 1.2-kb A R S doublet sequence, which is mainly responsible for the characteristics of this vector, can easily be recovered. This sequence is smaller than the arsl and the stb fragment (1.1 and 1.3 kb, respectively), which are the only k n o w n sequences which, when combined, enable a Sp vector to be stably maintained and transmitted. The A R S doublet sequence is, thus, a g o o d candidate for further plasmid construction. We expect that pDblet, available u p o n request along with its reconstituted nucleotide sequence, will be a useful tool for further Sp studies.

(e) Conclusions

ACKNOWLEDGEMENTS

We have described a new multipurpose shuttle vector for the fission yeast Sp. pDblet was designed to overcome the deficiencies of previously designed Sp vectors. O u r comparative study showed that pDblet (i) transforms Sp with higher efficiency than the other tested vectors, p F L 2 0 and pSP2, (ii) is transmitted more stably t h r o u g h

We are grateful to Shelley Sazer for having inspired this w o r k by pointing out to us the need for new Sp vectors. T h a n k s are due to GuiUaume Cottarel and Mich+le Minet for the gift of pSP2 and pFL20, respectively. We also t h a n k Peter Fantes, Philippe Fournier,

177 Paul Nurse and Mitsuhiro Yanagida for their kind responses to our initial survey. We are grateful to Deborah Carlson for testing the ability of pDblet to transform Sc and to Deborah Carlson, Deborah Mahoney, Karuna Sharma and Martin Weinberger for their constructive comments on the manuscript. This work was supported by a NIH grant (RO1-GM49294) to J.A.H. and by a Human Frontier Science Program Organization long-term fellowship to C.B.

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