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Copy Number Modulation in an Autoselection System for Stable Plasmid Maintenance in Saccharomyces cerevisiae C. Compagno, A. Tura, B. M. Ranzi, L. Alberghina, and E. Martegani* Diparthento di Fisiologia e Biochimica Generali, sez. di Biochimica Comparata, via Celoria 26, UniversitA di Milano, 20133 Milano, Italy
Efficient expression of a foreign gene requires a stable vector present a t a high number of copies per cell. We have constructed an autoselection system for the stable maintenance of expression vector in the yeast Saccharomyces cerevisiae that uses the fructose 1,6bisphosphate aldolase gene (FBAl) t o stabilize plasmids in cells bearing a disruption of the chromosomal FBAl gene. This system allowed us to obtain stable production of a reporter heterologous enzyme (Escherichia coli &galactosidase) in rich media. By using an inducible promoter to regulate the expression of FBAl gene, we have also obtained the modulation of plasmid copy number by carbon source.
Introduction The expression of foreign genes in microorganisms and the ensuing possibility of obtaining high levels of valuable heterologous proteins is one of the major achievements of biotechnology. Two factors are of paramount importance for the efficient and stable expression of heterologousgenes in microorganisms: (1)the geneticstability of transformed strains and (2) the gene dosage, which could be increased by cloning into high copy number vectors. Plasmid stability and the number of plasmid copies in a cell strongly depend on the type of plasmid, the selection marker, and the growth conditions (Romanos et al., 1992). The high copy number expression vectors usually employed for heterologous gene expression in yeast are all derivatives of the native 2-pm plasmid; they are stable enough, but they can be maintained only under selective conditions (Futcher, 1988). This type of selection requires the growth of transformants in the minimal synthetic media suitable for the available auxotrophic selective marker, but which are usually unsuitable for industrial fermentations. The ideal plasmid vector for industrial purposes should be stable and maintained in high copy number in less expensive complex media that can, in addition, support fast growth rates and give higher biomass concentrations, thus increasing the overall efficiency of the process. The autoselection systems, in which the expression vector can be stably maintained independently of the growth medium composition,are of particular interest in biotechnology as this feature permits cell growth in the relatively inexpensive complex media preferred by industry for commercial-scale fermentations. Recently, a few procedures for the development of these systems have been described (Loison et al., 1986; Unternahrer et al., 1991; Rech et al., 1992;Napp and Da Silva, 1993). The first was developed by Loison et al. using the double mutant ura3,furl that cannot utilize external uracil and therefore requires a URA3 gene, present on a plasmid, for cell viability; this was recently improved by Napp and Da Silva. This system has been successfullyapplied, using a defective URA3 gene promoter to increase the copy number, to obtain a high level of heterologous gene expression in both batch and continuous cultures (Loison et al., 1989).
* Author to whom correspondence should be addressed. 87567938/93/3009-0594$04,00/0
In this article, we report the development of an autoselection system that uses the fructose 1,g-bisphosphate aldolase gene (FBAl) to stabilize plasmids in Saccharomyces cereuisiae cells bearing a disruption of the chromosomal FBAl gene. We show that this system allows one to obtain a stable production of a reporter heterologous enzyme (Escherichia coli 8-galactosidase) in complex rich media. It has been observed that the level of expression of the (auto)selectivemarker influences the plasmid copy number and stability (Erhart and Hollemberg, 1983; Piper and Curran, 1990; Loison et al., 1989). By using an inducible promoter to regulate the expression of FBAl gene, we have also developed a system which allows us to modulate the plasmid copy number.
Materials and Methods Strains and Growth Conditions. Saccharomyces cerevisiae haploid strain W303-1A (MAT a, ade 2-1, can 1-100,ura 3-1,leu2-3,112, trp 1-1,his 3-11,151 and W303 D homozygous diploid were obtained from M. A. Teste (CNRS, Gift-sur Yvette, France). Yeast cells were grown in YEP complete medium containing 2% peptone and 1%yeast extract or in defined medium SM containing 0.67% yeast nitrogen base without amino acids (Difco Laboratory, Detroit, MI) supplemented with appropriate additives (50 pg/mL). The carbon source was 2 % glucose (YEPD), 2% galactose (YEPGAL), or 2% glycerol plus 2% ethanol (YEPGE). E. coli JMlOl ((lacpro), thi,strA, supE, endA, sbcB, hsdR-, F'traD36, proAB, laclq, Z M15) was used for plasmid amplification. Gene Disruption. The disruption of the FBAl gene was described in Compagno et al. (1991). Disruption was tested by Southern blot and tetrad analysis. Plasmid Construction. Standard DNA manipulations were performed according to Sambrook et al. (1989). Plasmid DNA was isolated from E. coli cultures by the alkaline lysis method and, when required, further purified by centrifugation on cesium chloride-ethidium bromide density gradient. Plasmid p13A was constructed by cloning into the vector YEpl3 (Broach et al., 1979) the 5.6-kb BamHI fragment containing the FBAl gene (Compagno et al., 1991). Plasmid pYATl was constructed from YEplacll2 (Gietz and Sugino, 1988) digested with BamHI and SmaI and
0 1993 American Chemical Society and American Institute of Chemical Engineers
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ligated to the 3-kb BamHI-DraI fragment containing the FBAl gene. Plasmid pYATl was subsequently digested with XbaI and AatII, and the 5.6-kb fragment, which containsthe FBAl gene, the 2-pm sequences,and the TRPl gene, was ligated to the 8-kb XbaI-StuI fragment of pLGSD5 (Guarente et al., 19821, containing the lac2 gene under the control of the UASGALICYCIpromoter, obtaining the plasmid pCCS5. The 2.8-kb HindIII-Aut11 fragment of pE1(-462), containing the deleted FBAl promoter (Compagno et al., 1991),was ligated to theHindII1-Aut11 fragment of pYATl containing the FBAl gene, obtaining the plasmid pYAT462. For the construction of plasmids pIAl0 and pIA12, we first inserted the EcoRI-XhoI fragment (from pLGSD5) containing the URA3 gene and UASGAL in the EcoRI site of the plasmid YEplacll2, obtaining the PIA vector. Then we recovered the Sad-BamHI fragments containing the FBAl gene and 5' sequences up to 136bp (pIA12) and 200 bp (pIA10) from a series of plasmids obtained by subcloning in pGEM-blue vector DNA fragments from pEl(-200) and pE1(-136) containing the deleted FBAl promoter (Compagno et al., 1991). These fragments were inserted in SacI-BamHI-digested PIA vector, obtaining pIAlO and pIA12 plasmids. DNA Analysis. Total yeast DNA was prepared by the method of Nasmyth and Reed (1980). DNA transfer from agarose gels to Hybond-N membranes (Amersham) was performed according to the supplier's instructions. 32Plabeled DNA probes were obtained by nick-translation and hybridization according to Sambrook et al. (1989). Plasmid copy number was determined by comparing the density of the hybridization band of plasmid DNA with that of chromosomal DNA (FBAl gene) as the internal control (1copy per cell), using a scanning densitometer. ContinuousCultures. Yeast continuouscultures were run in a Bioflo C30 chemostat (New Brunswick, Edison, NJ). The medium reservoir was filled with 10 L of 1% (w/v) galactose, 1%yeast extract, and 2% peptone. In the chemostat, the volume of the culture was 0.5 L, the temperature was maintained a t 30 "C, and the agitation speed was 400 rpm. The culture medium was aerated with a flow of 1L/min. Enzyme Assays. Aldolase was extracted from cell pellets with glass beads in lysis buffer (50 mM K2HPO4, 10 mM 8-mercaptoethanol, and 2 mM PMSF). After centrifugation, the supernatant was used for aldolase and protein determinations. The aldolase assay was performed in 50 mM triethanolamine hydrochloride, 10 mM MgC12 (pH 7.4), 0.2 mM NADH, 4 mM fructose 1,6-diphosphate, 0.1 M potassium acetate, 10 units of glycerol-3-phosphate dehydrogenase, and 1 unit of triosephosphate isomerase (Boehringer Mannheim). Aldolase specific activity is expressed as units/milligram of yeast proteins (1unit of aldolase activity is defined as the quantity of enzyme catalyzing the cleavage of 1pmol of fructose bisphosphate per minute). 8-Galactosidasewas extracted from cell pellets with glass beads in Z buffer (0.1 M Na2P04,O.l M NaHP04, 10 mM KC1,l mM MgS04, and 50 mM ,&mercaptoethanol). After centrifugation, the supernatants were used for 8-galactosidase and protein assay as described previously (Lotti et al., 1988). One unit of enzyme activity equals the production of 1nmol of o-nitrophenolper minute. Protein assays were performed using a Bio-Rad kit. Plasmid Stability. To determine plasmid stability in batch or in continuous culture, cells were diluted, sonicated, and spread on YEPD, YEPGE, or YEPGAL-X-GAL (50 pm/mL) plates to obtain about 300 colonies/plate. Cells
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Table I. Structure of Different Plasmids Used in This Study parental yeast FABl gene heter plasmid vector marker promoter gene p13A YEP13 LEU2 FBAl DCCS6 YEPlacll2 TRPl FBAl lacP ~YATZ YEPlacll2 TRPl FBAl pYAT462 YEPlacll2 TRPl FBAlb YEPlacll2 TRPl UASGL~ pIalO PIA12 YEPlacll2 TRPl UASGL~ The lac2 gene is under the U A S G ~ ~ J C Y promoter. C~ FBAl is the abbreviation for the truncated FBAl promoter which lacks a portion of UAS. UASGAL is 75 bp upstream from the FBAl TATA box. UASGL is 12 bp upstream from FBAl TATA box.
from individual colonies were transferred to minimal selective plates. The fraction of plasmid-containing cells was calculated as the number of colonies grown in selective conditions divided by the total number of colonies transferred or the fraction of blue colonies in YEPGALX-GAL plates. Analysis of Cell Volume Distributions. Samples of culture were mildly sonicated and counted after appropriate dilution with Isoton (Coulter Electronics, England) with a Coulter Counter ZBI equipped with a 70-pm orifice. Cell volume distributions were obtained with a Coulter Channelyzer C-1000, previously calibrated with 8.79-pm latex beads. The volume distributions were acquired and analyzedwith a personal computer (IBM-AT)to calculate mean and variance.
Results Construction of the WA6[p13A] Strain. Since the glycolytic enzyme fructose 1,6-diphosphate aldolase is essential for both fermentative and gluconeogenicgrowth, we performed the FABl gene disruption in homozygous diploid Saccharomyces cerevisiae strain W303-D by insertion of the URA3 gene in the FBAl locus (Compagno et al., 1991). Several transformants were checked by Southern blotting and by tetrad analysis. One of them has been transformed by using the p13A plasmid, a derivative of the YEpl3 vector, containing a functional copy of the FBAl gene (Table I). Transformed diploid cells were induced to sporulate, and Leu+/Ura+ haploid segregants were isolated and characterized, obtaining the strain WA6[p13A] which carries a disrupted chromosomal copy of the FBAl gene and a functional copy of the same gene in p13A plasmid. All media, whatever the carbon source used, are selective for plasmid maintenance, so that it is very difficult to test the stability of p13A plasmid. The rise in revertant plasmid-free cellswas checked by growing the WA6[p13Al strain for about 50 generations in glucose (YEPD) or glycerol/ethanol (YEPGE) rich media and by replica plating on minimal glucose and glyceroVethano1 media lacking leucine and uracil. All colonies grew on minimal medium and were Leu+ and U r d , showing good stability of the plasmid and gene disruption. WA6[p13Al transformants overexpress fructose 1,6-diphosphate aldolase (2.25 units/mg) relative to wild-type haploid cells with a single copy of the FABl gene (0.5 units/mg), indicating that plasmid p13A was present at several copies. Indeed, a Southern blot analysis showed that the p13A vector was maintained at about five copies per cell in cells grown in both rich media and minimal media selective for the auxotrophic marker (leu2); in addition, this copy number was found to be stable after about 50 generations in YEPD (Figure 1).
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-cr
- PI
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Time(ht-) Figure 2. &Galactosidase specific activity (units/mg) and biomass concentration in continuous culture of the WAG[pCCS5] strain a t D = 0.2 h-l on rich galactose medium. Figure 1. Analysis of the copy number of the plasmid p13A. Total DNA (10 pg) was isolated from two different colonies of WA6[p13A] grown in minimal medium lacking leucine (lanes A and D), in minimal nonselective medium (lanes B and E), and in rich medium after 50 generations (lane G ) and digested with BamH1. The Southern blot was hybridizedwith a nick-translated FBAl probe. cr: Chromosomal disrupted copy of the FBAl gene. pl: Plasmid fragment bearing the same gene.
This result indicates that an FBAI-bearing YEP vector, in an fba- genetic background, remains fully stable a t several copies, independent of the growth conditions. Heterologous Gene Expression and Stability in Batch and in Continuous Culture in Complex Media. In order to develop a stable system for heterologous gene expression based on our autoselection procedure, we constructed the plasmid pCCS5, a YEp vector bearing the E. coli lac2 gene under the control of hybrid promoter UASGA~CYCI and the FBAl and TRPl genes as selective markers (Table I). We used plasmid pCCS5 to transform WA6[p13A] cells, and plasmid shuffling was induced (Mann et al., 1987),selecting Trp+/Leu-cells in which the p13A plasmid was substituted by the pCCS5 plasmid. Batch experiments were performed to study plasmid stability, cell growth, and heterologous gene expression. The specific growth rate of the WA6[pCCS5] strain on galactose selective medium was 0.7 h-l, and biomass concentration in the stationary phase was low, about 3 x lo7cells/mL. The kinetics of @-galactosidaseproduction showed that the highest levels of enzyme activity were reached in the late exponentialphase, accordingto previous reports from our laboratory using the same inducible promoter (Compagno et al., 1987; Lotti et al., 1988). The maximum level of 8-galactosidasespecificactivityobserved was about 2.2 units/pg. In richgalachemedium (YEPGAL),thespecificgrowth rate of the WA6[pCCS5] strain was higher (0.34 h-l), and in the stationary phase a 10-foldhigher biomass concentration (3 X lo8 cells/mL) was reached. The time course of &galactosidase production and the specific activity of the enzyme were the same as in selective medium; therefore, a 10-fold higher absolute productivity was obtained. Again, even after 50 generations in YEPGAL medium we never detected the presence of revertant plasmid-free cells. Southern blot analysis also showed that pCCS5 plasmid was maintained a t about five copies per cell, both in induced (YEPGAL) and uninduced (YEPD)conditions of growth (data not shown). Continuous cultures in galactose-rich medium were performed a t a dilution rate of 0.2 h-l, galactose being the
limiting nutrient. The biomass and &galactosidaselevels observed during the continuous fermentation of WA6[pCCS5] are shown in Figure 2. The chemostat was inoculated with 5 X lo7 cells/mL already induced by pregrowth in batch cultureon YEPGAL medium. Biomass increased until the steady-state level was reached, and @-galactosidasespecific activity showed a small decrease. Small fluctuations in biomass concentration and 8-galactosidase specific activity were also observed after the steady state was established. The residual galactose concentration during the cultivation was 0.12 g/L. Samples were taken after the steady state was reached, starting from 60 and continuing up to 230 h (about 80 generations), to determine the stability of recombinant cells by plating on YEPGAL-X-GAL plates. All of the colonies were blue, indicatingagain the absence of reversion until the end of the continuous growth in rich medium. A stable retention of plasmid is also suggested by the constant @-galactosidaseactivity level observed during continuous culture growth. Copy Number Amplification. In order to obtain a plasmid with a higher copy number, we constructed the plasmid pYAT462 bearing a deleted FBAl promoter gene cutting off a portion of UAS, which results in a reduced level of gene expression (Compagno et al., 1991). As a control, we used the plasmid pYAT1, which contains the whole FBAl promoter. Both plasmids were used to transform the WA6[p13A] strain, and plasmid shuffling was induced, selecting Trp+/Leu- cells in which p13A plasmid was substituted by the pYATl or pYAT462 plasmid. Lower levels of aldolase were indeed obtained in WA6[pYAT462J transformants (2.1 units/mg) in comparison with the aldolase levels expressed in WA6[pYATl] transformants (4 units/mg). However, we observed that the copy number of pYAT462 was the same as that of pYATl in both glucose and glycerol/ethanolmedia (about 8-10 copies per cell; data not shown). This result can be explained by consideringthat the deleted promoter is still strong enough to produce a high level of aldolase in a low copy number plasmid, and therefore there is a lack of selective pressure to increase the copy number. We then tested the possibility of obtaining a carbon source dependent copy number amplification by putting the FBAl gene under the control of U A S G ~ .We constructed two plasmids, pIAl0 and pIA12, in which the 365-bp fragment of the pLGSD5 plasmid containing GALI-IOIUAS was placed 75 bp (pIA10)and 12bp (pIA12)
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Table 11. Physiological Parameters of WAG[pIA10], WAG[pIA12], and WAG[pYATl] Grown in Glucose and in Galactose glucose galactose growthrate' 0.27 cellnumberb 5.5 buddedcells 46% aldolase' 0.21 copynumber 80
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0.33 18 38% 0.57 40
0.41 30 35% 4 5
Specific growth rate (hour'; In 2/29.
0.16 15 11% 4.8 5
0.23 20 15% 4.6 3-5
( X lo6cells/mL).
A
I
2000
0.16 20 25% 3.8 5
1000
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upstream from the TATA box of the FBAl gene (Table I). Both plasmids were used to transform the WA6[p13Al strain and plasmid shuffling was induced, selecting again Trp+/Leu- cells on galactose selective medium. Some colonies of WAG[pIAlOI and WAG[pIA121 transformants were transferred to glucose plates, and we observed growth in all of these cases. In Table I1 the physiological parameters of the pIAlO and pIA12 transformants are compared with the data from pYATl transformants. As expected, the aldolase level of pIAl0 transformants in glucose medium was lower than the wild-typelevel (0.5unit/mg), but was enough to support the growth. Instead, in galactose medium the effect of induction on FBAl gene expressionwas evident. The same levels of aldolase specificactivity were observed when cells were grown in rich media, but higher biomass concentrations were obtained (data not shown). The WA6[pIA101 strain showed a low growth rate in glucose and arrested growth a t an unusually low cell density (5 X lo6cells/mL). The analysis of cell volume distribution during growth on glucose showed an unusually large volume of WAGCpIAlO] cells (Figure 3A), which also agrees with absorbance data. Further, we observed a high fraction of budded cells at the end of growth. In parallel, we measured the copy number in transformed strains. The plasmid pIAl0 was present in glucose medium a t a larger copy number than plasmid pIA12, whereas in galadose medium both plasmids maintained the same copy number as pYATl (copy numbers are reported in Table 11). This indicates that, in response to weak FBAl expression, the cell needs high plasmid copy number to reach the minimum level of aldolase sufficient for cell growth. Moreover, these results, in relation to the aldolase level expressed, indicate greater glucoserepression for the pIAlO plasmid, probably due to the greater distance of UAS from the TATA box and so better conditions for the mechanism of UAS regulation. In order to study the modulation of plasmid copy number, we performed carbon source shift experiments. Cells of the WAG[pIA101strain growing in glucose medium were shifted in galactose medium at a density of 5 X lo6 cells/mL. After the lag time required for induction of the GAL system, aldolase levels increased up to a specific activity of 38 units/mg, which corresponds to 40 ?6 of total yeast proteins (Figure 4). At the same time, the plasmid level decreased again, reaching the low copy number characteristic of growth on galactose. After 20 h from the shift, the cell number was about 2 X lo7cells/mL, and no further increase was observed until the end of the experiment. This high productivity level should be due to the initially high copy number and the stability of the enzyme that allows its accumulation. Figure 3C shows that during the shift the unusual cell volume distribution observed in glucose medium returned to that in galactose (Figure 3B), indicating a correlation between large cell volume and high plasmid copy number.
0 0
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Channel number Figure 3. Cell volume distributions of WAG[pIAlO] (-) and WAG[YEPlacll2] (- - -1 grown on glucose (A) and galactose (B).
(C) Cell volume distribution of the WAG[pIAlOI population during the shift from glucose to galactose: the distributions have been acquired in glucose (-) at the moment of the shift and after 22 h from the shift in galactose (- - -1. Each channel, referred to cell volume distribution, corresponds to 1.44 fim3 in our experimental conditions.
Discussion The results presented in this article provide evidence for an autoselection system for stable maintenance of
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stable production of heterologous proteins in yeast, and studies are in progress along these lines.
Acknowledgment This work was supported by Consiglio Nazionale delle Ricerche Target Project “Biotechnology and Bioinstrumentation-, Sottoprogetto 3, Grant No. 91.01191.70.115.16078 t0E.M. Part of this work is covered by Italian Patent Application No. MI92 A 001425.
Literature Cited
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Time(hr) Figure 4. Time course of plasmid copy number and aldolase level (units/mg)during the shift of WA6[pIAlOJ cells fromglucose to galactose.
exprewion vectors in yeast in which it is also possible to modulate the plasmid copy number by the carbon source. This finding was achieved in two steps. First, we constructed a yeast strain dependent on the plasmid by disrupting the essential glycolytic gene FBAl, coding for fructose l,&diphosphate aldolase, and providing at the same time the FBAl gene on a plasmid. This manipulation makes unnecessary the presence of a selective growth medium: even after prolonged growth in batch or in continuous culture on rich media, no plasmid-free cells can be detected. In this way cultures can be grown in media that support fast growth rates and give higher biomass concentrations, thus increasing the yield of heterologous product. Afterward, we constructed a vector in which FBAl gene expression is regulated by UASGAL. In this way, the carbon source modulates the plasmid copy number, allowing one to obtain different gene dosages according to the carbon source utilized. Thisfeature makes our system more flexible than others in which a high copy number of plasmids was obtaining by using defective marker genes (Loison et al., 1989). In glucose medium, the low expression level of the autoselective marker gives selective growth advantage to cells with a higher gene dosage and thus promotes cells with a higher plasmid copy number. The analysis of cell volume distributions shows that the high plasmid copy number causes a large cell volume and a high fraction of budded cells in the stationary phase. The nuclear mutation nib1 (Holm, 19821,which results in an elevated 2-pm plasmid copy number, also originates a similar phenotype. The same effect was observed in other cases in which the regulation of the 2-pm copy number has been altered by overexpressing 2-pm gene products (Murray et al., 1987; Reynolds et al., 1987). After the shift in galactose, we observed the induction of FBAl gene expression, which produces a large amount of aldolase and, consequently, a gradual decrease in plasmid copy number a t the level present in cells growing in galactose medium, The decrease in plasmid copy number can be explained by two mechanisms: by DNA degradation or by the selective advantage that cells with a lower plasmid copy number present in the situation of strong induction of the selective marker. The mechanism of copy number modulation can be studied by monitoring the plasmid copy number distribution in subpopulations in different physiological conditions by flow cytometry (Wittrup et al., 1990). Furthermore, we believe that the selection system described herein could be used for the
Broach, J. R.; Strathern, J. N.; Hicks, J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 1979,8,121-133. Compagno, C.; Coraggio, I.; Ranzi, B. M.; Alberghina,L.; Viotti, A.; Martegani, E. Translational regulation of the expression of zein cloned in yeast under an inducible GAL promoter. Biochim. Biophys. Res. Commun. 1987,146,809-814. Compagno, C.; Ranzi, B. M.; Martegani, E. The promoter of SaccharomycescerevisiaeFBAl gene contains a single positive upstream regulatory element. FEBS Lett. 1991,293,97-100. Erhart, E.; Hollenberg, C. P. The presence of a defective LEU2 gene in 2 pm DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J. Bacterid. 1983, 156, 625-635. Futcher, A. B. The 2 I.tm circle plasmid of Saccharomyces cerevisiae. Yeast 1988, 4, 27-40. Gietz,D. R.; Sugino, A. New yeast-Escherichiacoli shuttle vectors constructed with in vitro mutagenized yeast genes lackingsixbase pair restriction sites. Gene 1988, 4, 527-534. Guarente, L.; Yocum, R. R.; Gifford, P. A. GALlO-CYCI hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc. Natl. Acad. Sci. U.S.A. 1982, 79,74107414. Holm, C. Clonal lethality caused by the yeast plasmid 2 p DNA. Cell 1982,29, 585-594. Loison, G.; Nguyen-Juilleret, M.; Alouani, F.; Marquet, M. Plasmid-transformed URA3 FUR1 double-mutant of S. cerevisiae: an autoselection system applicable to the production of foreign proteins. BiolTechnology 1986, 4, 433-437. Loison, G.; Vidal, A.; Findeli, A.; Roitsch, C.; Balloul, J. M.; Lemoine, Y. High level of expression of a protective antigen of schistosomesin Saccharomyces cerevisiae. Yeast 1989,5, 497-507. Lotti, M.; Porro, D.; Martegani, E.; Alberghina,L. Physiological and geneticmodulation of inducible expressionof Escherichia coli &galactosidase in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 1988,28, 160-165. Mann, C.; Buhler, J. M.; Treich, I.; Sentenac, A. RPC40, aunique gene for a subunit shared between yeast RNA polymerase A and C. Cell 1987,48,627-637. Murray, J. A. H.; Scarpa, M.; Rossi, N.; Cesareni, G. Antagonistic controlsregulate copy number of theyeast 2p plaamid. EMBO J. 1987, 6, 42054212. Napp, S. J.; Da Silva, N. A. Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae. Biotechnol. Bioeng. 1993, 41, 801-810. Nasmyth, K. A.; Reed, S. I. Isolationof genes by complementation in yeast, molecular cloning of a cell-cycle gene. h o c . Natl. Acad. Sci. U.S.A. 1980, 77, 2119-2123. Piper, P. W.; Curran, B. P. G. When a glycolytic gene on a yeast 2r ORI-STBplasmid is made essential for growthita espression level is a major determinant of plasmid copy number. Cum. Genet. 1990,17,119-123. Rech, S. B.; Stateva, L. I.; Oliver, S. G. Complementation of the Saccharomyces cerevisiae srbl-1 mutation: an autoselection system for stable plasmid maintenance. Curr. Genet. 1992, 21,339-344. Reynolds, A. E.; Murray, A. W.; Szostak, J. W. Roles of the 2 pm gene products in stable maintenance of the 2 pm plasmid of Saccharomycescerevisiae. Mol. Cell. Biol. 1987,7,35663573.
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Romanos, M. A.; Scorer,C. A.; Clare,J. J. Foreign gene expression in yeast: a review. Yeast 1992,8,423-488. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY,1989. Untemahrer, S.;Pridmore, D.; Hinnen, A. A new system for amplifying 2 pm plasmid copy number in Saccharomyces cerevisiae. Mol. Microbiol. 1991,5, 1539-1548.
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Wittrup, K. D.; Bailey, J. D.; Ratzkin, B.; Patel, A. Propegation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae. Flow cytometry studies and segregated modeling. Biotechnol. Bioeng. 1990,35,565-577. Accepted July 8,1993.' e Abstract published in Advance ACS Abstracts, September 1, 1993.
