Mol Gen Genet (1988) 211:155-159 © Springer-Verlag 1988

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae G.R. Zealey 1'2'*, A.R. Goodey 2, J.R. Piggott 2, M.E. Watson 2, R.C. Cafferkey 2, S.M. Doel 2, B.L.A. Carter 2, and A.E. Wheals 1 1 School of Biological Sciences, University of Bath, Bath BA2 7AY, UK 2 Department of Molecular Genetics, GD Searle and Company Ltd., Lane End Road, High Wycombe, Bucks HP12 4HL, UK Summary. A 2 gm circle-based chimaeric plasmid containing the yeast L E U 2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the L E U 2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 T K gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the T K gene, and the increase in T K gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined. "High-copy" number cells showed a greater mitotic stability. The relationship of T K expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression. Key words: Saecharomyces cerevisiae - Yeast - Copy number - Thymidine kinase

Introduction Yeast chimaeric plasmids based upon the 2 ~tm circle are mitotically unstable during non-selective growth and one of the major factors controlling stability is the selective marker present on the plasmid (Fu!Lcher and Cox 1984). A common means of selecting for yeast transformants is complementation of a leucine auxotroph with a plasmidborne L E U 2 gene. Two yeast L E U 2 genes have been independently isolated (Beggs 1978; Broach et al. 1979). One allele is only expressed at 5% of the wild-type L E U 2 gene and has been designated leu2-d (Erhart and Hollenberg 1983). This poor expression has been attributed to a deletion of sequences 5' to the gene. Plasmids containing the leu2-d gene are maintained at a high copy number presumably to compensate for the poor expression. It has been demonstrated that plasmids containing the leu2-d allele are substantially more stable than plasmids containing the wildtype L E U 2 allele (Futcher and Cox 1'984) and one explana* Present address: Department of Molecular Genetics, Connaught Research Institute, 1755 Steeles Ave West, Willowdale, Ontario M2R 3T4, Canada Offprint requests to: G.R. Zealey

tion for this increased stability is the initial high copy number of the plasmid. By controlling plasmid copy number it should be possible to show a relationship between plasmid copy number and mitotic stability. Saccharomyces cerevisiae does not possess a thymidine kinase (TK) gene (Grivell and Jackson 1968) and yeast D N A synthesis is inhibited in the presence of the folate antagonists amethopterin and sulphanilamide (Fig. 1). Expression of a T K gene in yeast provides an alternative supply of deoxythymidine monophosphate (dTMP) and the T K gene from Herpes Simplex Virus type 1 (HSV-1 TK) was first expressed in yeast by McNeil and Friesen (1981). In the present work a plasmid borne HSV-1 T K gene was used to select for an increase in T K expression. The increase in thymidine kinase activity in cell-free extracts was concomitant with both an increase in plasmid copy number and mitotic stability.

Materials and methods Strains. Escherichia coli D H I F - endA1 hsdRI7 ( r k - m k +) recA1 supE44 thi-1 gyr96 relA1 (Hanahan 1983) was the host for bacterial transformations. Saccharomyces cerevisiae C B l l 63 [Cir °] a adel leu2-63 was obtained from B.S.

Cox. Media. Bacteria were grown in L-broth (1% tryptone, 0.5%

yeast extract, 0.5% NaC1, 0.1% glucose) and yeast in either YEPD (1% yeast extract, 2% peptone, 2% glucose) or Y E G E (1% yeast extract, 3% glycerol, 2% ethanol). DAST media was YEPD with 100 t*g ml-1 amethopterin and 400 gg m l - 1 thymidine. GST media was Y E G E with thymidine (400 gg ml-1). Sulphanilamide was added to GST and DAST media at the concentration shown in brackets (mg ml-1). The defined medium for yeast was SD (0.67% yeast nitrogen base, 2% glucose) and auxotrophic growth requirements were added as described (Sherman et al. 1979). Transformations, D N A isolation and cloning procedures. Restriction enzymes and D N A modifying enzymes were purchased from BRL or Biolabs and used in accordance with the manufacturers recommendations. E. eoli DH1 was transformed by the method of Hanahan (1983) and S. cerevisiae as described by Beggs (1978). 2 I*m circle was extracted from S. eerevisiae by the method of Guerineau et al. (1974).

156 Thymidine IS ,, dUMP

TK (thymidine kinase)

(thymidylate synthetase)

N-5, N-10 methylene IHFA '

• dTMP

DH FA

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,

DNA

AMETHOPTERIN

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DR (dihydrofolate reductase)

THFA

SULPHANILAMIDE O O

Thymidine kinase assay. Thymidine kinase activity was measured in cell-free extracts of S. cerevisiae transformants by the method of Cheng and Ostrander (1976) as described by Goodey et al. (1986). Total protein was determined with a Biorad protein estimation kit. Estimation of plasmid copy number in yeast transformants. Plasmid copy numbers in yeast transformants were estimated by extracting total cellular D N A (Goodey et al. 1986) and comparing the staining intensity of ribosomal R N A gene-specific and plasmid-specific restriction fragments as described (Futcher and Cox 1984). Agarose gels were photographed and the photographs scanned with a Joyce-Loebl Chromoscan-3. Determination of mitotic stability of yeast transformants. Transformants were grown in selective media to a density of about 5 x 1 0 7 cells m l - 1. One hundred microlitre of this culture were then transferred to 100 ml of YEPD (nonselective media) and grown to a cell density of about 5 x 107 cells ml-1. One hundred microlitre of the culture were transferred to a further 100 ml of YEPD and a sample plated onto YEPD to determine cell density. Two hundred colonies were transferred to SD-Leu medium to determine the percentage of Leu + (Plasmid +) cells. Results

Construction of a chimaeric plasmid for expression of the HSV-1 TK Gene in S. cerevisiae

A chimaeric plasmid (pAYE56) for expression of the HSV-1 TK gene in S. cerevisiae was constructed by the protocol shown in Fig. 2. The HSV-I TK gene was isolated from the plasmid p T K I (Wilkie et al. 1980) and fused to the yeast promoter-like BglII fragment close to the yeast HIS3 gene (McNeil and Friesen 1981). The plasmid pAYE56 transforms S. cerevisiae CB11 63 [Cir °] at a very high frequency (about 105 transformants per microgram of plasmid

Fig. 1. Inhibition of DNA synthesis in Saccharomyces cerevisiae by the folate antagonists amethopterin and sulphanilamide. dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid

H2" ZY-COOH] ~amino benzoic acid ]

DNA) despite its large size. No rearrangements of pAYE56 were seen during propagation of the plasmid in E. coli or S. cerevisiae. Inter- and intra-molecular rearrangements of 2 gm circle-based plasmids are greatly increased by the activity of the 2 gm circle FLP gene product and inactivation of this gene leads to a 99% reduction in recombination events (Broach 1981). Insertional inactivation of the FLP gene at the NdeI site may therefore account, in part, for the structural stability of plasmid pAYE56. The HSV-1 TK gene was functionally expressed in S. cerevisiae CBll 63 [Cir°]; pAYE56 transformants. Transformants could be selected directly on DA(5)ST regeneration media, Leu ÷ transformants grew on DA(5)ST media and thymidine kinase activity could be detected in cell-free extracts of transformants. Transformants grown under selection for the HSV-I TK gene showed an altered cell morphology. The cells were extensively elongated and distorted and each cell had several buds. This aberrant cell morphology is very similar to temperature-sensitive (ts) D N A synthesis mutants shifted to the restrictive temperature (Pringle and Hartwell 1981) and presumably reflects cell growth with delayed budding because D N A synthesis is limited by the supply of dTMP. Continued growth of S. cerevisiae CBI 1 63 [Cir°]; pAYE56 transformants on DA(5)ST media, where mitochondria are dispensable, leads to the generation of petite colonies. The cell morphology and petite formation indicate that whilst HSV-1 TK expression can rescue yeast cells when other routes to dTMP synthesis are blocked, the extent of complementation is poor and cell growth is still severely limited by the supply of dTMP. Plasmid copy number of transformants grown under selective conditions for the yeast LEU2 and the HSV-1 TK genes S. cerevisiae CBll 63 [Cir°]; pAYE56 transformants were grown non-selectively (YEPD media) and selectively for the yeast LEU2 (SD-leu media) and HSV-1 TK genes (G[5]ST media) and the plasmid copy numbers and the percentage

157 1o0

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100

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50

80

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0-2

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1

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of s u i p h a n i l ~ m l d e

Fig. 3. The relationship of sulphanilamide concentration to plasmid copy number, percentage Leu ÷ cells and intracellular thymidine kinase activity in Saccharomyces cerevisiae CBll 63 [Cir°]; pAYE56 transformants. • Percentage Leu ÷ cells; o plasmid copy number; • thymidine kinase activity

ligase B

Cm r

U

4= -J

N

Fig. 2. Construction of a chimaeric plasmid (pAYE56) for expression of the HSV-1 TK gene in Saccharomyces cerevisiae. P, PstI; RI, EcoRI; B, BamHI; N, NdeI; Bg, BglIT~; Cm, chloramphenicol resistance

o--e

Table 1. Plasmid copy number in Saccharomyces cerevisiae CBII 63 [Cir°]; pAYE56 transformants grown under different selective conditions Genes selected for on plasmid

% Leu ÷ cells in culture

Plasmid copy number

None LEU2 HSV1-TK

54 78 100

15 15 100

of Leu + cells determined (Table 1). Selection for the HSV-1 T K gene leads to a substantial increase in copy n u m b e r and percentage of Leu + (Plasmid +) cells. To determine the concentration of sulphanilamide required to produce this increase in plasmid copy number, S, cerevisiae C B l l 63 [Cir°]; pAYE56 transformants were grown in G S T media containing various concentrations of sulphanilamide, and plasmid copy numbers, percentage Leu + cells and intracellular thymidine kinase activities determined (Fig. 3). Although 5 mg ml 1 of sulphanilamide is required to inhibit

1

'0

1;

210 3i0 4[0 No. of cell generations

510

610

Fig. 4. The effect of plasmid copy number upon mitotic instability of Saccharomyces cerevisiae CBll 63 [Cir°]; pAYE56 transformants during non-selective growth. Transformants were pre-grown in [a] SD-leu, [v] G(2)ST, or [n] G(5)ST media and maintained in logarithmic growth in non-selective (YEPD) media as described. The initial plasmid copy numbers prior to transfer were 15 (m), 20 (v) and 100 (D) the growth of yeast cells, a concentration as low as 0.5 mg ml 1 leads to a substantial increase in plasmid copy n u m b e r in yeast transformants despite little effect upon growth rate and cell morphology. The percentage of Leu ÷

158 cells also increased dramatically during selection for the HSV-1 T K gene. The effect o f plasmid copy number upon the mitotic stability o f chimaeric plasmids in S. cerevisiae

Yeast chimaeric plasmids based upon the 2 gm circle are mitotically unstable during non-selective growth (Futcher and Cox 1984). The ability to maintain the same plasmid at different copy numbers in the same yeast strain by selection for HSV-I T K gene with various concentrations of sulphanilamide allowed the effect of plasmid copy number upon mitotic stability to be tested. Transformants were therefore grown in SD-Leu media and in GST media with two different concentrations of sulphanilamide to generate cells with different copy numbers, and the mitotic instabilities were determined (Fig. 4). Discussion

The HSV-1 T K gene has been expressed in S. cerevisiae by a variety of workers (McNeil and Friesen 1981; Zhu et al. 1984; G o o d e y et al. 1986). In the present work a study was made of the effect of T K expression upon plasmid copy number. Expression of the T K gene leads to an amplification of plasmid copy number and this is reflected in a proportional increase in intracellular thymidine kinase activity. Expression o f the T K gene in yeast therefore shows similarities to the D H F R selection system in mammalian and yeast systems (Kaufman and Sharp 1982; Scahill et al. 1983; Zhu et al. 1985; Miyajima et al. 1984). The ability to control plasmid copy number by T K expression allowed a study to be made of the effect of copy number upon mitotic instability of chimaeric plasmids, and it was shown that an initially high copy number leads to an increased mitotic stability during non-selective growth. The mode of control of plasmid replication and partition is still unclear. The simplest model is that (i) a cell has n copies of a plasmid, (ii) each copy replicates once per cell cycle and (iii) these copies are partitioned at random at cell division. Zakian et al. (1979) produced evidence in favour of point (ii) but Sigurdson et al. (1981) showed that over-replication could occur under circumstances where a cell had a low plasmid copy number. Futcher (1986) has proposed that a recombination event at the F R T site, both within and controlled by the F L P gene, is necessary for this over-replication and evidence in support of this view has been obtained (Volkert and Broach 1986). In an effort to avoid recombination events that could complicate the analysis of the plasmids used in this study, we chose to insert heterologous genes within the F L P gene, thus inactivating it. Since over-replication can not occur, we must presume that the high plasmid copy numbers obtained in this work were the result of unequal partition. Indeed Futcher and Cox (1984) showed that point (iii) of the model was incorrect since the frequency of loss of chimaeric plasmids (as opposed to 2 gm circle itself) was orders of magnitude greater than expected on a random partition model. It is therefore likely that plasmid partition is highly asymmetric (but not necessarily directional as was discovered for A R S plasmids during a pedigree analysis [Murray and Szostak 1983]) and that cells with high and zero plasmid copy numbers are generated naturally at high rates. The selective system is thus not forcing up copy number but

merely enabling high copy number segregants to proliferate faster. On this hypothesis, stability is increased not by reducing partition asymmetry but by increasing plasmid copy number and hence reducing the rate at which Plasmid ° cells are produced. It seems likely that the effect upon plasmid stability produced by T K selection is due to changes in copy number since cells with approximately the same number of plasmids by T K or L E U 2 selection have similar instabilities. S. cerevisiae has become a host for the expression of heterologous genes (Kingsman et al. 1985). The ability to increase gene dosage by T K selection may be useful for regulating or increasing expression of foreign genes in yeast. Gene dosage has been shown to be a limiting factor for interleukin-2 production in yeast where plasmid copy number was controlled by D H F R expression (Zhu et al. 1985). The control of plasmid copy number control in bacteria has been extensively studied and many mutants have been isolated (Scott 1984; Nordstrom et al. 1984). The dose-dependent expression of the T K gene may make this a suitable marker for the isolation of plasmid copy number mutants and the elucidation of any control mechanisms in yeast. Any such mutants may also be useful in heterologous gene expression. Acknowledgements. GRZ received a CASE award from the SERC. References

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159 thymidine kinase gene in Saeeharomyces cerevisiae. Mol Gen Genet 184:386-393 Miyajima A, Miyajima I, Arai KI, Arai IN (1984) Expression of plasmid R388-Encoded Type II Dihydrofolate Reductase as a Dominant Selective Marker in Sacchclromyces cerevisiae. Mol Cell Biol 4:407-414 Murray AW, Szostak JW (1983) Pedigree analysis of plasmid segregation in yeast. Cell 34:961 970 Nordstrom K, Molin S, Light J (1984) Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the R1 system. Plasmid 12:71-90 Pringle JR, Hartwell LH (1981) The Saccharomyces cerevisiae cell cycle. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces, metabolism and gene expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 97-142 Scahill SJ, Devos R, Van der Heyden J, Fiers W (1983) Expression and characterization of a human eDNA gene in Chinese hamster ovary cells. Proc Natl Acad Sci USA 80:4654M658 Scott J (1984) Regulation of plasmid replication. Mierobiol Rev 48 : 1-23 Sherman F, Fink GR, Lawerence CW (~979) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

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