.JOURNAL OF CELLULAR PHYSIOLOGY i42:194-200 ti99n)

Expression and Amplification of Cloned Rat Liver Tyrosine Aminotransferase in Nonhepatic Cells KULWANT K. KOHL1 AND ROBERT H. STELLWAGEN* Department of Hiochemistry, University of Southern California School of Medicine, [OF Angeles, California 90033

A full-length cDNA tor the rat liver enzyme tyrosine aminotransferase has been used to construct manimalian expression vectors by recombindnt D N A techniques. These vectors, which have employed either a simian viru5 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse 1 cells, hamster Wgl a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoidinduced hepatoma cells were achiewd in some CHO clones by this technique. The tyrosine aminotransferase produced a t these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.

Tyrosine aminotransferase (TAT; EC 2.6.1.5) is a liver-specific enzyme of tyrosine catabolism, which is subject to a variety of hormonal and other controls (Hargrove and Granner, 1985).Both the enzyme itself (Hershko and Tomkins, 1971) and its mRNA (Lee et al., 1970) are short-lived, permitting rapid responses to physiological signals. We have been investigating the structural and cellular requirements for rapid macromolecular turnover in the TAT system. As part of this study we have sought to express the enzyme a t high levels in nonhepatic cells. When we started this investigation, a full-length cDNA for the TAT was already available from the work of Pictet and colleagues (Grange et al., 1985). However, since the expression of endogenous TAT is known to be suppressed in nonhepatic cells by a trans-acting genetic locus (Killary and Fournier, 1984), it was unclear whether successful expression could be achieved from the cDNA. This paper describes two expression vectors we have constructed for TAT and documents their successful use in several mammalian nonliver cell lines. It also reports the expression of the structurally normal enzyme at extremely high levels in a system in which gene amplification was forced to occur. An abstract describing some of this work has recently appeared (Kohli and Stellwagen, 1988).

MATERIALS AND METHODS Vector constructions A full-length cDNA for TAT in a pUC-derived vector called pcTAT123 was kindly provided by Dr. R. Pictet (Grange et al., 1985). The vector pSVL was obtained 'G 1990 WILEY-LISS. INC.

from Pharmacia and contains the simian virus 40 (SV40) late promoter and VP1 intron followed by a multiple cloning site and a n SV40 poly-A site. A vector called pRSV was provided by Dr. C. Gorman of Genentech. This vector has a multiple cloning site located between the Rous sarcoma virus (RSV) long terminal repeat (LTR) and a poly-A site. It also contains a dihydrofolate reductase (DHFR) gene linked to a n SV40 promoter. Both the vectors with mammalian promoters contain the SV40 origin of replication, and all three vectors contain pBR322 sequences, which enable them to replicate in Escherichia coli and which confer resistance t o ampicillin. A 1.6 kb fragment containing the complete coding sequence for TAT was removed from pcTAT123 by cleaving with the restriction enzymes Xmn 1and Sac 1 (Fig. 1).This Xmn M a c 1DNA fragment was purified by electrophoresis in low-melting-temperature agarose as described by Maniatis et al. (1982) and ligated into the Sma liSac 1 site of pSVL to give the expression vector we call pSVL-TAT1. The new construction was cloned in E. coli HB 101 in the presence of ampicillin, and the structure of the cloned plasmid was verified by restriction mapping (Maniatis et al., 1982). A second mammalian expression vector, to be called pRSVTAT1, was constructed by removing the TAT coding region from pSVL-TAT1 with restriction enzymes Xho 1 and Sac 1 and inserting it, after gel purification, into

Received July 24, 1989; accepted September 21, 1989

*To whom reprint requestsicorrespondence should be addressed.

EXPRESSION OF CLONED TYROSTNE AMINOTRANSFERASE

the Xho 1/Sma 1site of pRSV. In making this construction the Sac 1 cut in pSVL-TAT1 was made first and converted to a blunt end with the use of T4 DNA polymerase (Maniatis et al., 1982) before the Xho 1 cleavage was made. The resulting construct was cloned in E . coli and its structure confirmed by restriction mapping. Plasmids were purified by CsCl centrifugation (Maniatis et al., 1982) or by a column procedure (Zervos et al., 1988). See Figure 1for diagrams of the TAT cDNA and the two expression vectors just described.

Growth and transfection of mammalian cells Monkey COS cells were obtained from Dr. James Ou; mouse L cells (thymidine kinase-deficient) were provided by Dr. P. Jones; and hamster Wgla lung fibroblasts (Roscoe et al., 1973) were from Dr. A. Lee. Drs. R. Moran and S. Taylor supplied the Chinese hamster ovary (CHO) cells used in these experiments, including both the CHO-K1 and CHO-DG44 lines. The latter cell line was derived by Dr. L. Chasin and colleagues and completely lacks DHFR genes (Urlaub et al., 1983). All the above cells were grown in monolayers in a humidified 37°C incubator in the presence of 5% C02/95%air. The CHO lines were grown in a-minimum essential medium supplemented with nucleosides and 10%’(viv) fetal bovine serum. For growth of CHO-DG44 cells under selective conditions, this medium was modified by omitting the nucleosides and using dialyzed serum. The other cell lines mentioned above and also HTC hepatoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g glucoseiliter and 10% (viv) heat-inactivated calf serum. In most experiments, penicillin and streptomycin were added t o the culture media. Cells were removed from the plastic substratum by treating with 0.25% (wiv) trypsin in the presence of 1 mM EDTA. Media, antibiotics, and trypsin were obtained from Gibco Laboratories. Sera were from Gibco Laboratories or HyClone Laboratories. Transfections were carried out by calcium phosphate precipitation methods using 3-20 g of plasmid DNA per 10 cm culture dish of about 10 cells (Davis et al., 1986; Chen and Okayama, 1987). Transient expression was followed over a period of 3 days. To select for stably transfected clones, the plasmid pNE03 (obtained from Dr. A. Lee), which contains a neomycin resistance gene, was generally included, and selections were carried out in the presence of 0.4 mg G418 (100% potency)/ ml (Chen and Okayama, 1987). Surviving clones were then isolated and screened for TAT expression. In the case of the CHO-DG44 line, which lacks DHFR, no cotransfecting plasmid was used, and the selection simply involved growth in the absence of nucleosides. Amplification of the pRSV-TAT1 vector in CHO-DG44 transfectants was done by adding gradually increasing concentrations of methotrexate in steps of 20,100,500, and 2,000 nM. Individual colonies were isolated by trypsinizing within a sterile plastic chimney placed around the colony on the surface of the culture dish.

I-

Measurement and characterization of expression Cells for enzyme activity assays were harvested by trypsinization as described above, washed in 10 mM potassium phosphate buffer at pH 7.6 containing 0.14 M NaC1, and stored frozen. For TAT activity measure-

195

ments, frozen cell pellets were disrupted by thawing in cold TAT lysing buffer: 50 mM potassium phosphate, pH 7.6, containing 10 mM 2-oxoglutarate and 0.2 mM pyridoxal phosphate. The lysates were centrifuged for 10 min a t 1,7OOg, and the supernatants were used for TAT activity and protein measurements. TAT enzymatic activity was determined by the method of Diamondstone (1966) a s described previously (Kohli et al., 1988).One unit of activity corresponds to production of 1 Fmole of product per minute in the assay at 37°C. Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Characterization of TAT by reaction with a polyclonal rabbit antiserum was carried out essentially as described before (Stellwagen et al., 1977). TAT heat stability, kinetic constants, and subunit size upon sodium dodecyl sulfate (SDSI-polyacrylamide gel electrophoresis were also determined by previously published methods (Stellwagen and Tomkins, 1971; Stellwagen et al., 1977; Kohli et al., 1988). Extracts for measurement of DHFR activity were made by lysing frozen cell pellets in cold 10 mM Tris HC1 buffer, pH 7.4, containing 0.15 M KC1 and 3 mM 2-mercaptoethanol. When methotrexate was present, the extracts were dialyzed overnight vs. 1,000 volumes of the lysing buffer before assaying (Urlaub et al., 1983). DHFR assays were performed essentially a s described by Frearson et al. (1966) except that the pH used was 7.6. Protein was measured on the uncentrifuged lysates by the same method used above for TATspecific activity determinations, and a unit of activity has been defined in the same way as for TAT above.

RESULTS Expression of TAT in nonhepatic cells A map of the TAT cDNA based on its published sequence (Grange et al., 1985) is shown in Figure 1along with the structures of the two mammalian expression vectors made for this study. Both expression vectors use the full protein coding region of the TAT cDNA. Each includes the sequence from the Xmn 1site to the first of two Sac 1 sites in the cDNA. Use of this 1.6 kb fragment excludes about 75 nucleotides of untranslated sequence from the 5’ end and about 700 nucleotides of untranslated sequence (including the poly-A sites) from the 3 ’ end of the full-length cDNA. The vector pSVL-TAT1 utilizes the SV40 late promoter to direct transcription of the TAT coding sequence whereas pRSV-TAT1 employs the RSV LTR as the promoter. Each vector supplies its own poly-A site following the TAT coding sequence. The pSVL-TAT1 vector contains a n SV40 intron within the TAT transcription unit, whereas the pRSV-TAT1 vector has no introns. The pRSV-TAT1 vector includes the DHFR gene in a separate transcription unit to permit forced amplification in the presence of methotrexate (Schimke, 1988). These expression vectors were introduced into mammalian cells in culture by calcium phosphate transfection methods. Since we were interested in production of functional enzyme, expression was monitored initially by TAT activity measurements. Figure 2 depicts a n experiment in which pSVL-TAT1 expression was monitored for 3 days following transfection into monkey COS cells. TAT activity increased to a maximum in

196

KOIILI AND STELLWAGEN ~ A A T AA A ~

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KILOBASES TAT.

Promoter

Late Promoter and VPI lntron

DHFg

PSVL - TAT 1 6.4 kb

pRSV - TAT 1 6.3 kb

Fig. 1. Structure of the cDNA and expression vectors for TAT. The upper part of the figure shows the cDNA for TAT and indicates key restriction sites, translation start (ATG) and stop ITAA) sites, and

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Fig. 2. Time course of TAT expression after transfection of vectors into monkey COS cells. The control vector pSVL (0) or the TAT expression vector pSVL-TAT1 ).( was introduced into COS cells by calcium phosphate-mediated transfection. T A T activity was measured in lysates of cells harvested a t the times shown following transfection.

about 2 days. The level achieved was substantially above the background seen in the case of the control pSVL vector. It should be noted that the low background activity detected in enzymatic assays of these and other nonhepatic cells is due to other transaminases that have some ability to use tyrosine as a sub-

putative poly-A sites (AATAAA) based on Grange et al. (1985). The lower part of the figure summarizes the structures of the two expression vectors made for this study.

strate (Spencer and Gelehrter, 1974; Hargrove and Mackin, 1984). We have found that this background activity is not inhibitable by antibodies against TAT and is more sensitive to heat than authentic TAT (shown later). Table 1 summarizes the results of several transient expression studies in different cell lines. The monkey COS cells, which showed easily detectable levels of TAT activity, have the advantage of allowing replication of the vectors to increase expression. Mouse L cells also exhibited TAT expression levels severalfold above the nonspecific background in these assays, perhaps due to their high efficiency of transfection (Chen and Okayama, 1987). The levels of TAT activity were lower during transient expression with the other cells tested. For example, CHO-K1 cells gave a value only about twofold above background (Table 1).Use of the control vectors pSVL or pRSV, which do not contain TAT coding sequences, produced no increase in activity above the nonspecific background levels (Fig. 1, Table 1).In addition, a vector in which the TAT coding sequence was reversed yielded no significant expression of activity (not shown). To select for stable transformants in most of the cell lines used, cotransfections were carried out with a neomycin-resistance plasmid. The individual colonies obtained from cloning in the presence of the antibiotic G418 were tested for expression of TAT activity. About 2 5 5 0 % of the clones were found t o be positive for TAT,

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EXPRESSION OF CLONED TYROSINE AMINOTRANSFERASE

TABLE 2. Exaression of TAT in isolated clones

TABLE 1. Transient expression of TAT activity TAT specific activity'

TAT specific activity (rnUimg) Total Minus control 1.5 2.9 1.4 3.2 1.7 5.8 4.3 0.8 0.8 1.6 6.0 6.8 0.7 25.0 24.3 1.2 5.3 4.1 14.0 12.8

(rnuimgl

Cell line Monkey COS Monkey COS

Mouse L CHO-K1

Vector None pSVL pSVL-TAT1 pRSV pRSV-TAT1 pSVL-TAT1 None pSVL-TAT1 None pRSV pRSV-TAT1

Total 0.8 0.9 6.2 04 2.7 2.5 1.9 7.2 0.5 0.6 1.2

Minus control -

-

Cell line Mouse L

5.3

~RSV-TATI

-

2.3 2.1

Hamster W "e l a

-

5.3

CII 0- K 1

-

CHO-DG44

0.6

'Measured 2 days after transfection

and their levels of expression varied substantially. The levels observed in several TAT-positive clones obtained with different vectors and cell lines are shown in Table 2 . In the case of the pRSV-TAT1 plasmid and the CHODG44 cell line, the selection method utilized the DHFR gene in the plasmid itself rather than a cotransfected neomycin-resistance plasmid. In this case all the colonies obtained by growth under selective conditions (absence of nucleosides) showed TAT activity levels above the nonspecific control. The elevated TAT activity produced in transfected cells was inhibitable by specific antibodies against TAT, as shown in a later section for one case.

Amplification of TAT expression The TAT-specific activities observed in the various clones isolated in the experiments described above ranged from 1 to 24 mUimg above the controls (Table 2 ) . In our previous work with the HTC rat hepatoma cell line, we generally found uninduced TAT levels to be in the range of 5-20 mUimg (Stellwagen and Tomkins, 1971; Stellwagen et al., 1977). Thus the transfected clones from nonhepatic cells express levels of TAT t h a t are comparable t o the levels observed in uninduced hepatoma cells. In our experience, when the HTC cell line is treated with a n optimal concentration of a glucocorticoid inducer, TAT levels typically increase to around 100 mUimg. None of the transfected cells that we have tested for responsiveness to glucocorticoids show any effect of the steroid inducer on TAT. This is not surprising in that the 5'-flanking region of the TAT gene, which contains the glucocorticoid-response elements (Jantzen et al., 1987), is not present in our vectors. To determine whether TAT can be expressed to high levels in nonhepatic cells, we used the powerful methotrexate selection method that has been applied successfully to certain other transfected genes (Ringold et al., 1981; Kaufman and Sharp, 1982; Haynes and Weissman, 1983). Some of the clones of CHO-DG44 cells that expressed TAT from the pRSV-TAT1 vector were treated with a series of increasing concentrations of methotrexate over a period of several weeks. Certain of the clones showed a greater increase in TAT than others. Table 3 shows results from one such clone. TAT and DHFR were both monitored and found to increase in parallel during the selection. A more extensive selection in which TAT alone was

Vector pSVL pSVL-TAT1 pRSV-TAT1 None

pSVL-TAT1 pSVL-TAT1 pRSV ~RSV-TATI DRSV pRSV-TAT1 pRSV-TAT1

TABLE 3. Comparison of TAT and DHFR levels during amplification' Methotrexate concentration (nM) 0 100 500

TAT mUimtr 10.9 274 1133

Normalized 1

25 104

rnUimg 0.75 20.4 72.9

DHFR Normalized 1

27 97

'Enzyme levels were measured after about 3 weeks of sequential treatment with each methotrexate concentration.

monitored is summarized in Table 4.In this case TAT levels in the population increased to over 3,800 mUi mg. Some of the cells from the next to the last stage in the selection were cloned in the presence of 500 nM methotrexate, and five clones were selected a t random for growth to mass populations in the absence of methotrexate. A wide range of specific activities from 80 to 3,700 mUimg was found. This diversity probably reflects differences in starting levels in the clones as well a s differential loss of the amplified copies during subsequent growth to mass populations in the absence of methotrexate. The highest expressor among these clones was fairly stable and has maintained a level of TAT between 3,700 and 5,000 mUimg for about 1 month in culture without methotrexate. The TAT protein levels in the high expressing clones should be nearly 1%of soluble protein. This estimate is based on the observation t h a t highly purified TAT has a specific activity of approximately 600,000 mU!mg (Hargrove and Granner, 1980) or only about 120 times the highest value reported above. In addition, a high level of DHFR is being produced in these cells. In spite of these facts, the cells appear healthy and grow well. The growth rate is only slightly reduced in comparison to the original CHO-DG44 cells. However, there may be some long-term selective disadvantage to high TAT expression as suggested by results observed with one mass population. When this particular population was being grown in the presence of increasing methotrexate concentrations, the TAT level first increased and then declined dramatically while the DHFR level continued to increase. A possible interpretation of this observation is that a DNA rearrangement might have separated the two genes in some of the cells and that those cells, which were no longer forced to amplify both genes, then outgrew the others.

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KOHL1 AND STELLWAGEN

TART,E 4. Amplification of TAT expression Methotrexate concentration (nM)

TAT specific activity’ (mUimg)

n

5

20 100 500 2,000

18 371 2,300 3.800

‘Measured after about 3 weeks of sequential treatment with each concentration o f mcthotrexate.

n

Characterization of the TAT produced in nonhepatic cells We chose to examine the TAT produced at high levels in CHO-DG44 cells to determine whether i t had the expected properties. First, its reaction with a specific antiserum was studied. As is illustrated in Figure 3, there is no significant difference between the TAT from CHO-DG44 cells and that from HTC cells in terms of sensitivity to inactivation by this antiserum. Figure 3 also shows that the background enzymatic activity in untransfected CHO-DG44 cells does not react with the antiserum against TAT. In addition, we have found that radiolabeled TAT produced from the amplified gene in CHO-DG44 cells has an apparent subunit molecular weight of 53,000 when analyzed by SDS-polyacrylamide gel electrophoresis after immunoprecipitation (not shown). This is identical within experimental error to the value observed for native TAT from r a t liver or HTC cells under the same electrophoretic conditions using the same molecular weight standards (Stellwagen and Kohli, 1981; Kohli et al., 1988). We determined the Km for tyrosine of the cloned TAT to be 1.4 mM, which is also the same as that of the native enzyme (Kohli et al., 1988). Finally, Figure 4 shows that the cloned enzyme has the same degree of resistance to inactivation by heating in the presence of pyridoxal phosphate and 2-oxoglutarate that is characteristic of the enzyme from hepatoma cells (Stellwagen and Tomkins, 1971). DISCUSSION This study has shown that functional liver TAT can be expressed from the coding portion of its cDNA in a variety of nonhepatic mammalian cell lines. Expression has been observed both transiently during the first 3 days after transfection and in stable clones, which presumably contain integrated copies of the artificial TAT gene. Thus the suppression mechanism that normally keeps the endogenous TAT gene from being expressed in nonhepatic cells (Schneider and Weiss, 1971) fails to prevent expression from the constructs used in our study. This finding suggests that the portion of the cDNA used in our experiments is not a target for the putative trans-acting factor (Killary and Fournier, 1984) or other mechanism (Becker et al., 1987) t h a t suppresses endogenous TAT gene expression in nonliver cells. TAT expressed from the cDNA is also unresponsive to induction by glucocorticoids. These results are consistent with the finding of glucocorticoid-response elements (Jantzen et al., 1987) and tissue-specific structural differences (Becker et al.,

1

0; 1

10

1

ANTlBODY

100

ADDED [ n \ / m C )

Fig. 3. Antibody titrations of TAT activity. The enzyme extracts were obtained from CHO-DG44 cells in which the pRSV-TAT1 vector had been amplified (01, from control untreated CHO-DG44 cells (m’l, and from HTC hepatoma cells in which TAT had been induced with hydrocortisone (A). Incubations with a polyclonal rabbit antiserum against rat liver TAT (Kohli et al., 1977) were carried out for 2 hr a t 37°C in TAT lysing buffer containing 5 mg bovine serum albuminiml. The TAT activity remaining was measured and is expressed as a percentage of the corresponding activity of a control incubation without antibodies. The amount of undiluted antibody solution present has heen expressed relative to the mU of TAT activity in each incubation.

L 5 10 15 20

‘OO

TIME AT 75°C (rnin) Fig. 4. Heat inactivation of TAT activity. Extracts were obtained from CHO-DG44 cells in which the pRSV-TAT1 vector had been amplified (*), from HTC hepatoma cells in which TAT had heen induced with hydrocortisone (A),and from untreated mouse L cells (m). Samples were heated a t 75°C for the indicated times in TAT lysing buffer containing 5 mg bovine serum albuminiml. The TAT activity remaining is expressed as a percentage of that present in unheated controls.

1987) in the untranscribed genomic 5‘-flanking region of the endogenous TAT gene. This investigation also demonstrates that the TAT gene in the pRSV-TAT1 vector can be extensively amplified along with the DHFR gene in CHO-DG44 cells by selection in methotrexate. This type of selection has been applied successfully by others to various genes (Ringold et al., 1981; Kaufman and Sharp, 1982; Haynes and Weissman, 1983; Schimke, 1988). In the case of TAT, we have obtained increases in expression as much as 1000-fold above the levels before amplifi-

EXPRESSION OF CLONED TYROSINE AMINOTRANSFERASE

cation, and even higher levels could probably he reached by further increasing the methotrexate concentration. The highest TAT levels achieved so far (about 5,000 mUimg) are approximately 50-fold higher than we typically find in HTC hepatoma cells following induction with a glucocorticoid (Stellwagen and Tomkins, 1971; Stellwagen et al., 1977). Based on the reported specific activity of purified TAT (Hargrove and Granner, 1980), these levels correspond to about 1% of soluble protein in CHO-DG44 cells vs. about 0.02% in glucocorticoid-induced HTC cells. Since the CHO-DG44 cells remain viable and continue to grow well with highly elevated TAT levels, the enzyme appears to have no serious deleterious effects on metabolism. Presumably, these cells lack the full pathway for tyrosine catabolism, which is characteristic of the liver, and thus cannot degrade tyrosine beyond the p-hydroxyphenyl pyruvate produced by TAT. Although metabolite levels have not been measured, it is likely that having TAT without the rest of the pathway enables the cells to achieve a n equilibrium between tyrosine and p-hydroxyphenyl pyruvate, allowing normal cell growth. The highly amplified TAT produced in CHO-DG44 cells appears to be indistinguishable from the normal rat liver enzyme by all the tests we have applied so far. Thus the expression vector appears t o produce a normal translation product. We have not tested rigorously for possible differences in the posttranslational processing events known t o occur with TAT, namely, Nterminal acetylation (Hargrove and Granner, 1981) or serine phosphorylation (Lee and Nikol, 1974). The former modification is known to take place with many eukaryotic proteins (Brown and Roberts, 1976). Because N-terminal acetylation is catalyzed cotranslationally by a ribosome-associated enzyme present in most cells, it is not usually found to be sensitive to cell type or to the final level of protein synthesized (Arfin and Bradshaw, 1988). Phosphorylation, on the other hand, could be more sensitive to such variables. In the case of TAT, only a fraction of the r a t liver enzyme appears to be phosphorylated (Hargrove and Granner, 1981). In addition, i t is unclear which protein kinase phosphorylates TAT, and there is no proved function for the modification (Stellwagen and Kohli, 1981; Spielholz et al., 1987). It will be important to clarify the phosphorylation state of TAT in future studies of TAT degradation in nonhepatic cells. The ability to express TAT activity in many cells opens up the possibility of studying the degradation of the protein and its mRNA in nonhepatic environments. This approach should reveal whether there are tissuespecific components in the degradation pathway. It is also possible to modify the structure of TAT protein and mRNA by site-directed mutatagenesis of the cDNA, which will permit systematic exploration of the effects of defined structural changes on both activity and degradation. In addition, the extremely high levels of TAT that can be achieved in some cells through gene amplification should be useful for studies of degradation. Investigations are in progress along these lines.

ACKNOWLEDGMENTS The authors thank Dr. Raymond Pictet for the plasmid containing the cDNA for TAT, Dr. Cornelia Gor-

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man for the pRSV plasmid, and Dr. Lawrence A. Chasin for permission to use the CHO-DG44 cell line. We are also grateful t o the following colleagues a t USC for providing cell lines, plasmids, and advice: Drs. Amy S. Lee, Peter Jones, Raymond D. Mosteller, James Ou, Richard Moran, and Shirley Taylor. This work was supported in part by grants from the National Science Foundation (DMB 84-17736), the National Institutes of Health (BRSG Grant 2 SO7 RR05356-27 to USC), and the USC Faculty Research and Innovation Fund.

LITERATURE CITED Arfin, S.M., and Bradshaw, R.A. (19881 Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry, 21 :79797984. Becker, P.B., Ruppert, S., and Schutz, G. (1987) Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors. Cell, 51t435-443. Brown, J.L., and Roberts, W.K. (1976) Evidence that approximately eighty per cent of the soluble proteins from Ehrlich ascites cells are N"-acetylated. J. Biol. Chem., 251t1009-1014. Chen, C., and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol., 7t2745-2752. Davis, L.G., Ilihner, M.D., and Rattey, J.F. (1986) Basic Methods in Molecular Biology. Elsevier Science Publishing Co., New York. Diamondstonc, T.I. (1966) Assay of tyrosine transaminase activity by conversion of p-hydroxyphenylpyruvate to p-hydroxybenzaldehyde. Anal. Biochem., 16t395-401. Frearson, P.M., Kit, S., and Dubbs, D.R. (1966) Induction of dihydrofolate reductase activity by SV40 and polyoma virus. Cancer Res., 26rl653-1660. Grange, T., Guenet, C . , Dietrich, J.B., Chasserot, S., Fromont, M., Befort, N., Jami, J., Beck, G:, and Pictet, R. (1985) Complete complementary DNA of rat tyrosine aminotransferase messenger RNA: Deduction of the primary sequence of the enzyme. J . Mol. Biol., 184t347-350. Harmove. J.L., and Granner. D.K. (1980) Purification of the native form of tyrosine aminotransferase from rat liver. Anal. Biochem., 104t231-235. Hargrovc, J.L., and Granner, D.K. i1981) Physical properties, limited proteolysis and acetylation of tyrosine aminotransferase from rat liver. J. Biol. Chem., 256r8012-8017. Hargrove, J.L., and Granner, D.K. (1985) Biosynthesis and intracellular processing of tyrosine aminotranxferase. In: Transaminases. P. Christen and D.E. Metzler, eds. John Wiley and Sons, New York, pp. 511-532. Hargrove, J.L., and Mackin, R.B. (1984) Organ specificity of glucocorticoid-sensitive tyrosine aminotransferase: Separation from aspartate aminotransferase isoenzymes. J. Riol, Chem., 2591386-393, Haynes, J., and Weissman, C. (1983) Constitutive, long term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene. Nuclcic Acids Res., flt687-706. Hershko, A,, and Tomkins, G.M. (1971) Studies on the degradation of tyrosine aminotransferase in hepatoma cells in culture: Inf hence of the composition of the medium and adenosine triphosphate dependence. J . Biol. Chem., 246t710-714. Jantzen, H.M., Strahle, U., Gloss, B., Stewart, F., Schmid, W., Boshart, M., Miksicek, K., and Schutz, G. (1987) Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransfcrase gene. Cell, 49:29-38. Kaufman, R.J., and Sharp, P.A. (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase cDNA gene. J. Mol. Biol., 159.501-621. Killary, A.M., and Fournier, R.E.K. (1984) A genetic analysis of extinction: Trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells. Cell, 38t523-534. Kohli, K.K., Obuch, A,, and Stellwagen, R.H. (1988) Protection of tyrosine aminotransferase against proteolytic digestion by nucleot.ide derivatives. Biochim. Biophys. Acta, 956t77-84. Kohli, K.K.. and Stellwagen, R.H. (1988)Expression and degradation of tyrosine aminotransferase in non-hepatic cells. J. Cell Hiol., 107: 613a. Lee, K.L., and Nikol, J.M. (1974) Phosphorylation of tyrosine aminotransferase in vivo. J. Biol. Chem., 249t6024-6026. Lee, K.L.. Reel, J.R., and Kenney, F.T. (1970) Regulation of tyrosine alpha-ketoglutarate transaminase in rat liver: IX. Studies of the

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