Bioprocess Engineering 4 (1989) 75-80

Bi0pr0cessEngineering 9 Springer-Verlag 1989

Effect of plasmid size and medium on growth kinetics and plasmid copy number in Saccharomyces cerevisiae S.J. Coppella, Baltimore, and P. Dhurjati, Newark

Abstract. Saccharomyces cerevisiae cultures were selected to determine the effects of plasmid size on host growth kinetics and plasmid copy number. A complete synthetic medium was verified and the effects of yeast nitrogen base without amino acids medium and leucine selection were established for the strain. The dependence of copy number on the plasmid size, medium, and oxygen availability was also measured. This study was part of a comprehensive effort to elucidate the behavior of recombinant yeast.

1 Introduction

Recombinant DNA technology has provided the tools for producing many biological compounds that otherwise were recovered in only minute quantities, if at all. These products have uses ranging from the treatment of human genetic disorders and cancer, to the production of bulk chemicals. Before benefits of this technology can be realized, an understanding of the reaction system to be utilized must be obtained followed by a mathematical description of the system's kinetics to aid in production control and scheduling. Two variables of a recombinant system that needed to be addressed before production were the host/plasmid interactions, and the product protein production and secretion kinetics and its effect on the host. Host/plasmid interactions included the effect of the plasmid on host growth rate and substrate yield, plasmid stability during host replication, and the effects of plasmid size and the environment on plasmid copy number (number of plasmids per cell or per host ge-

nome). This work deals with the first question of host/plasmid interactions in the yeast Saccharomyces cerevisiae (Baker's or Brewer's Yeast). By selecting a host and using a control with the two different size plasmids (6.3 and 14.3 Kbp) both with the same origin of replication (2 gin), the dependence of growth kinetics, plasmid stability, and plasmid number on plasmid size are determined. 2 Strain and cultivation

Three microorganisms were used: ABI03 (Mate cir + leu2-3, -112 ura3-52 his4-580 pep4-3), ABI03.1 (ABI03 cir~ and

AB103.1 pYeEGF-25 [1]. ABI03 contained the 2 gm plasmid (6.3 Kbp) endogenous to yeast cir + which conferred no known phenotype. The second plasmid pYeEGF-25, 14.3 Kbp) used the full 2 gm plasmid for its origin of replication, and contained the leu2 selective marker. The heterologous protein expressed by this plasmid was the human epidermal growth factor (hEGF), but since it is secreted into the broth and produced to less than 0.5 g/m 3 there was no toxicity to the host. A cured host was used with pYeEGF-25, which eliminated competition and recombination with the 2 gm plasraids and also eliminated the transacting F L P gene product which facilitated covalent bonding between homologous sequences. Recombination, interconversion, and associated multimerization resulting from the inverted repeats (IRI and IR2) whithin the 2 gm replicon were eliminated by using a REC- host and p C l / l (derived from pJDB219), in which the F L P gene was interrupted. Also this construct conserved the REP1, REP2, and REP3 loci which maintained high plasmid copy number and segregation stability. Without the F L P gene product, recombination occurred approximately 1% in 30 generations [2]. Plasmid integrity was verified with agarose gel-electrophoresis [3]. These cultures were obtained from the Chiron Corp. (Emeryville, CA.) on petri dishes with the recombinant culture under selective pressure. Colonies were transferred to the complete synthetic liquid medium given in Table 1, grown at 30 ~ and refrigerated for seeding future i n o c u lums. This medium was modified from that used by Lievense [4] and the L e u - supplements (Table 2) were provided by Chiron. Batch and shake flask fermentations were qualitatively used to develop the complete medium and all the L e u - supplements and the high phosphate concentration were required by the strains (data not shown). Recombinant cells were kept under selective pressure unless otherwise stated. The yeast nitrogen base without amino acids (YNB) medium contained 3.35 g YNB without amino acids, 0.2 g threonine, and 2.5 g L e u - supplements per 10 g glucose. Petri dish solid medium recipe was presented in Table 3 and the two previous recipes were also provided by Chiton. All media were filter sterilized with a GVWP 142 50 0.22 gm

76

Bioprocess Engineering 4 (1989)

Table 1. Nonselective complete synthetic medium Compound ~

Stock concentration [kg/m 3]

1 (NH4)zSO 4 2 NaCI 3 MNSO4- H20 4 ZnSO~- 7H20 5 CuSO~. 5HzO 6 KHzPO 4 7 MgSO 4.7HzO 8 CaC12.2H20 9 FeSO 4. 7 H20 10 HsBO 3 11 MoO 3 12 CoCI z . 6 H 2 0 13 NiSO 4. 6H20 14 KI 15 b myo-inositol 16 Thiamine-HCL 17 Ca-pantothenate 18 pyridoxine 19 d-biotin 20 glucose 21 threonine 22 L e u - supplements 23 ~ l-Leucine

200 10 0.4 0.3 0.08 100 200 140 8 0.02 0.01 0.01 0.01 0.01 20 20 10 5 0.05

Medium concentration [kg/m 3] 2.0 0.1 0.004 0.003 0.008 0.5 0.20 0.14 0.08 0.00002 0.00001 0.00001 0.00001 0.00001 0.02 0.02 0.01 0.005 0.00005 10 0.2 2.5 0.652

a Stock solutions: 1-5, 6, 7, 8, 9, 10-14, 15-18, stored at 4~ b 15--18 Adjusted to pH = 5 ~ Only used in nonselective medium Table 2. L e u - supplements Compound

Weight [g]

Adenine Uridine 1-tryptophan 1-histidine l-arginine 1-methionine 1-tyrosine 1-1ysine 1-phenylalanine

4.0 3.0 2.0 2.0 2.0 2.0 3.0 3.0 5.0

filter in a 142 m m stainless steel Filter Holder (Millipore Corp.) for volumes greater than I d m 3. Volumes less than I d m 3 were filtered with GA-8 0.2 gm filter in a 25 m m Delrin in-line Filter H o l d e r (Gelman Sciences). Both filters were low protein binding. Batch fermentations were conducted at 30~ 5.0 pH, 400 m i n - 1 agitation, air flow of 0.5 dmS/min, and 0.5 psig, with breathing air supplied to 4 d m 3 of medium. A 7 d m 3 MA-100 Magnaferm F e r m e n t o r with large impellers was used. Mass transfer limitations were measured to be insignificant. A PH-40 p H Controller (New Brunswick Scientific) controlled p H and used a Ingold p H probe (465-25-90-K9, Ingold Electrodes Inc., Wilmington, MA), with 7520-35 and 7553-20 Masterflex peristaltic pumps and silicon tubing (Cole-Palmer Instrument Co.) for controlled aseptic addition of 2.5 M N H 4 O H and 1.0 M H2SO 4. The fermentor was part of the Off-Gas and D a t a Acquisition System described earlier [5]. F o a m i n g problems were avoided by adding 10 cm 3 of autoclaved 20 kg/m 3 D o w Corning 1520 Silicon Antifoam (Dow C o m i n g Corp.) and dissolved oxygen always remained above 50% saturation. All equipment and liquids were sterilized for 45 rain at 121 ~ Closed containers were vented with G e l m a n 4210 Bacterial Air Vents (4550-C90 T h o m a s Sci.), which were also used to filter the air supply and the acid and base. Sterilization procedure was evaluated by maintaining medium clarity after 4 d in a blank fermentation. Shake flasks were covered with two 16.5 cm N o n Gauze Milk filters (Kendall Co., Boston, MA) and secured with at least two rubber bands before autoclaving. C o n t a m i n a t i o n was tested for by examination of b r o t h color and odor, colony appearance on plated samples, and visual inspection of stained slides using a 1500 fold inverted phage contrast microscope.

3 Analytical measurements

Table 3. Solid medium" Compound

Amount

Basal salts L e u - supplements 2 M sorbitol b Agar Distilled water

100 cm 3 0.5 g 500 cm 3 20 g 350 cm 3

Basal salts c Yeast nitrogen base without amino acids Succinic ~icid Sodium hydroxide

66.8 g 100 g 60 g

a Makes approximately 30 10 cm plates; mix all compounds, add agar last and do not mix; autoclave for 30 min at 212 ~ cool, pour into dishes b Store at 4 ~ Dissolve succinic acid and sodium hydroxide first q.s. to t dm 3 with distilled water, filter, sterilize, store at 4 ~

An in-house-constructed galvanic probe [6] was used to m o n i t o r dissolved oxygen, Optical Density was measured on-line after passing through a bubble trap and using a dilution tube [7] and Bausch & L o m b Spectronic 20 Spectrophotometer at 600 rim. D r y cell weight was obtained by filtering 50 cm 3 of broth through a W h a t m a n G F / A 5.5 cm filter disk in a Buchner funnel, washed twice with 25 cm 2 of distilled water, then drying for 24 h at 65 ~ Optical Density of the filtrate was zero and all media gave zero dry cell weight. Off-line analysis was described earlier [3] as was the copy number assay [8]. Ethanol analysis had a relative error of + 12%.

4 Results

4.1 Dry cell weight~optical density correlation Figure 1 contained the D r y Cell Weight/Optical Density (OD) correlation for b o t h off-line undiluted samples from a

Coppella et al.: Effect of plasmid size and medium on growth kinetics and copy number in Saccharomyces cerevisiae

5 kglrn3I

/

5 [kg Ira?

5 kglm'

............... i(

77

-100 %

i

80 3

~3_~ z~

t:

i

50 #

x:z

'=" 2

4o

-2 .~

1! 0 ~--~ 0

~--4--+-+--~~---<--~b- 0 0.5 1.0 1.5 2.0 Optical densityat 000 nm

Fig. 1. Dry Cell Weight/Optical Density Correlation o ABt03.I pYc~EGF-25, nonselective complete medium zx ABt03.1 pYc~EGF-25, nonselective YNB medium o AB103 nonselective complete medium o AB103.1, nonselective complete medium v ABt03.t pYc~EGF-25, nonselective defined medium

DCW

0

r

dcw kg/m 3 = - 0 . 0 6 9 4 + 3.48 ( O D ) - 1.16 (OD) 2 4-1.01 (OD) 3

M a x i m u m measurable sample D r y Cell Weight concentration had increased over four times and with greater sensitivity when the sample was diluted. Also the correlation was unchanged by the presence of either plasmid or the m e d i u m used as shown by the superposition of the four d a t a sets. This result was expected as the plasmids represented a small weight fraction of the total cell and p r o d u c e d only small levels of heterologous protein. Also the m e d i u m should not have affected the correlation, since both media had zero O D against distilled water. Verifying the correlation to all cases was required to avoid misinterpretation of on-line data.

'~

+

l:r:: ~- 50

mmol/(dm3.h)

mmol/(drn3.h)

!

40

1

co

/i

30

cPR/ ~~ ~;

N

i'

N 10~

// ..........."

Undiluted d a t a was fit with a quadratic to yield:

dcw kg/m 3 = 0.173 + 0.548 ( O D ) - 0.260 (OD) 2

:

50 1

30 Bausch & L o m b Spectronic 2000 Spectrophotometer, and the on-line diluted samples measured with a Spec 20 Spectrophotometer, both at 600 nm. A third order correlation was adequate to fit the diluted d a t a and yielded:

20

h u~+--~-~

.y

.:

~3 g 10 ~=

'~

~l,i~ - .........

E ', ', ', [ ;--+-4 s ,--r~

,

:

t0

1~t Tf ii

6-

o~

f o

4.2 Cell growth Figure 2 showed typical on-line fermentation d a t a taken from the 14.3 K b p plasmid bearing strain grown in nonselective complete medium. D r y Cell Weight d e m o n s t r a t e d the diauxic growth of yeast in batch fermentation with glucose. After a short lag phase, growth was exponential while fermenting glucose to ethanol and carbon dioxide. After glucose exhaustion, exponential growth resumed as the ethanol was oxidized to water and carbon dioxide. After ethanol

N2

~ ~-+ ~ ,-+-q--+-c , 20 30 h 413 Time l Fig. 2. a On-line data from AB103.1 pYeEGF-25, nonselective complete medium, b On-line data from ABI03.t pYeEGF-25, nonselective complete medium, e On-line data from ABt03.1 pYctEGF-25, nonselective complete medium e

04 , ~ E ~ ~ - ~ , 0 10

78 exhaustion stationary phase was entered. Dissolved oxygen mirrored this growth with an initial decrease then rise-back at glucose exhaustion, a faster decrease with ethanol oxidation, then rise-back to 100% saturation at ethanol exhaustion. Riseback to 100% indicated a low oxygen maintenance requirement for the yeast. Off-gas data illustrated the different catabolisms and also followed the Dry Cell Weight. During glucose fermentation the Carbon Dioxide Production Rate (CPR) increased exponentially while the Oxygen Uptake Rate (OUR) increased slowly and the Respiratory Quotient (RQ) was greater than 1. The change in CPR and RQ at glucose exhaustion was abrupt as the CPR decreased, then again increased, but at a slower rate, and the RQ then changed to 0.4-0.7 during ethanol oxidation. The OUR did not display an abrupt change at the diauxic lag, but increased at a quicker rate during the second growth phase. A positive OUR during glucose fermentation and lack of a sharp change at the switch in catabolisms indicated continuous activity of the TCA cycle during glucose utilization. Ethanol was produced during glucose utilization followed by ethanol utilization after glucose exhaustion. Dry cell weight yield and ethanol yield on glucose correlated well (data not shown). A value of 0.21 g dry cell weight and 0.44 g ethanol per g glucose consumed was measured. For AB103.1 pYeEGF-25 in nonselective medium the maximum specific growth rate was 0.46 h - a. Also the glucose saturation constant calculated was 2.4 kg/m 3 and compared favorably with the literature value of 2.0 kg/m 3. Other correlations gave equally good fits. Maximum specific growth rates in Tables 4 and 5 were obtained from Lineweaver-Burk plots for glucose because of the high K s , and from log (dry cell weight) plots for ethanol because of the high ethanol measurement error and low K s value. Carbon balances for all experiments yielded between 93 and 130% carbon recovery.

Bioprocess Engineering 4 (1989) Table 4. Effect of medium and AB103.1 pYeEGF-25 Glucose fermentation

Ethanol oxidation

Overall

AB103.1 pYc~EGF-25

/~. yb [h- ~] [g/g]

~tm~ y~ [h- ~] [g/g]

yb [g/g]

Complete medium Leu + YNB medium Leu + Complete medium Leu -

0.47 0.21

0.07

0.50

0.49 0.21

0.02 (0.21)

-

0.43 0.21

0.03 (0.40)

-

Host literature (2) values with complete minimal or rich mediums

0.46 0.15

0.12

0.50

0.69

0.6-0.7

a 15% Relative error b 7% Relative error ~ 12% Relative error

Table 5. Effect of plasmid size on host AB103.1 Glucose fermentation

Ethanol oxidation

Overall

Complete medium

#maxa yb

Leu +

[h- x] [g/g]

/lm,, yc [h- 1] [g/g]

yb [g/g]

ABI03.1 ABI03.1 (6.3 Kbp) AB103.1 pYc~EGF-25 (14.3 Kbp)

0.45 0.21 0.48 0.20 0.47 0.21

0.08 0.67 0.07 0.71 0.07 0.69

0.51 0.52 0.50

" 15% Relative error b 7% Relative error ~ 12% Relative error

4.3 E f f e c t o f m e d i u m

Table 4 contained the maximum specific growth rate and substrate yield for both glucose fermentation and ethanol oxidation and overall yield results to determine the effect of medium on the cell and for comparison to literature values for host Saccharomyces cerevisiae in complete carbon substrate limited medium. In all three experimental data sets the AB103.1 pYeEGF-25 system was used. The control case was complete medium without selection (Leu+) which compared favorably to literature values except for a lower growth rate on ethanol and a slightly higher glucose and ethanol yield. The lower growth rate indicated that ABI03.1 had a small respiratory deficiency, this deficiency was also seen on YEPD [3]. The smaller substrate yield was explained by the presence of L e u - supplements (nucleotides and amino acids) that increased the amount of carbon substrate which was not accounted for in the yield on glucose calculation. The nonselective YNB medium was equal to the complete medium during glucose fermentation, but exhibited

significantly slower growth on ethanol. Due to the slow growth, ethanol yield calculations were suspect and an overall yield could not be determined for growth on the undefined medium. Obviously the Yeast Nitrogen Base w/o amino acids lacked a component essential to oxidation (the TCA Cycle) in yeast. This deficiency could have been due to an inadequate concentration or to the loss of vitamin activity. The selective (Leu--) complete medium had the same glucose yield as expected, since leucine represented a small weight fraction of the total cell carbon source requirement. However, without leucine the cell exhibited lower growth in both phases, which was explained by the relatively weak expression of the plasmid leu2 gene, which made leucine also growth limiting. This weak expression is exploited to select high copy number and thus highly stable recombinant cultures under selection [9, 10], like pY~EGF-25. The effect

Coppella et al.: Effect of plasmid size and medium on growth kinetics and copy number in Saccharomyces cerevisiae of leucine selection was more pronounced during ethanol oxidation and was possibly due to a lower intracellular pyruvate concentration because of glucose exhaustion and subsequent inactivity of the Embden-Meyerhof pathway. Leucine synthesis was slowed and further limited cell growth. As Fig. 3 showed, leucine was synthesized from pyruvate (4 mole pyruvate/mole leucine), which was an intermediate in the glycolysis pathway, but not an intermediate in the oxidation of ethanol [11]. Therefore leucine synthesis required pyruvate synthesis only during ethanol oxidation, thus lowering the intracellular pyruvate concentration. This synthesis and the high ratio of pyruvate required to synthesize leucine magnified the effect of the leu2 gene by further limiting cell growth. Again, due to the slow ethanol growth rate the ethanol yield was suspect and an overall yield not obtainable.

4.4 Effect of plasmid size Table 5 showed the effect of the plasmid size on host growth kinetics and substrate yield for the AB103.1 host and with plasmid sizes 6.3 and 14.3 Kbp, all with the same 2 Mm replicon. Relative errors for yields were 7%, and 15% for growth parameters (/l. . . . Ks). These cultures were a mixture of colonies selected for over 30 generations in L e u - complete medium. The fermentations were all with nonselective (Leu +) complete medium. For the three cases substrate yield for both glucose and ethanol remained unchanged, as did the overall yield and the Maximum Specific Growth Rate on both glucose and ethanol. Obviously the heterologous protein expressed by pYc~EGF-25 had little to no effect on the host, as speculated earlier. This is due to its low expression rate (< 0.5 g/din 3 at stationary phase) and complete secretion into the broth. The plasmid size had little to no effect on the growth rate of the host or substrate yields. No plasmid instability was observed in both selective and nonselective fermentations with AB103.1 pYc~EGF-25. To determine longer range plasmid stability, six nonselective medium shake flasks were serially inoculated (i.e. the first was inoculated with 5 cm 3 of stationary phase seed, grown, then 5 cm 3 of the first were used to inoculate the second, etc.) and grown at 30~ in 150 cm 3 nonselective medium for two d. After approximately 30 generations no instability was observed. Since the 2 Mm plasmid was naturally occurring it was assumed to be 100% stable. Therefore plasmid size had no effect on stability in the regions tested. The results compared favorably with those of Walmsley et al. [12] and Hollenberg [9], who found pJDB219 (used in construction of pYc~EGF-25) in Saccharomyces cerevisiae to remain nearly 100% recombinant in nonselective medium after 45 generations.

4.5 Plasmid copy number Copy number results (35% relative error) in Table 6 for AB103.1 pYeEGF-25 showed no dependence on the physio-

Glucose l ...".........................."%...... Pyruvote Ethanol ~ F ix /"

#

Leucine

79

Glucose catabolism Ethanol oxidation Leucinesynthesis

Aoety[- CoA TCA !ycle

Fig. 3, Leucine biosynthesis and catabolism pathways in yeast Table 6. Copy number behavior of 2 gm Replicon Complete m e d i u m

Inoculum a

Diauxiea lag

Stationary a

AB103.1 pYeEGF-25 (14.3 Kbp) Leu + Leu YBN medium Leu + AB103.J (6.3 Kbp) Leu +

50

68

47

50 50

41 37

40 51

52

64

78

" 36% Relative error logical state of the cell, 0 2 availability or medium nutrients (leucine or the essential respiration nutrient not contained in YNB). This behavior was also observed with other media [13] where the copy number did not change with fermentor time or catabolism. The only two copy number data in Table 6 that had a statistically significant deviation (37 and 78) were believed to be due to excessive errors resulting from cell density measurement. Throughout the fermentation the plasmid copy number was maintained as observed earlier by Gerbaud and Guerineau [13]. Copy number conservation is also consistent with the action of the REP1 and REP2 products that override normal replication to maintain a high plasmid content. Oxygen dependence was determined by comparing aerated fermentor samples with the inoculum which was grown in shake flasks. These flasks had oxygen transfer limitations as evidenced by a decreased ethanol utilization capacity. Effects of medium were observed from three fermentations: Leu + Complete Medium, Leu- Complete Medium, and Leu + YNB Medium. Results of these media on growth were discussed earlier. The lack of effect of environment on the copy number was consistent with the report of Hollenberg [9] that copy number of the 2 lam plasmid was independent of environment and the physiological state of the cell. It was also supported by the observed controlled multiple replications of the 2 gm plasmid as a result of the REPI and REP2 gene products to maintain a high and constant copy number. Comparison of measurements using the pYc~EGF-25 (14.3 Kbp) and the 2 gm (6.3 Kbp) from Leu + Complete Medium fermentations showed no dependence of plasmid copy number on plasmid size (Table 6).

80

5 Discussion Thus it appeared that a mechanism existed to maintain the number of plasmids constant per cell and was independent of environmental stress or plasmid size. Results from Futcher and Cox [10] elude to a mechanism that conserved the number of 2 ~tm origins in yeast cells, even though a distribution of copy numbers resulted from variances in the plasmids and strains. They have speculated that this control mechanism may actually be an equilibrium between opposing forces that increase and decrease plasmid copy number and that colony variations resulted from variations in these forces. One of the forces involved would be the products of the R E P I and REP2 genes that override normal replication to maintain high copy number. Another force would be the cis-acting REP3 sequence which has been shown necessary for stable propagation of the 2 ~tm plasmid by facilitating partitioning during cell division [14]. REP3 may also function with the R E P I and REP2 products to control plasmid copy number like the "cer" sequence in the E. coli plasmid ColE1 which led to the "origin counting model" [15]. Thus, mechanisms existed that actively control plasmid copy number and segregation stability and that this control was both plasmid and host dependent. 6 Summary A host with two different plasmid sizes (6.3 and 14.3 Kbp) was used with the same origin of replication (2 gm) to determine host/plasmid interactions and the effects of medium on growth kinetics and plasmid copy number in Saccharomyces cerevisiae. The nonselective complete synthetic medium gave a higher growth rate than both the nonselective YNB medium, which lacked a respiratory requirement of the strain, and the selective complete medium, which significantly lowered growth during ethanol oxidation due to a limiting supply of leucine. Plasmid size had no effect on host growth parameters, or plasmid stability in the region tested. This result indicated that a region on the 2 gm plasmid was used to conserve the plasmid copy number. Plasmid copy number was not affected by the physiological state of the cell, substrate limitations, or plasmid size.

Acknowledgements The authors wish to thank the Chiron Corp. (Emeryville, CA) for their generous donation of the cultures used for this work. We especially acknowledge the help of Dr. James P. Merryweather, and Dr. Carlos George-Nascimento for their continuous support. Also we would like to acknowledge the help and advice of Carolyn Acheson, Department of Chemical Engineering, Cornell University, Ithaca, NY.

References 1. Brake, A. J.; Merryweather, J. P.; Coit, D. G.; Herberlein, U. A.; Masiar, F. R.; Mullenbach, G. T.; Urdea, M. S.; Valenzuela, P.; Barr, P. J.: c~-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81 (1984) 4642-4646

Bioprocess Engineering 4 (1989) 2. Broach, J. R.: The yeast plasmid 2 gm circle. In: Strathern, J. N.; Jones, E. W.; Broach, J. R. (Eds): The molecular biology of the yeast Saccharomyces - life cycle and inheritance, pp. 445-470. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1981) 3. CoppeUa, S. J.: Growth kinetics, plasmid stability, and foreign gene expression in Saceharomyces cerevisiae. Ph.D. Thesis, Dept. Chem. Eng., University of Delaware (1987) 4. Lievense, J. C.: An investigation of the aerobic, glucose-limited growth and dynamics of Saccharomyees cerevisiae. Ph.D. Thesis, Dept. Chem. Eng., Purdue University (1984) 5. Coppella, S. J.; Dhurjati, P.: Low cost computer-coupled fermentor off-gas analysis via Quadrupole Mass Spectrometer, Biotechnol. Bioeng. 29 (1986) 679-689 6. Borkowski, J. D.; Johnson, M. J.: Long-lived steam-sterilizable membrane probes for dissolved oxygen measurement. Biotechnol. Bioeng. IX (1968) 635-639 7. Lee, C.; Lira, H.: New device for continuously monitoring the optical density of concentrated microbial cultures. BiotechnoL Bioeng. 22 (1980) 639-642 8. Coppella, S.J.; Aeheson, C. A.; Dhurjati, P.: Isolation of high moleuclar weight nucleic acids for copy number analysis using HPLC. J. Chrom. 402 (1986) 189-199 9. Hollenberg, C. P.: Cloning with 2 ~tm DNA vectors and the expression of foreign genes in Saccharomyces cerevisiae. Current Top Microbiol. Immunology 96 (1982) 119-144 10. Futcher, A. B.; Cox, B. S.: Copy number and the stability of 2 ~tm circle-based artificial plasmids of Saccharomyces cerevisiae. J. Bacteriol. 157 (1984) 283-290 11. Harris, G.: Nitrogen metabolism. In: Cook, A.H. (Ed.): The chemistry and biology of yeasts, pp. 469. New York: Academic Press Inc. 1958 12. Walmsley, R. M.; Gardner, D. C. J.; Oliver, S. G.: Stability of a cloned gene in yeast grown in ehemostat culture. Mol. Gen. Genet. 192 (1983) 361-365 13. Gerbaud, C.; Guerineau, M.: 2 gm plasmid copy number in different yeast strains and repartition of endogenous and 2 gm chimeric plasmids in transformed strains. Curr. Genetics 1 (1980) 219-228 14. Wu, L. C.; Fisher, P.; Broach, J. R.: The REP1 protein of the 2 micron circle is associated with the nuclear matrix. In: Hicks, J. (Ed.): Yeast Cell Biology, pp. 323-344. New York: Alan R. Liss, Inc. 1986 15. Summers, D. K.; Sherratt, D. J.: Multimerization of high copy number plasmids causes instability: ColE1 encodes a determinant essential for plasmid monomerization and stability. Cell 36 (1984) 1097-1103 Received Dec. 7, 1987

Steven J. Coppella (corresponding author) Assistant Professor Department of Chemical Engineering University of Maryland Baltimore County Baltimore, MD 21228 and Medical Biotechnology Center of the Maryland Biotechnology Institute University of Maryland Baltimore, MD 21201 Prasad Dhurjati Associate Professor Department of Chemical Engineering University of Delaware Newark, De 19716 USA