induction and reversion of asparagine auxotrophs in cho-k1 and v79 cells

Somatic Cell and Molecular Genetics, Vol. 12, No. 5, 1986, pp. 459-466

Induction and Reversion of Asparagine Auxotrophs in CHO-K1 and V79 Cells

Morgan Harris

Department of Zoology, University of California, Berkeley, California 94720

Received 1 April 1986--Final 2 June 1986

A b s t r a c t i T w o asparagine-dependent clones (2002-103 and 2002-109) were obtained from CHO-K1 cells by treatment with EMS followed by a BrdU-black light selection procedure; an additional ASP- clone (2293-343) was isolated similarly from V79-56 cells. All three clones show a low rate of spontaneous reversion which is increased somewhat by exposure to EMS. Two of the variant clones (2002-103 and 2002-109) are converted in high frequency to asparagine independence by treatment with 5-azacytidine, while 2293-343 cells show no significant induction after similar exposure. All three ASP- clones were found by hybrid analyses to belong to the same complementation group. Treatment of hybrids constructed between 2002-103 or 2002-109 x 2293-343 with 5-azacytidine resulted in high-frequency induction of asparagine independence. Thus, the potential for response to 5-azacytidine in such hybrids by reversion to asparagine independence is dominant or codominant, with no suppressor effect in hybrids from the nonresponsive parent line.

INTRODUCTION

Auxotrophs as variants with specific nutritional requirements have been useful in the definition and analysis of intermediary metabolism in animal cells (1-3). Variants of this type can be shown to originate by gene mutation in microbial systems, and the assumption has been that auxotrophs also arise by genetic change in mammalian cells. But direct evidence is lacking, e.g., for the many purine or pyrimidine auxotrophs that have been isolated from CHO cells (3). And at least one well-known auxotrophic marker in this lineage--proline dependence--appears now to result from changes in DNA methylation rather than alteration in coding sequence (4).

It is possible, however, that proline dependence in CHO cells represents a special

case, while most other auxotrophs may be produced in mammalian cells by gene mutation. The careful experiments of Patterson (3) and by Worton and Grant (5) provide evidence which points in this direction. Patterson found treatment with the demethylating agent 5-azacytidine (5-aza-CR) did not revert URD-A CHO populations to uridine independence, although a separate selection with the same cells revealed that they had been converted in high frequency to proline independence. Worton and Grant similarly observed reversion at the pro- locus in CHO cells after exposure to 5-aza-CR, but failed by this means to alter auxotrophy for a number of variants in the glycine and adenosine biosyn- thetic pathways. These results make clear that direct conversion of auxotrophs to protro- trophs by 5-aza-CR is an unusual process, but it does not follow that genetic changes must be

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0740-7750/86/0900-045955.00/0 �9 1986 Plenum Publishing Corporation

460 Harris

assumed for nonresponders to 5-aza-CR on this basis alone. The effects of 5-aza-CR in different cell systems are capricious, and it may be that DNA demethylation is a neces- sary but not sufficient condition for the reexpression of many quiescent wild-type genes (6).

Studies with specific gene probes are needed to characterize auxotrophy and proto- trophy in molecular terms. One system that has provided useful information is based on functional auxotrophy for thymidine, which arises when normal animal cells are grown in HAT medium. Chinese hamster cells lacking thymidine kinase ( tk-) do not grow in HAT but can be converted in high frequency to the HAT + state by treatment with 5-aza-CR (7). Thymidine kinase activity reappears in HAT + revertants, and recent experiments with DNA probes from V79 cells indicate that reexpression may be linked to demethylation at specific sites near the 5' end of the tk gene (8).

In the present paper we describe asparagine-dependent lines with variant features that vary in response to 5-aza-CR, and which may therefore be useful in determining the primacy of DNA methylation changes for transitions to the prototrophic state. Variants auxotrophic for asparagine and deficient in asparagine synthetase were obtained earlier by Goldfarb and coworkers (9) from DON cells, and by Waye and Stanners (10) with CHO cells. Asparagine-independent revertants arose at low rates in both systems, and these infrequent occurrences were attributed to gene mutation. Jensen rat sarcoma cells are also asparagine-dependent, and the basis for auxotrophy in this system seems to be epigenetic, since asparagine prototrophs arise in high frequency after treatment with 5-aza- CR (11). This disparity in the preceding studies is bridged by data from our current experiments. We show here that two auxotrophs obtained from CHO-K1 cells show low spontaneous reversion after exposure to EMS, but high reversion frequencies following treat-

ment with 5-aza-CR. On the other hand, an asparagine auxotroph obtained by the same procedure from V79 cells is unresponsive to 5-aza-CR, with little or no increase in reversion frequency above spontaneous background levels. Such variants, when used in combina- tion with DNA probes for asparagine synthetase (12), could provide useful models in assessing the causal relationships between DNA methylation changes and gene expres- sion.

MATERIALS AND METHODS

Cells and Cell Culture. CHO-K1 cells used for this study were obtained from the American Type Culture Collection, Rockville, Maryland. V79-56 (13) is a subline of V79 cells received originally as a gift from Dr. Ernest Chu. Stock cells of each line were maintained as monolayers in 10% fetal calf serum plus 90% alpha modification of Eagle's minimum essential medium (10FCS-~- MEM). Ribosides and deoxyribosides were omitted, and the concentrations of glucose, bicarbonate, and glutamine adjusted to 4.5 mg/ml, 3.7 mg/ml, and 600 #g/ml, respec- tively. For selection of asparagine-independent (asp + ) variants, cells were grown in 10% dialyzed fetal calf serum + 90% Dulbecco's modification of Eagle's medium fortified with L-proline at 50 ug/ml (ASP- medium). The serum was dialyzed with agitation in 50-ml aliquots for three days at 4~ with seven changes of Hanks' saline, sterilized by filtra- tion, and frozen until needed. The ASP- medium was completed by supplementation with pyruvate, L-alanine, L-aspartic acid, and L-glutamic acid to the same concentrations as in a-MEM. Petri dish cultures were maintained in a humidified CO2 incubator with fluid changes after five days, and once or twice a week subsequently as needed. When colonies were well-formed, experiments were terminated by staining the cultures for 30 rain in a saturated solution of crystal violet in

Asparagine Auxotrophs in CHO-K1 and V-79 Cells 461

0.85% NaC1, after which the dishes were rinsed in tap water and air dried.

Isolation o f Asparagine Auxo- trophs. Asparagine-dependent variants were isolated from CHO-K1 and V79-56 cells by a BrdU-black light selection procedure, based on the methods of Puck and Kao (1, 2) as modified by Chu et al. (14). To increase the incidence of auxotrophic variants, stock populations were first treated with ethyl methane sulfonate (EMS) at 300 #g/ml in 10FCS- a -MEM for 18 h and were allowed to recover for five days in normal medium. The survivors were inoculated into Petri dishes and incubated for 16 h in ASP medium to deplete intracellular pools of asparagine, after which BrdU was added to 10 #g/ml for a further 24-h period. The cells were then rinsed with phosphate-buffered saline (PBS) and exposed for 30-60 min to a black light source (Blak- Ray, Ultraviolet Products Co.) mounted 5 cm above the culture dishes. Subsequently, the cultures were charged to 10FCS-a-MEM for the growth of survivor colonies, which were either recycled through BrdU-black light or screened directly for asparagine dependence. Further details of these procedures are given in later sections.

Isolation of Hybrids between Aspara- gine Auxotrophs. To facilitate selection between asparagine-dependent lines, markers for thioguanine and ouabain resistance were added to one partner. The cells were first treated with EMS at 300 #g/ml and resistant subclones then selected serially in appropriate concentrations of these agents. Fusions were carried out in 15-ml centrifuge tubes containing a pellet with 2.0 x 10 6 cells of each auxotrophic line. To this was added 1.0 ml PEG (ATCC, mol wt 1300-1600) in a 1:1 solution with a-MEM. After 1 min, 5 ml o~-MEM were added rapidly, the suspension centrifuged, and the supernatant replaced with 4.0 ml 10FCS-o~-MEM. After incubation for 30 min at 37~ the cells were plated out in HAT + 1000 #g/ml ouabain for selection of

hybrids. A similar procedure was used for isolation of hybrids between asparagine auxotrophs and wild-type cells carrying thioguanine and ouabain resistance markers.

Preparation o f Cell Extracts. Log- phase cells were inoculated into roller bottles at 5.0 x 10 6 cells in ASP- medium (for asp + revertants and wild-type cells) or 10FCS- a -MEM (for asp- auxotrophs) and incubated for three days to yield 100-200 x 10 6 total ceils. After trypsinization, the cells were rinsed twice at 0~ with PBS and the final pellet resuspended at 100 • 10 6 cell/ml in 10 mM Tris HC1, pH 7.4, containing 0.5 mM /3-mercaptoethanol. This mixture was dis- rupted by freezing in a CO2-methanol bath and thawing twice, after which the suspension was centrifuged at 0~ and 16,000g for 20 min. Aliquots of the final supernatant were frozen and used within a few days for assays. Protein determinations were made by the method of Lowry et al (15).

Assays for Asparagine Synthetase. As- paragine synthetase (AS) activity in cell extracts was determined by the procedure of Gantt et al. (16) with modifications as described below. In this procedure, labeled aspartic acid is converted to asparagine which is then separated from other compounds on an ion-exchange column. All AS assays were per formed with L-[U-14C]aspartic acid (Amersham, 224 mCi/mmol), using 0.5 #Ci per sample. A standard assay mixture of 200 #1 contained 150 mM Tris HC1, pH 7.5, 8 mM MgC12, 8 mM ATP, 30 mM L-glutamine, and about 125-250 ug cell protein. Samples were incubated at 37~ for 60 min and the reaction stopped by heating at 56~ After cooling, 50 #1 of 100 mM cold L-asparagine was added to each tube. Fractionation of the samples was accomplished with 2.0-cm columns in Pasteur pipets, prepared with AG1-X8 ion-exchange resin, 200-400 mesh, acetate form. A total of 200 ~1 from each sample was placed on top of a column and washed with 3.0 ml double- distilled water. The eluate was collected and

462 Harris

100 #l added to each of two scintillation vials with 15 ml Aquasol. The vials were counted in a Beckman LS 7000 liquid scintillation system. Individual extracts were assayed in quadruplicate and blanks were run with extrac- tion buffer in place of cell extract.

R E S U L T S

Development of Asparagine-Dependent Sublines from CHO-K1 and V79 Cells. Since direct selection of asparagine auxotrophs was not feasible, we used the BrdU-visible indi- rect selection light procedure devised by Kao and Puck (1, 2) with modifications for substi- tution of black light as recommended by Chu et al. (14). Stock cultures were first exposed to EMS and then placed in ASP- medium containing BrdU. Wild-type cells incorporate BrdU and are sensitized for killing on subse- quent exposure to black light. Asparagine auxotrophs, which are unable to grow or incorporate BrdU, can survive this procedure (see Materials and Methods for further details).

In a preliminary experiment with CHO- K1 cells we isolated 18 survivor clones, some of which were wild-type cells that had escaped selection, while others were auxotrophic for asparagine at low population densities but grew at higher densities without this supple- ment. We therefore recycled mass populations three times through BrdU-black light and isolated two colonies from separate Petri dishes in ASP- medium. These variants, designated 2002-103 and 2002-109, were sub- cloned twice more in 10FCS-a-MEM. The final derivatives showed no population density effects, had no detectable asparagine synthetase activity (see later section), and gave rise to asp + revertants by spontaneous reversion at frequencies of 10 -6 o r less.

V79 stock populations were treated similarly, and with a single BrdU-black light selection we obtained an asparagine auxotroph which, after two subclonings in 10FCS- a-MEM, was designated 2293-343. These

cells showed no diminution in asparagine requirement at high population densities and gave rise to spontaneous asp + revertants only at frequencies of 10 7 or less in ASP- medium. In contrast to the CHO variants, 2293-343 cells retained a low but measurable level of asparagine synthetase activity, as will be detailed subsequently.

Frequency of Spontaneous and Induced Reversion from Auxotrophy to Prototro- phy. Table 1 gives the results of comparative studies performed for lines 2002-103, 2002- 109, and 2293-343. Selection of prototrophic colonies in ASP- medium was readily accomplished for all three variants, without population density effects. Spontaneous variants were less frequent in cultures of 2293-343 than in either of the 2002 lines. Asp + colonies of all three grew progressively in the presence or absence of asparagine and showed similar high plating efficiencies when tested in ASP- medium with or without added asparagine. Pretreatment with EMS led to an increase of about 250• over background variation for the two auxotrophs from CHO-K1 (2002-103 and 2002-109), while a similar exposure to EMS was significantly less effective for the V79 derivative (2293-343). Much greater disparity was observed when the three lines were compared for induction of asparagine independence after a standard 24-h treatment with 5-aza-CR. In such studies, high- frequency induction of asp + colonies was con- sistently observed in cultures of 2002-103 and 2002-109, with one-step increases in frequency of more than 100,000 x over the spontaneous background level (see. Table 1). This clear-cut result is closely similar to the high level induction of asp + colonies which we have previously described in experiments with Jensen rat sarcoma cells (11).

In sharp contrast to these results, there was little or no induction of asparagine independence when 2293-343 cells were exposed similarly to 5-aza-CR (Table 1). However, 2293-343 cells are capable of spontaneous reversion and also show a significant elevation in the frequency of revertants after treatment


Table 1. Frequency of Reversion from Asparagine Dependence to Independence in Chinese Hamster Cells

Auxotrophs and lines of origin

2002-103 2002-109 2293-343 Treatment a CHO-K1 CHO-K1 V79-56

None Total cells plated 12.4 • 106 4.2 • 106 33.0 • 106 asp + revertants 4 1 2 Relative PE 0.88 0.93 0.70 Reversion frequency 3.3 • 10 7 2.6 • 1 0 -7 8.6 • 10 -s

EMS Total cells plated 4.2 • 106 4.2 • 106 36 • 106 asp + revertants 223 171 90 Relative PE 0.66 0.65 0.62 Reversion frequency 8.1 • 10 5 6.3 • 10 -5 4.1 • 10 -6

5-aza-CR Total cells plated 4.2 • 103 4.2 • 103 26.4 • 106 asp + revertants 84 102 3 Relative PE 0.54 0.39 0.27 Reversion frequency 3.7 • 10 2 6.3 • 10 -2 4.2 • 1 0 -7

aTreated cells were plated in graded numbers into 10FCS-c~-MEM to determine relative survival and into ASP- medium to measure incidence of revertants. Spontaneous reversion frequencies were determined by direct plating of stock populations from 10FCS-cc-MEM into ASP- medium. Cultures exposed to EMS (300 #g/ml in 10FCS- ~-MEM, 18 h) were assayed after recovery for five days in normal medium. Cells treated with 5-aza-CR (1.0 ~g/ml in 10FCS-a-MEM, 24 h) were assayed after a two-day recovery period in 10FCS-a-MEM.

with E M S . Thus, fa i lure to respond to 5 - aza -CR cannot be explained on the basis of genet ic deletion. Wha teve r the explanat ion (see Discussion), the differential in inducibi l- i ty with 5 - a z a - C R between 2293-343 and the 2002 clones spans near ly five orders of magni- tude. Thus, even between lines or iginat ing from the same species, the act ion of 5 - aza -CR can be unpred ic tab le (6).

A s p a r a g i n e S y n t h e t a s e A c t i v i t y in Ce l l

E x t r a c t s . Asparag ine synthetase has been observed to be a regula ted ra ther than consti- tutive enzyme in C H O - K 1 cells (12, 17). S imi lar ly , we found in p re l iminary runs tha t levels of asparagine synthetase act ivi ty were higher in extracts of C H O - K 1 cells tha t had been grown in A S P - med ium than for those p ropaga t ed in 1 0 F C S - ~ - M E M . However , asparagine synthetase act ivi ty in ext rac ts from V79 cells seemed independent of the asparagine concentra t ion in growth medium, as had been repor ted ear l ier for D O N Chinese hamster cells (9). W e have not invest igated these differences in regula t ion between cell lines but, to assure uniformity , all cells used

for asparag ine synthetase assays were grown in A S P medium.

Table 2 provides a compar ison of asparagine synthetase act ivi ty in ext rac ts from C H O - K 1 and V79 lines, their respect ive auxotrophs, and aspa rag ine - independen t rever- rants arising spontaneously or a f te r induct ion by E M S and 5 -aza -CR. Stock V79-56 cells have a high overall level of a sparag ine synthetase, while a low but measurab le level is found in the derivat ive auxotroph, 2293-343. Aspa r - agine synthetase act ivi ty is much lower in C H O - K 1 cells, and none could be de tec ted in the auxot rophs 2002-103 and 2002-109 by the procedure used.

Aspa rag ine synthetase was consis tent ly elevated in protot rophic rever tants of all types. Very uni form levels of act ivi ty were observed for rever tants arising spontaneously f rom 2002-103 cells, or af ter exposure of these cells to E M S . Values obta ined for four clones of each type were qui te s imilar and close to wi ld- type levels for the progeni tor C H O - K 1 cells (Table 2). W i d e r var ia t ion was observed among rever tants induced from 2002-103 and

464 Harris

Table 2. Asparagine Synthetase Activity in Chinese Hamster Cell Lines

Line of Revertants, AS activity (clones) Cells origin Phenotype inductor" (cpm/#g protein) b

CHO-K1 a s p + 61.3 2002-103 CHO-K1 a s p - 0 .0

2002-103 a s p + (62.2), (60.4), (66.4), (67.3) (68.9), (69.6), (65.2), (62.7)

(131.3), (94.5), (127.4), (79.8), (68.0) 2002-109 CHO-K1 a s p 0 .0

(65.1), (99.7), (64.0) V79-56 a s p + 199.6 2293-343 V79-56 a s p - 8.5

2293-343 a s p + Spontaneous (397.0), (447.1), (109.2), (110.2)

aTreatment with 5-aza-CR: 1.0 #g/ml for 24 h; with EMS: 300 t~g/ml for 18 h. Variar/ts were isolated from single colonies in separate petri dishes and maintained in ASP- medium for enzyme assay.

bAll values shown are averages for two independent assays, each performed in quadruplicate.

Spontaneous EMS

5-aza-CR

5-aza-CR

2002-109 cells by exposure to 5-aza-CR. Asparagine synthetase activity in eight clones tested ranged from wild-type levels to double this value. Divergent and apparently discrete levels of asparagine synthetase were also found in spontaneous revertants from the V79 auxotroph, 2293-334. The basis for these interclonal differences is not clear. Conceiv- ably ploidy differences could account for the roughly even multiples of wild-type levels, but there was no indication of this among the variants in terms of an increase in mean cell size. Differences in the level of reexpression could occur, or nondisjunction after treatment with 5-aza-CR might give rise to clones with 1, 2, 3, or 4 active asp + genes. Further study of revertant clones will be required to assess the merit of these and other possible explana- tions, e.g., gene amplification at the asparagine synthetase locus (12).

Hybrids between Asparagine Auxo- trophs and with Wild-Type Cells. To determine whether the asparagine auxotrophs under investigation were members of one or more complementation groups, we prepared preliminary mixtures in all combinations from stock populations of 2002-103, 2002-109, and 2293-343 cells. These mixtures were then treated with PEG and plated out in ASP medium following the standard procedures for hybrid formation as described in Materials

and Methods. The frequency of the occasional asp + colonies obtained did not exceed the low incidence of spontaneous revertants in the parent lines (Table 1). In control mixtures with other cell lines, large numbers of hybrids appeared when tk- x hgprt- cells fused by the same procedure were plated in HAT medium.

Our data thus suggest that all three asparagine auxotrophs belong to the same complementation group. This conclusion is further supported by the study of hybrids made with asp- cell lines in other combinations (Table 3). These hybrids were isolated in HAT medium to avoid selective pressure on the asp marker during the hybridization procedure. For fusions of asp- x asp- cells this was made possible by the earlier incorpo- ration of OUA R and TH G R markers in one partner (2436-1). In other series, an asp + subline of V79-56 also possessing OUA R and TH G R markers was used (128-1 ). From these results, it is clear that asparagine auxotrophy is recessive at the phenotypic level.

More quantitative data were obtained by measuring the background incidence of spontaneous reversion for three clones each from the 2510 and 2511 hybrid series (Table 4). Although clonal differences can be noted and the general level of spontaneous reversion is slightly higher than that of the parents, such


Table 3. Cell Fusions between Asparagine Auxotrophs and with Wild-Type Cells

Parental lines Hybrids

Pt Markers P2 Markers Cell line Markers

2436-1 OUA R, THG R 2002-103 ASP- 2510 ASP , PRO +

2002-109 ASP- 2511 CHO-K1 ASP-, PRO- 2627

128-1 OUA rt, THG R 2002-103 ASP-, PRO 2628 ASP + , PRO + 2002-109 ASP-, PRO- 2629

HAT + , ASP-

HAT + , ASP HAT + , ASP-, PRO + HAT + , ASP + , PRO + HAT + ASP + , PRO +

differences in background are minor. We therefore performed tests to see whether asp- hybrids can respond by high-frequency reversion of asp colonies after exposure to 5-aza CR. This is an interesting point since one parent of the 2510-1 1 hybrids (2002-103 or -109) had shown clear-cut and strong induction of asp + variants with 5-aza-CR, while the other parent (2436-1, from 2293- 343) did not. The data shown in Table 4 provide an unequivocal result. All hybrid clones tested showed a one-step increase in r e v e r s i o n f r e q u e n c y of a p p r o x i m a t e l y I00,000, very similar to that of the 5-aza-

CR-responsive parent (see Table l). Thus, the potential for response to 5-aza-CR by reversion to asparagine independence seems to be dominan t or codominant , and there is no indication of a suppressor effect in hybrid combinat ions from the nonresponsive parental line.

D I S C U S S I O N

Asparagine dependence has been treated here in the context of genetic or epigenetic change at the asparagine synthetase locus, but it should be noted that asparagine auxotrophy can also arise as a pleiotrophic effect in respi- ratory-deficient mutan ts (18, 19). Such variants were obtained by Scheffler and his col- leagues from Chinese hamster cells after t rea tment with EMS and a specialized selection procedure. The survivors were unable to utilize galaetose in place of glucose and arose in high frequency, especially from V79 cells; up to 65% of some survivor populations were

g a l . These gal- cells showed also a low oxygen consumption, were strictly dependent on glucose for even temporary survival, and were auxotrophic for carbon dioxide and asparagine. In genetic studies, the gal mutan ts could be sorted out into eight complementa-

Table 4. Frequency of Spontaneous Reversion of Asparagine-Dependent Hybrids to Asparagine Independence and Effect of 5-aza-CR on Reversion Frequency ~

Frequency of reversion

Hybrid Spontaneous After 5-aza-CR clones Parental lines b (x 10 6) (x 10 z)

2510-1 2293-343 • 2202-103 1.0 2.8 -4 <0.7 0.74 -5 10.2 3.4

2511-1 2293-343 • 2202-109 1.4 4.1 -4 14.1 1.9 -6 1.6 1.8

aSpontaneous reversion frequencies were determined by direct plating of stock populations from 10FCS-a-MEM into ASP- medium. Cultures exposed to 5-aza-CR (1.0 ~g/ml in 10FCS-a-MEM, 24 h) were assayed after recovery for two days in normal medium.

b~;ee Table 3 for origin of hybrids.

466 Harris

tion groups representing specific defects in oxidative energy metabolism. No assays for asparagine synthetase were performed, and the authors stated that variants defective in this enzyme would not be picked up in their selective system. Consistent with this view, our asparagine-dependent lines (2002-103, 2002-109, and 2293-343) differ significantly from variants of the Scheffler types. Thus, the three lines showed normal survival for short periods in the absence of glucose, and all grew progressively when galactose was substituted for glucose, maintaining an asp phenotype throughout. This suggests that in spite of the phenotypic overlap of asparagine dependence, our variants do not share the general inhibi- tion of Krebs cycle activity common to the mutants described above.

The results we have described show that some asp auxotrophs, such as the 2002 lines obtained from CHO-K1 cells, can respond to 5-aza-CR treatment with high-frequency induction of revertants, very much as has been described for the transition of CHO-K1 cells from proline dependence to independence (4). The inability of 2293-343 asp- cells to show any significant induction by 5-aza-CR however is equally clear-cut. Such a result does not signify any general lack of responsiveness in the progenitor V79 line, since thymidine kinase can be activated in high frequency when t k - derivatives of V79 are treated with 5-aza-CR (7). Conceivably, gene mutation in 2293-343 cells may be required for reversion to asparagine independence, while in the 2002 lines the asparagine synthetase gene is merely suppressed and can be reactivated by DNA demethylation after 5-aza-CR treatment. But it is possible that asparagine dependence in 2293-343, as in the 2002 lines, is caused by a silent wild-type gene, with the difference that one or more changes in addition to demethylation are required for reexpression to occur. Our present data do not distinguish between these alternatives. It seems clear, however, that auxotrophs similar in phenotype, but differing in response to 5-aza-CR, are the

material of choice for resolution of the prob- lem.

ACKNOWLEDGMENTS

I thank Patrick Link and Celia Simpson for excellent technical assistance. This work was supported by U.S. Public Health Service grant CA 12130 from the National Cancer Institute.

LITERATURE CITED

1. Kao, F., and Puck, T.T. (1967). Proc. Natl. Acad. Sci. U.S.A. 58:1227-1234.

2. Puck, T.T. (1985). In Molecular Cell Genetics (ed.) Gottesman, M.M. (Wiley-lnterscience, New York), pp. 65-74.

3. Patterson, D. (1985). In Molecular Cell Genetics, (ed.) Gottesman, M.M. (Wiley-Interscience, New York), pp. 267-309.

4. Harris, M. (1984). Somat. Cell Mol. Genet. 10:615-624.

5. Worton, R.G., and Grant, S.G. (1985). In Molecu- lar Cell Genetics, (ed.) Gottesman, M.M. (Wiley- Interscience, New York), pp. 831-867.

6. Riggs, A.D., and Jones, P.A. (1983). Adv. Cancer Res. 40:1-30.

7. Harris, M. (1982). Cell29:483-492. 8. Wise, T. (1985). PhD Thesis (Zoology), University

of California, Berkeley. 114 pp. 9. Goldfarb, P.S.G., Carritt, B., Hooper, M.L., and

Slack, C. (1977). Exp. Cell Res. 104:357 367. 10. Waye, M.M.Y., and Stanners, C.P. (1979). Somat.

Cell Genet. 5:625-639. 11. Sugiyama, R.H., Arfin, S.M., and Harris, M.

(1983). Mol. Cell Biol. 3:1937-1942. 12. Andrulis, I.L. (1985). In Somatic Cell Molecular

Genetics (ed.) Gottesmann, M.M. (Wiley-Inter- science, New York), pp. 489-518.

13. Harris, M. (1984). Somat. Cell Mol. Genet. 10:275- 28 l.

14. Chu, E.H.Y., Sun, N.C., and Chiang, C.C. (1972). Proc. Natl. Acad. Aci. U.S.A. 69:3459-3463.

15. Lowry, O.H., Rosebrough, J.A., Farr, A.L., and Randall, R.J. (1951). J. Biol. Chem. 193:265 275.

16. Gantt, J.S., Chiang, C., Hatfield, G.W., and Arfin, S.M. (1980). J. Biol. Chem. 225:4808-4813.

17. Arfin, S.M., Simpson, D.R., Andrulis, I.L., and Hatfield, G.W. (1977). Proc. Natl. Acad. Sci. U.S.A. 74:2367-2369.

18. Ditta, G., Soderberg, K., Lardy, F., and Schemer, I.E. (1976). Somat. Cell Genet. 2:331 344.

19. Soderberg, K., Mascarello, J.T., Breen, G.A.M., and Schemer, I.E. (1979). Somat. Cell Genet. 5:225 240.

induction and reversion of asparagine auxotrophs in cho-k1 and v79 cells

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