THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 256, No. 14, Issue of July 25, pp. 7311-7315, 1981

Elevated Levels of Asparagine Synthetase Activity in Physiologically and Genetically Derepressed Chinese Hamster Ovary Cells Are Due to Increased Rates of Enzyme Synthesis*

(Received for publication, February 13, 1981)

J. Stephen Gantt4 and Stuart M. Arfin From the Denartment of Biolopical Chemistrv. California College of Medicine, University of California, Zruine, Iruine,

I -. . California 9271 7

The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J Biol. Chem 254, 10629-10633). CHO cells resistant to fl-aspartylhydroxamate have up to &fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J Biol. Chem 255, 4808- 4813). We have investigated the basis for these differ- ences in enzyme activity by combined radiochemical and immunological techniques.

The asparagine synthetase of beef pancreas was pu- rified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the aspar- agine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the differ- ence in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attrib- uted solely to an increased rate of enzyme synthesis.

Cultured Chinese hamster ovary cells modulate the specific activity of asparagine synthetase in response to the degree of aminoacylation of a number of species of tRNAs (1). CHO’ cell mutants affected in asparagine synthetase activity were previously isolated by selection for resistance to @-aspartyl- hydroxamate (2). The activity of asparagine synthetase in some of these /3-aspartylhydroxamate-resistant lines is 5-fold higher than that in the parental line and is no longer subject to regulation by either the asparagine content of the medium or the extent of aminoacylation of tRNAtze“. Initial studies, which compared some physical and catalytic properties of the enzyme from mutant and parental lines, failed to show any

* This investigation was supported by Research Grant GM 26014 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of United States Public Health Service Predoctoral Trainee Fellowship GM 07134.

I The abbreviations used are: CHO, Chinese hamster ovary; PMSF, phenylmethylsulfonyl fluoride; a-ME medium, an enriched Eagle’s minimal essential medium; SDS, sodium dodecyl sulfate.

significant differences in these properties. This suggests that the differences between these lines are the result of differences in the number of enzyme molecules rather than differences in catalytic activity per molecule.

In addition to mutants containing constitutively elevated levels of asparagine synthetase activity, Chinese hamster cell lines containing low or undetectable levels of activity have been isolated as asparagine auxotrophs (3, 4). Thus, the as- paragine synthetase system appears to be an attractive one for genetic and biochemical studies of the regulation of enzyme levels in animal cells.

In this study, we describe the purification of asparagine synthetase from beef pancreas and the preparation of rabbit antibody to asparagine synthetase. Using this antibody, we show that the differences in enzyme activity levels resulting from both physiological manipulation and genetic alteration are due to differences in the content of asparagine synthetase molecules. Elevated levels of asparagine synthetase are achieved through alterations of the rate of enzyme synthesis with no change in the rate of enzyme degradation.

EXPERIMENTAL PROCEDURES

Materials-Hydroxylapatite (Hypatite C) was purchased from Clarkson Chemical Co., and agarose-linked Cibacron blue 3GA (Ma- trex gel blue A) was purchased from Amicon Corp. PMSF, ATP, and dithiothreitol were from Sigma. Freund’s complete and incomplete adjuvants were from Grand Island Biological Co. Formaldehyde-fixed Staphylococcus aureus was prepared by the method of Kessler (5). L- [U-’4C]Aspartic acid (222 mCi/mmol) and ~-[~~S]methionine (600 to 1400 Ci/mmol) were purchased from Amersham Corporation, Arling- ton Heights, IL. The sources and purity of all other chemicals were identical to those described earlier (1, 2, 6).

Cell Culture-The properties of the parental CHO cell line, tsH1, which contains a temperature-sensitive leucyl-tRNA synthetase, and lines AH-2 and AH-5, which contain increased asparagine synthetase activity, have been described (1,2). Cells were grown in monolayer or suspension culture in a-ME medium (7) as previously described (I, 2, 6).

All cell lines were tested periodically and found to be free of mycoplasma contamination.

Enzyme Assay-Asparagine synthetase activity was measured by following the conversion of [I4C]aspartic acid to [“C]asparagine, as previously described (2). Labeled aspartic acid was purified in order to establish low assay backgrounds. [“CIAspartic acid was adsorbed to a Dowex 1-acetate column and washed thoroughly with water. The adsorbed aspartic acid was eluted with 2 N HC1 and neutralized with Tris base. One unit of activity catalyzes the formation of 1 pmol of asparaginehin. Protein was assayed by the method of Lowry et al. (S), or by the dye-binding method described by Bradford (9), using crystalline bovine serum albumin as a standard.

Enzyme Purification-All steps were carried out at 0-4 “C unless otherwise noted. Beef pancreata, obtained fresh from a local slaugh- terhouse, were trimmed of fat, minced, and homogenized in 2 volumes of 10 mM Tris, 1 mM dithiothreitol, 5 mM EDTA, 20% glycerol, 0.5 mM PMSF, pH 7.5 (Buffer A), using a Brinkmann Polytron. The

731 I

7312 Synthesis of Asparagine Synthetase in CHO Cells

homogenate was centrifuged at 15,000 X g for 30 min and then at 105,000 X g for 60 min, and the supernatant solution was saved (Fraction I) . Cetyltrimethylammonium bromide (1%) was added drop- wise while stirring to a final concentration of 0.1%. After 10 min, the precipitate was removed by centrifugation. The supernatant (Fraction 11) was mixed with concentrated solutions of sodium ATP, MgC12, and sodium aspartate to achieve final concentrations of 5, 10, and 3 mM, respectively. This solution was rapidly raised to 55 "C, held at that temperature with gentle stirring for 8 min, and then rapidly chilled in an ice bath. Heat-denatured protein was removed by centrifugation. (NH4)2S04, saturated at 4 "C and adjusted to pH 7.5 with NH40H, was added to the supernatant solution while stirring to a final saturation of 60%. After stirring for an additional 15 min, the precipitate was collected by centrifugation and dissolved in Yi of the original homogenate volume of buffer A (Fraction 111). Hydroxylap- atite, previously equilibrated with buffer A, was added to Fraction I11 in the ratio of 10 mg of hydroxylapatite/mg of protein. After stirring for 15 min, the hydroxylapatite and adsorbed protein were collected by centrifugation for 10 min at 10,000 rpm in a Beckman JA-IO rotor. The pellet was suspended in ?4 of the original homogenate volume of buffer A containing 12 mM ATP and stirred for 15 min. The suspension was centrifuged for 10 min at 15,000 X g and the supernatant was saved. The protein was precipitated at 60% (NH4)2S04, as before, and the pellet was dissolved in 20 to 30 ml of buffer A (Fraction IV). Fraction IV was applied to a column (3.0 X 25 cm) of MZtrex gel blue equilibrated with Buffer A. The column was washed with Buffer A until no more protein was removed and then with Buffer A containing 16 mM sodium ATP and 16 mM MgCL. The active fractions eluted by this buffer were pooled and the proteins reprecipitated at 60% satu- rated (NH4)2S04. The pellet obtained after centrifugation was redis- solved in Buffer A minus glycerol (Fraction V), and 0.1-ml aliquots

A. The tubes were centrifuged at 47,000 rpm for 20 h in a Spinco SW were layered onto 5.0-ml 10 to 25% linear glycerol gradients in Buffer

50.1 rotor. Thirty-five 0.15-ml fractions were collected from the bot- tom of the tube. The bulk of the asparagine synthetase activity is found in fractions 10 to 12 (Fraction VI). The enzyme was stored in small quantities at -20 "C.

Acrylamide Gel Electrophoresis-SDS-acrylamide gel electropho- resis, using 7.5% separating gels and 3% stacking gels, was carried out according to Laemmli (IO). Proteins were visualized as described by Laemmli (10). Gels containing radiolabeled proteins were cut into 1.2- mm slices, digested with 0.5 ml of 30% Hz02 for 2 h a t 60 "C, and counted as described by Hanford and Arfin (11).

Preparation of Anti-Asparagine Synthetase Antibody-Homoge- neous beef pancreas asparagine synthetase (200 pg/ml in Buffer A) was mixed with an equal volume of complete Freund's adjuvant. Three New Zealand white rabbits were each injected with about 100 pg of protein divided into four equal portions, two given intramuscu- larly in the haunches and two subcutaneously along the backbone. Each rabbit was also given three additional injections (100 pg of protein in incomplete adjuvant) at 3-week intervals. Tests for circu- lating antibody during this time were negative. Twelve weeks after the immunization schedule was begun, each rabbit was similarly injected with 1 mg of protein in complete adjuvant. Maximal antibody titers were obtained 10 days after this injection. The rabbits were bled from the ear vein every 2 weeks after this final injection. A y-globulin fraction was prepared from the serum as described by Palacios et al. (12), adjusted to a protein concentration of 20 mg/ml and stored at -20 "C. A control y-globulin fraction was prepared in an identical manner from preimmune serum.

Immunotitration of Asparagine Synthetase-Cell extracts were prepared by 2 cycles of freeze-thawing in Buffer A, followed by centrifugation at 30,000 x g for 15 min. Protein concentration was adjusted to 10 mg/ml. Fifty pl of cell extracts were incubated with various dilutions of antiserum for 30 min at 4 "C. A sufficient amount of 20% (w/v) formaldehyde-fixed S. aureus suspended in Buffer A was then added to each tube and incubated for 60 min at 4 "C. The suspension was then centrifuged for 1 min in an Eppendorf 5412 centrifuge and the activity remaining in the supernatant was deter- mined by the usual assay procedure.

Measurement of Relative Rates of Synthesis of Asparagine Syn- thetase"tsH1 cells were grown in either complete a-ME medium, a-ME medium containing 40 p~ leucine (%o of the normal leucine concentration), or a-ME medium lacking asparagine for 10 days to allow the enzyme to reach its steady state specific activity. AH-2 cells were grown in complete a-ME medium. Prior to labeling, cells were grown for several hours in these media containing a reduced amount of methionine (15 p ~ ) . The rate of protein synthesis and the specific

activity of asparagine synthetase in cells grown for 2 days under these conditions is unchanged and identical with that in cells grown in a- ME medium containing 100 p~ methionine. [35S]Methionine was then added to a final specific radioactivity of 500 mCi/mmol (7.5 pCi/ml). After 2 h of further incubation, cells were harvested by centrifugation and washed once with cold 10 mM potassium phosphate, 0.15 M NaC1, pH 7.4. Cell extracts were prepared as described above, and the protein concentration was determined. Asparagine synthetase was precipitated as described above, using twice the amount of antiserum calculated to be sufficient. The fixed bacteria, to which the asparagine synthetase-antibody complex is bound, were washed as described by Kaplan et al. (13). Bound protein was prepared for SDS-gel electro- phoresis by boiling for 5 min in 100 p1 of 60 mM Tris-HCl, 2% SDS, 10% glycerol, 5% mercaptoethanol, pH 7.5. The gels were processed as described above.

[35S]Methionine incorporation into total protein was determined by precipitating a small aliquot of cell extract in cold 10% trichloro- acetic acid. Samples were collected on glass fiber filters and washed 5 times with cold 10% trichloroacetic acid. The filters were dried, and the 35S activity was determined in a liquid scinitllation spectrometer. Relative rates of synthesis are presented as the ratio of ,%3 counts per min in asparagine synthetase and in total cytosolic protein.

Measurement of Rate of Degradation of Asparagine Synthetase- The degradation rate of asparagine synthetase was determined by pulse-chase analysis. Cells were labeled for 2 h with [3sS]methionine, as described above, pelleted, and resuspended in the appropriate a- ME medium containing 200 p~ methionine, to minimize isotope reutilization. Extracts were prepared from cells harvested at various times during the following 75-h period. 35S remaining in asparagine synthetase during this time was determined by immunoprecipitation and SDS-acrylamide gel electrophoresis as described above.

RESULTS

Enzyme Purification-A typical purification of asparagine synthetase from beef pancreata is summarized in Table I. This method of purification results in a 300- to 400-fold enrichment of asparagine synthetase from the high speed supernatant fraction used as starting material, with an overall yield of approximately 5%. Electrophoresis of the purified enzyme on SDS-acrylamide gels shows the presence of only a single band (Fig. 1).

When the purified enzyme is compared with proteins of known molecular weight by SDS-acrylamide gel electropho- resis, a subunit molecular weight of 57,000 is obtained. A native molecular weight of 108,000 is obtained by the method of Hedrick and Smith (14). These results suggest that the asparagine synthetase of beef pancreas is a dimer of identical subunits.

Characterization of Antibody Directed Against Aspara- gine Synthetase-Initial evidence for an anti-asparagine syn- thetase antibody was obtained using inactivation of bovine asparagine synthetase as an assay. Cross-reactivity of this antiserum with the enzyme from CHO cells was tested by adding increasing amounts of antiserum to fixed amounts of

TABLE I A representative purification scheme of bovine asparagine

svnthetase

mg unitso-l; x units/mg -fold

I. 100,000 X g superna- 11,414 4.1 358 (1)

II.O.l% cetyltrimethyl- 8,384 5.2 617 1.7

111. Heat denaturation: 55 2,120 5.2 2,448 6.8

IV. Hydroxyapatite 316 1.3 4,114 12 V. Cibacron blue 3GA 5.7 0.42 74,300 208

0.6 0.09 150,000 420

tant

ammonium bromide

"C, 8 min

VI. Glycerol gradient agarose

Synthesis of Asparagine Synthetase in CHO Cells

FIG. 1. SDS-acrylamide gel electrophoresis of bovine aspar- agine synthetase. 30 pg of Fraction VI protein were loaded onto a 6-cm, 10% gel and electrophoresed as described under "Experimental Procedures." The gel was stained with Coomassie brilliant blue R- 250.

cell extracts and removing the resulting complex with fixed S. aureus. These titration experiments (Fig. 2) show that all CHO cell asparagine synthetase activity is removed by the antibody. The specificity of the antiserum preparation is demonstrated by SDS-acrylamide gel electrophoresis analysis of the immunoprecipitate from ["S]methionine-labeled cells (Fig. 3). A single, sharp peak of radioactivity is observed. This peak can be eliminated by preincubation of the antiserum with an excess of purified bovine asparagine synthetase. No peaks of radioactivity are observed when an equivalent amount of preimmune serum is used. When the mobility of this band is compared with proteins of known molecular weights, a subunit molecular weight of 56,500 is determined.

Immunotitration of Asparagine Synthetase from Physio- logically and Genetically Derepressed Cells-We have pre- viously demonstrated that the specific activity of asparagine synthetase is increased in CHO cells grown under conditions leading to decreased aminoacylation of several tRNAs (1). We

10 20 30 40 50 60 VOLUME ANTISERUM (VI x 100)

240 be

73 13

VOLUME ANTISERUM ( P I x 100)

FIG. 2. Immunotitration of asparagine synthetase. 50 pl of cell extracts containing 500 pg of protein were mixed with the indi- cated volume of antiserum and incubated a t 4 "C for 30 min. Following precipitation with fixed S. aureus, asparagine synthetase activity remaining in the supernatant was assayed. In A, extracts were pre- pared from tsHl cells growing in complete a-ME medium (A), a-ME medium containing 40 PM leucine (0). or a-ME medium lacking asparagine (0). In B, extracts were prepared from tsHl (A), AH-2 (O), and AH-5 (0) cells grown in complete a-ME medium.

FIG. 3. SDS-acrylamide gel electrophoresis of immunoad- sorbed 36S-labeled CHO cell asparagine synthetase. tsHl cells were grown for 48 h in the presence of ["'S]methionine. Asparagine smthetase was immunoprecipitated and electrophoresed in a 7.5% gel. '% activity was determined following digestion of the gel slices with H202.

have also shown that cell lines AH-2 and AH-5, which are resistant to the toxic effects of P-aspartylhydroxamate, have constitutively elevated asparagine synthetase activity (2). Ex- tracts from tsH1 cells grown under different conditions leading to elevated enzyme activity (Fig. 2 A ) and extracts from tsH1, AH-2, and AH-5 cells (Fig. 2B) were titrated with rabbit anti- asparagine synthetase antiserum to determine whether this

7314 Synthesis of Asparagine Synthetase in CHO Cells

increase in enzyme specific activity results from an increase in catalytic efficiency or an increase in enzyme protein.

Fig. 2A shows that the amount of antiserum required for complete neutralization of asparagine synthetase activity in extracts from cells grown in complete medium, medium lack- ing asparagine, or medium containing %a of the normal leucine concentration is proportional to the specific activity of the enzyme. Similarly, Fig. 2B shaws that for the tsH1, AH-2, and AH-5 cell lines, the amount of antiserum required to neutralize the enzyme activity is also directly proportional to the specific activity of asparagine synthetase in these cell lines. Incubation of these extracts with control serum had no effect on enzyme activity. These results demonstrate that the increased enzy- matic activity results from an increase in the amount of an immunologically identical enzyme protein and is not due to an alteration in catalytic efficiency.

Turnover of Asparagine Synthetase-The increased aspar- agine synthetase content in the physiologically and genetically derepressed cells could result from changes in the rate of enzyme synthesis or degradation, or a change in both. To properly assess the relative contribution of rates of synthesis and degradation in determining the steady state concentration of asparagine synthetase, it is necessary to avoid conditions which may alter these rates. Since asparagine synthetase levels are known to be affected by the degree of aminoacyla- tion of a number of tRNAs (l) , labeling conditions which changed the degree of acylation of any tRNA were avoided. The concentration of methionine used during the labeling period allowed cells to grow normally for at least 48 h. In addition, asparagine synthetase specific activity remained un- changed throughout this period. It is, therefore, unlikely that the labeling conditions change the steady state rate of synthe- sis or degradation.

Turnover studies were performed on tsHl and AH2 cells growing in complete medium and tsH1 cells growing in me- dium lacking asparagine or medium containing a reduced amount of leucine. To measure the relative rate of enzyme synthesis, short term incorporation of [”5S]methionine into asparagine synthetase and total cytosolic proteins was deter- mined as described under “Experimental Procedures.” [35SJMethionine is incorporated into total cytosolic proteins and asparagine synthetase in a linear fashion for at least 8 h under these conditions. Two h after addition of label, cell extracts were prepared and incubated with rabbit antiserum. Following binding to fixed S. aureus and washing, the 35S- labeled enzyme was subjected to SDS-acrylamide gel electro- phoresis. The gels were sliced, and the :j5S activity was quan- titated as described under “Experimental Procedures.” The relative rate of 35S incorporation into asparagine synthetase is expressed as the ratio of the counts per min in the enzyme to the counts per min in total cytoplasmic proteins. Table I1 shows that the differences in asparagine synthetase specific activities are completely accounted for by increases in the relative rates of enzyme synthesis.

The rate of degradation of asparagine synthetase was in-

vestigated by pulse-decay analysis. Cells were labeled using the same medium as described for the experiments used to determine the rates of enzyme synthesis. Fig. 4 shows the decrease of radioactivity in asparagine synthetase due to degradation after transfer to a medium containing an excess of unlabeled methionine. Loss of radioactivity in asparagine synthetase followed f i s t order exponential kinetics. The cal- culated half-life of asparagine synthetase in tsHl and AH-2 cells growing in complete medium is 43 and 46 h, respectively. Very similar half-lives are found for asparagine synthetase in tsHl cells growing in medium lacking asparagine (49 h) or in medium containing limiting concentrations of leucine (52 h).

DISCUSSION

The asparagine synthetase activity of CHO cells is elevated when wild type cells are grown in a medium lacking asparagine or when mutants containing altered aminoacyl-tRNA synthe- tases are grown under conditions that lead to decreased ami- noacylation of their cognate tRNAs (1). This physiological derepression results in a 2- to 3-fold rise in the activity of asparagine synthetase. Mutants containing constitutively de- repressed levels of asparagine synthetase activity have been isolated on the basis of their resistance to the toxic effects of the asparagine analog B-aspartylhydroxamate (2). These ge- netically derepressed cells have about 5-fold more asparagine

0 20 40 60 80 TIME (h)

FIG. 4. Degradation of asparagine synthetase. The half-life of asparagine synthetase was determined in tsH1 (e) and AH-2 (A) cells grown in complete a-ME media and in tsH1 cells grown in a-ME medium lacking asparagine (0) or containing a limiting amount of leucine (0). CeUs were labeled with [35S]methionine for 2 h, divided into four equal fractions, and grown in the appropriate media. Cells were harvested at various times during the following 75 h, and the amount of 35S activity in asparagine synthetase was determined as described under “Experimental Procedures.”

TABLE I1 Relative rates of [35S]methionine incorporation into total protein and asparagine synthetase

tsH1 and AH-2 cells were grown and labeled in the indicated a-ME medium. Radioactivity in total protein and in asparagine synthetase was determined as described under “Experimental Procedures.”

Cell type Medium tase radioactivity Asparagine synthe- Total protein ra- co~~$:,$~~&. Specific activity of as- dioactivity paragine synthetase paragine synthetase

epm X IO-“ x 106 units/mg -

tsHl Complete 2,735 8.85 309 130 tsHl Lacking asparagine 3,934 6.45 610 278 tsHl Limiting leucine 4,166 5.41 770 316 AH-2 Comolete 10.778 7.05 1.529 568

Synthesis of Asparagine Synthetase in CHO Cells 7315

synthetase activity than wild type cells. No further derepres- sion can be achieved in these drug-resistant lines by limiting the extent to which tRNAhu is aminoacylated. In hybrids formed between wild type cells and P-aspartylhydroxamate- resistant mutants, elevated asparagine synthetase activity be- haves as a dominant marker (15).

We have investigated the basis for the physiological and genetic derepression of asparagine synthetase in CHO cells. Using antiserum specific for asparagine synthetase, we per- formed immunotitration experiments (Fig. 2) to quantitate the amount of asparagine synthetase antigen in wild type cells and in physiologically or genetically derepressed cells. The results demonstrate that the increased activity of asparagine synthetase arises from a corresponding increase in immuno- logically reactive protein.

Direct measurement of the relative rates of asparagine synthetase synthesis in physiologically derepressed tsHl cells and in the genetically derepressed cell line AH-2 shows that synthesis occurs at an increased rate (Table 11). Very similar half-lives were observed for asparagine synthetase under all conditions studied (Fig. 4). The increases in the rate of enzyme synthesis are sufficient to fully account for the elevated steady state asparagine synthetase content of these cells.

tRNAs have been implicated as having regulatory roles in a variety of cellular processes. In prokaryotes, these include the regulation of amino acid biosynthesis (16, 17), amino acid transport (18), and macromolecular synthesis (19). The mech- anisms by which tRNA participates in these processes have been studied in detail. There is now abundant evidence (20) that for several amino acid operons, regulation by tRNA occurs via attenuation of transcription. In this process, the rate of translation of a leader polypeptide on the nascent polycistronic mRNA determines whether further transcrip- tion will occur. The involvement of tRNA in macromolecular synthesis in prokaryotes, the stringent response, has been shown to be mediated through the compounds ppGpp and pppGpp (21). In both attenuation and the stringent response, the availability of mutations at critical steps has facilitated the elucidation of the regulatory mechanisms.

In the higher eukaryotes, tRNAs appear to play a role in regulating amino acid transport (22), protein degradation (23), and the levels of asparagine synthetase activity (1). The mechanisms through which tRNA exert their regulatory influ- ences in these cases are unknown. The evidence presented here shows that the increased levels of asparagine synthetase found in CHO cells grown under conditions leading to de- creased aminoacylation of tRNA are the result of an increased rate of enzyme synthesis. Whether this increased rate of synthesis reflects an alteration in the number of mRNA transcripts or an increased efficiency of transcript utilization is not yet known. A mechanism involving the coupling of transcription and translation, as in prokaryotic attenuation, seems unlikely in a eukaryotic cell, since transcription occurs within the nucleus, while translation occurs on cytoplasmic

ribosomes. The increased rate of asparagine synthetase synthesis in

strain AH-2 suggests that this mutant is altered in a regulatory element involved in controlling the rate of enzyme synthesis. The finding that this mutation behaves in a dominant manner in cell hybrids (15) suggests that a diffusible element may be involved. It is clearly too early to postulate any models re- garding the mechanism by which asparagine synthetase activ- ity is regulated. However, the ability to select for both struc- tural gene mutants (4) and mutants with defective regulatory mechanisms suggests that the further study of this system will provide insights into the mechanisms of the regulation of gene expression in mammalian cells.

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