nature of mutations conferring resistance to 8 …tissue hgprt was prepared to test a9 and rag for...

y. Ceii Sci. 14,235-251 (1974) 235

Printed in Great Britain

NATURE OF MUTATIONS CONFERRING

RESISTANCE TO 8-AZAGUANINE IN

MOUSE CELL LINES

SEUNG-IL SHIN

Basel Institute for Immunology, Grenzacherstrasse 487, 4058 Basel, Switzerland,and Department of Genetics*, Albert Einstein College of Medicine,Bronx, New York 10461, U.S.A.

SUMMARY

Two stable mouse cell mutants Ao and RAG, which are resistant to 8-azaguanine anddeficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT), have been studied inorder to establish the nature of molecular changes conferring the mutant phenotypes. A specificprecipitating rabbit antiserum was prepared against the normal HGPRT purified from mousetissues, and used to test for cross-reacting material (CRM) in the mutant lysates. Neithermutants contained detectable cross-reacting material, as demonstrated by precipitation in-hibition tests. However, the L cell-derived mutant A9 was shown to have a low but significantlevel of HGPRT activity which was clearly different from that of the normal enzyme in the wild-type parental cell line. Compared to the wild-type enzyme, the HGPRT in Ao is extremelyheat labile, and has an elevated substrate-binding constant in addition to distinct antigenicdifferences. Both A9 and RAG have been shown previously to revert to the normal phenotypewith low frequency, thus ruling out gene deletions as a possible cause of the 8-azaguanineresistance. It is suggested that RAG could involve a recessive regulatory mutation, while A9may contain a structurally altered HGPRT as a result of a missense mutation within thestructural gene for this enzyme.

INTRODUCTION

Somatic cell strains with stable variant phenotypes, whether arising spontaneouslyor induced by chemical agents, are now routinely isolated from a variety of culturedanimal cells. Resistance to synthetic analogues of nucleic acid precursors in particularhas been widely used as a selective marker for studies on mutation frequency, chemicalmutagenesis and cell hybridization. As exemplified by the classic system of Little-field (1964a), cellular mutants resistant to these drugs can be selectively eliminated orrescued from a population of mixed cells with high efficiency.

While the list of stable cellular variants that can be maintained in culture is growing,the nature of the mutations which are responsible for the expression of the variantphenotypes remains largely unknown. The apparent spontaneous frequency of varia-tion for a selected phenotype can be io"4 or higher, as in the case of temperature sensi-tivity in mouse cells (Thompson et al. 1971), loss of the capacity to synthesize a cell-specific product (Coffino & Scharff, 1971), or resistance to 8-azaguanine (Szybalski,Szybalska & Ragni, 1962; Littlefield, 1963; Chu & Mailing, 1968). One recent study

• Address to which requests for reprints should be sent.16 C E L 14

236 5. Shin

even reported that the recovery rate of 8-azaguanine-resistant colonies from a diploidhuman fibroblast culture was as high as io~2 per input cell (Van Zeeland, Van Diggelen& Simons, 1972). It has been shown in one of these studies that mouse L cells whichwere selected for partial resistance to 8-azaguanine merely contained reduced amountsof the enzyme concerned (inosinic acid pyrophosphorylase), while the enzymemolecule itself remained apparently unaltered (Littlefield, 1963). In addition, suchpartially resistant cells were recovered with a frequency of up to icr3 from untreatedcells depending on the concentration of the drug in the selection medium. It is highlyunlikely therefore that all of the phenotypic variations observed in these examplescould have been caused by gene mutations.

Furthermore, Harris (1971) and Mezger-Freed (1972) recently presented data whichindicate that the frequencies of the formation of surviving colonies of hamster or frogcells grown either in the presence of 8-azaguanine, bromodeoxyuridine (BUdR) orpuromycin, or after an acute thermal shock, are independent of the ploidy levels of thestarting populations. Harris therefore raised the question whether these variants couldreally be due to gene mutations on the DNA level, or whether some other mechanismsinvolving 'stable shifts in phenotypic expression rather than changes in the geneticinformation' might be responsible (Harris, 1971).

Here I describe some recent results which seem to provide a partial answer to thisquestion for at least 2 well known mutant cell lines of mouse. The cellular mutantsstudied here, A9 and RAG, have been selected originally by Littlefield (19646) andby Klebe, Chen & Ruddle (1970a) from an L cell subline and a renal adenocarcinomaculture, respectively, for complete resistance to 8-azaguanine. Both mutants prolifer-ate in media containing normally lethal concentrations of 8-azaguanine, have relativelystable heteroploid karyotypes, are sensitive to the HAT medium (medium containinghypoxanthine, aminopterin and thymidine: Szybalska & Szybalski, 1962) and maintainthe mutant phenotype indefinitely even in the absence of 8-azaguanine. Until recently,both lines were believed not to revert to the wild-type (Littlefield, 1966; Klebe et al.1970 a). Since their isolation, both A9 and RAG have been used extensively for cellhybridization studies by several laboratories, including those of Littlefield (1964a),Ruddle (see Ruddle, 1972), Harris (Harris, 1970; Schwartz, Cook & Harris, 1971; Klein,Bregula, Wiener & Harris, 1971), and Siniscalco (Miller et al. 1971; and Grzeschiket al. 1972). A significant body of genetic information has now accumulated from thesestudies, which bears on the questions of cellular differentiation, metabolic regulation,control of cellular malignancy, discovery of linkage groups in man, and mapping of theX-chromosome. The identification of the original molecular events which resulted inthe mutant phenotypes in A9 and RAG would therefore be useful for the correctinterpretation of the experimental data.

We have recently reported the isolation and characterization of a series of 10independently derived revertants from A9 and RAG, thus ruling out gene deletion as apossible cause for the mutant phenotype for these 2 mutant strains (Shin et al. 1973).The HGPRT activity in these revertants could not be distinguished from the normalmouse HGPRT activity by either electrophoretic mobility or heat sensitivity. Eventhough the number of revertants analysed was small, it seemed probable that both A9

Somatic mutation to drug resistance 237

and RAG might represent mutants in which the synthesis of HGPRT was repressedby a change or changes in regulatory function, while the structural gene for the enzymeremained unaltered. Therefore, a specific inhibiting antiserum against the mousetissue HGPRT was prepared to test A9 and RAG for the presence of immunologicallycross-reacting material (CRM).

MATERIALS AND METHODS

Cell culture

All cell lines were maintained in Dulbecco-Vogt-modified minimal essential medium (fromGIBCO), plus 10% heat-inactivated foetal calf serum and antibiotics. Mouse cell mutants A9(Littlefield, 19646) and RAG (Klebe et al. 1970a), and a human cell mutant D98/AH2 (Szy-balska & Szybalski, 1962), all of which are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.4.8), were maintained in the growth medium plus 8-azaguanine(10/ig/ml).

Assay of HGPRT activity

Cell lysates were prepared and HGPRT activity assayed essentially according to theDEAE-paper chromatography technique described previously (Shin, Meera Khan & Cook,1971). The reaction mixture contained, in a total volume of 100 p\: 55 mM Tris-HClbuffer, pH7"4; 500 nmol MgSO4; 55 nmol 5'-phosphoribosyl-I'-pyrophosphate (PRPP)(Calbiochem); 50 nmol [8-14C]hypoxanthine (Radiochemical Centre, Amersham) at specificactivity 1-89 mCi/mmol, and 10 /tl of cell lysate (1-3 mg total protein/ml). The reaction wasstarted by the addition of labelled hypoxanthine, continued for 20 min at 37 °C, and stopped byadding 1000 nmol of pre-chilled EDTA (20 fi\) and placing the tubes in an ice-bath. Aftermixing, 10 fil of reaction mixture were spotted on DEAE-cellulose paper (Whatman, DE-81),washed overnight by descending chromatography in o-i M Tris buffer, pH 9-5, and dried in air.Removal of the unreacted hypoxanthine by this method was better than 99-8 %. Disks of19 mm diameter were then punched out and counted in toluene plus Omnifluor (New EnglandNuclear Corp.). All assays were done in duplicates. Tubes with boiled lysate, or in whichEDTA was added before the substrates, served as background controls. Under these standardconditions, the conversion of hypoxanthine to inosinic acid was linear for at least 30 min, andlinearly dependent on enzyme concentration.

Purification of HGPRT from mouse tissues

Normal tissue HGPRT was extracted from pooled liver and kidney of adult C3H mice, andpurified by heat treatment, salt fractionation and DEAE-cellulose column chromatography(Krenitsky, Papaioannou & Elion, 1969). The final preparation contained at least 3 major bandsin SDS-polyacrylamide gel electrophoresis after staining with amido black, but represented300-fold purification from the crude extract. Details of the purification procedure and enzymo-logical data will be reported separately (S. Shin, in preparation).

Preparation of anti-HGPRT antiserumTwo 8-week-old female New Zealand rabbits were immunized by multiple subcutaneous

injections of the purified tissue HGPRT with complete Freund's adjuvant on days o, 8 and 29(Arnon & Neurath, 1969). Pre-immune sera from the same rabbits were collected beforehandand used as controls. The animals were bled 3 weeks after the last injection, and again 5 daysafter an intravenous boost given a week after the first bleeding. Of the 2 rabbits immunized,only one responded with a high level of anti-HGPRT antibody.

16-2

238 S. Shin

Precipitation of HGPRT activity with anti-HGPRT antiserum

To each 8 x 70 mm glass test tube, 10 /tl of cell lysate or test sample containing HGPRT(3 mg total protein/ml) and 40 /tl of Tris buffer (o-oi M MgSO4, o n M Tris-HCl, pH 7-4) wereplaced and 5 /tl of appropriately diluted antiserum were added. To maintain a constant totalrabbit globulin concentration, normal pre-immune rabbit serum was used as diluent for sometubes. After mixing, the suspension was incubated for 30 min at 37 °C to allow the formation ofthe enzyme-antienzyme complex. Then 5 /tl of undiluted sheep anti-rabbit IgG antiserum(kindly provided by Dr A. Kelus) were added, the mixture mixed, and again incubated for30 min at 37 °C. The tubes were kept for 16 hat4°Cand centrifuged in cold for 15 min to bringdown the precipitate. From the supernatant, so-/il aliquots were transferred to new tubes, andthe HGPRT activity still remaining in solution was assayed as described above after the additionof 10 /tl PRPP (ss x 10-3 M), 30 /tl distilled water, and 10 /tl [8-14C]hypoxanthine (5 x io"3 M,10/tCi/ml).

RESULTS

HGPRT activity in mouse cell lines

Mutant A9 was originally isolated from L929 cells in a single-step selection in2 fig 8-azaguanine/ml (Littlefield, 19646), and was initially reported to have no detect-able HGPRT activity. However, at least some sublines derived from A9 are known tocontain measurable enzyme activity (Harris & Cook, 1969), and subclones can beisolated after prolonged growth in normal culture medium which have between

Table 1. Specific activity of HGPRT

Source of HGPRT Specific activity*

1. Mouse tissuetCrude liver extract I I I - 6Crude kidney extract 65-8

2. Normal mouse cell linestL929 1288B82 1477MM-19 1385NB-41A3 39-9AR-s 1495RR-3 849

3. Mutant cell lines§A9S 7-64A9C 4-84RAG < 030BA829 < 0-30MM19-TG1 090D98/AH2 < 030

• Expressed as nmol IMP formed/mg protein/h.f Fresh extracts prepared from adult mouse tissue.X B82: thymidine kinase-deficient mutant derived from L929 (Littlefield, 1966); MM-19,

a mouse myeloma culture; NB-41A3, a mouse neuroblastoma cell line (Augusti-Tocco & Sato,1969); AR-5 and RR-3, HGPRT+ revertants isolated, respectively, from A9 and RAG (Shinet al. 1973).

§ BA829 and MM19-TG1, thioguanine-resistant mutants isolated from B82 or MM-19(Shin, unpublished data); other cell lines are identified in the text.


0-5-5-9% °f *ne normal specific activity (author's unpublished observation). For thestudy reported here, 2 such clones, designated A9C and A9S, containing respectively3-8 and 5-9% cf the specific activity found in the wild-type cell, have been selected.RAG was isolated after the conventional step-wise selection procedure in increasingdoses of 8-azaguanine (Klebe et al. 1970a). Unlike A9, no subclone with detectableHGPRT activity has so far been isolated from RAG. All wild-type (8-azaguanine-sensitive) cell lines have high levels of HGPRT activity, even though the specificactivity fluctuates somewhat depending on culture conditions. The specific activitiesof HGPRT in crude mouse tissue extracts, and in various mouse cell lines and mutantstrains are given in Table 1.

0-4 0-8 -4 0 8 0 20Antiserum, //I X101

40 80 100 200

Fig. 1. Precipitation of normal mouse HGPRT by anti-HGPRT antiserum in thepresence of sheep anti-rabbit immunoglobulin antiserum. Lysates of a subclone ofL929 (10/tl; 30 fig total soluble cell protein) were reacted first with increasingamounts of anti-HGPRT antiserum, the enzyme-antienzyme complex was thenprecipitated overnight with anti-rabbit IgG antiserum, and the remaining HGPRTactivity in supernatant was assayed as described in Materials and Methods.

Precipitation of HGPRT by anti-HGPRT antiserumAt antiserum saturation and in the presence of sheep anti-rabbit IgG antiserum,

mouse HGPRT was precipitated quantitatively by anti-HGPRT antiserum. Fig. 1shows a precipitation curve of HGPRT activity from a subclone of the mouse L cell,in which the enzyme activity remaining in solution after the precipitation of the enzyme-antibody complex is plotted against the antiserum concentration. The precipitation ofHGPRT by the antiserum was quantitatively complete at antiserum concentrationsabove 1/120 dilution (0-5 /A in Fig. 1).

240 5. Shin

Precipitation of the HGPRT activity from several mouse cell lines as well as ahuman cell line (HeLa) is presented in Table 2. The mouse cell lines tested hereinclude a fibroblast (clone L929), a globulin-secreting myeloma (MM-19), a neuro-blastoma (NB-41A3), revertants from A9 (AR-5) and RAG (RR-3), and four 8-aza-guanine-resistant mouse cell mutants. It is noteworthy that except for the smallfraction of the enzyme activity in the neuroblastoma culture, the residual activity foundin A9S is the only mouse-derived HGPRT activity which cannot be precipitated by theantiserum. This was the first indication that the residual HGPRT activity in A9 may bephysically distinct from the wild-type enzyme activity. This point will be discussedfurther below.

Table 2. Precipitation of cellular HGPRT by anti-HGPRT antiserum

Cell line

L929MM-19NB-41A3

AR-5RR-3A9SRAGBA829MM19-TG1

HeLa

HGPRT activityA

Control withoutantiserum

12261455

380

11601148

1430

13

25

1321

remaining, cpm

With antiserumat 1/60

0

34 0

0

0

1480

0

0

i°75

% HGPRTactivity

precipitated

1 0 0

1 0 0

89-5ICO

1 0 0

0

—

1 0 0

1 0 0

8-i

Test for cross-reacting material in RAG

The precipitating capacity of the anti-HGPRT antiserum against the normalHGPRT in L929 cells was now employed to test RAG for the presence of CRM. Acalibration curve was obtained for this purpose by titration of increasing amounts ofthe normal cell lysate against a fixed antiserum concentration, as shown in Fig. 2.In appropriate regions of the curve, the antibody concentration is limiting, so that theextent of precipitation of HGPRT is dependent only on the concentration of theenzyme protein in the precipitating mixture - the more protein, the less the fractionwhich is precipitated. Using 2 concentrations of HGPRT (1 and 2 /tl of L929 lysate),RAG lysate was tested for evidence of CRM by measuring the inhibition of precipitationof the normal enzyme by anti-HGPRT. Bovine serum albumin and the lysate of an8-azaguanine-resistant human cell line were used as controls. Data given in Table 3indicate that the addition of up to 3 weight equivalents of RAG cellular proteins to theprecipitating mixture did not significantly decrease the precipitation of the normalenzyme. It seems therefore that either the mutant cell does not contain any materialwhich antigenically resembles the normal mouse HGPRT, or, if the cell did indeedcontain a mutant HGPRT, the extent of its cross-reaction with the antiserum usedhere is not measurable.


100

2 80

01

a. 60

40

20

0-1 02 0 5 2 5L929 lysate, /A

10 20 30

Fig. 2. Precipitation of normal HGPRT by anti-HGPRT antiserum, with a constantantiserum concentration. The lysate of a L929 subclone was adjusted to contain 3 mgsoluble protein/ml, and the HGPRT activity remaining after overnight precipitationwith and without the precipitating antiserum was assayed in duplicates. A constanti :io ratio of total rabbit serum to sheep anti-rabbit IgG antiserum was maintainedby using the pre-immune serum from the same rabbit as diluent. Each experimentalpoint on the curve represents the difference between the HGPRT activities of thegiven volume of lysate in the presence and absence of the anti-HGPRT antiserum.

Nature of the residual HGPRT activity in Ag cells

The presence of low but measurable HGPRT activity in A9 subclones was shownearlier in Table 1. The fact that this HGPRT activity did not cross-react at all with theantibody against the normal mouse enzyme (Table 2) was quite surprising. We there-fore carried out the following analyses to establish the nature of this activity.

Heat stability and cross-reactivity to anti-HGPRT antiserum. Since it had beenshown earlier (Shin et al. 1973) that the normal mouse HGPRT is stable at 80 °C forat least 1 min, the heat stability of the residual HGPRT activity in A9 was tested.The effects of heating and of anti-HGPRT antiserum on the normal, mutant andrevertant HGPRT activities are summarized in Table 4. It is clear that the HGPRTactivities from the non-mutant mouse sources are unaffected by the heat treatmentbut completely precipitated by the antiserum, whereas the behaviour of the HGPRTactivity in two Ag-derived clones is exactly the opposite.

Catalytic product. The assay of HGPRT activity is based on the fact that theenzymic reaction yields a negatively charged product (inosinic acid, IMP) which bindsto the DEAE paper, from a neutral substrate (hypoxanthine) which does not. Therefore,it was necessary to establish whether the HGPRT-like activity found in A9 subcloneswas due to some other enzyme which could introduce negative charges onto the

242 5. Shin

Table 3. Effect of mutant cell lysates on precipitation of normal HGPRTby anti-HGPRT antiserum (CRM test)

Normal celllysate*, fil

i

i

i

i

2

2

2

2

2

Anti-HGPRTantiserum, fil

—O'O2

O O 2

0-02

O-O2

O O 2

O O 2

O O 2

Testt

BSAf, /tl

—

3———6

———

material addedA

D98/AH2RAG lysateJ,

lysate, fil fil

— —— —3 —

— 3— —— —2 —

6 —— 6

HGPRTactivity

remaining,cpm

1 2 1

2522

2 2

27791

n o115109

Activityexpectedif CRMpresent§,

cpm

——7070

——

144161

161

Test conditions were as in Fig. 2. All tubes contained a total of o-i fil of rabbit serum(anti-HGPRT antiserum and/or pre-immune serum) and 10 /tl sheep anti-IgG antiserum. Alllysates and bovine serum albumin solution were pre-adjusted to contain 3-0 mg soluble protein/ml.

• Lysate from L,929-subclone 1B1.f Crystalline bovine serum albumin (Miles-Seravac Co.).X Lysate from strain D98/AH2, a human cell derivative resistant to 8-azaguanine (Szybalska

& Szybalski, 1962).§ CRM expected to obtain if the test lysate contained material which cross-reacted 100%

with the antiserum and if the molar concentration of the material were comparable to that ofHGPRT in the normal lysate from L929; calculated from the calibration curve in Fig. 2.

purine ring by some other pathway. This possibility has been definitely ruled out bythe following observations. First, it was shown that the conversion of hypoxanthine tothe DEAE-binding material is PRPP-dependent and requires Mg2"1", as in the case ofthe genuine HGPRT (Krenitsky et al. 1969). Secondly, the catalytic end product ofthe reaction by the A9 enzyme was isolated from [8-14C]hypoxanthine, purified, andco-chromatographed on paper with authentic tritiated IMP in 3 different solvent sys-tems. As summarized in Table 5, the 14C-labelled catalytic product always migratedtogether with the tritiated standard, maintaining a constant isotopic ratio. These datatogether leave no doubt that the residual enzyme activity in A9 which convertshypoxanthine to a negatively charged material is indeed HGPRT.

Substrate binding. The Michaelis constant of the HGPRT in A9 with respect tohypoxanthine at non-limiting concentrations of PRPP was measured (Fig. 3). TheKm for the A9 enzyme was found to be 1 x \o~i M, nearly 5 times higher than thenormal value of 2-2 x io~6 M characteristic of the HGPRT in L929 and Ag-derivedrevertants.

Origin of the HGPRT activity in Ag. The residual HGPRT activity in A9 subclonesis unlikely to be due to contaminating micro-organisms. All of the mouse cell linesstudied here have been maintained routinely in culture media containing penicillin


Table 4. Effect of anti-HGPRT antiserum or heat-treatment on mouse HGPRT

Source of HGPRT

1. Tissue extractsPCCLCK

2. Normal cell lysatesL929MM-19AR-5RR-3

3. Mutant cell lysatesA9SA9CRAGBA829MM19-TG1

HGPRT

Untreatedcontrol

———

1566i4J31220

1398

9359

0

0

1 1

activity remaining

After 1 minat 70 °C

129822732997

159314621291

1452

60

0

0

0

, cpm

With antiserumat 1/60

0

0

86

0

30

0

1 0 2

7 i0

30

Assay conditions are described in Materials and Methods. PC: a 300-fold purified HGPRTpreparation from pooled mouse tissues, which had been used to obtain the anti-enzyme anti-serum. CL and CK: crude extracts from mouse liver and kidney, respectively. All tissue-derivedHGPRT preparations used here had been heat-treated previously at 67 °C for 3 min to inactivatethe nucleotide s'-phosphatases and to remove non-specific heat-labile proteins. Cell lines usedare identified in footnote to Table 1.

and streptomycin, and remained free of bacterial contamination. The A9 and L929subclones have been treated periodically with an anti-PPLO agent (Tylocine, GIBCO;60 /tg/ml). The HGPRT activity in A9 did not decrease significantly after a period ofgrowth in Tylocine-containing medium. Furthermore, the degree of conversion of[3H]uridine to piTJuracil by fresh lysates of A9 or L929 (Levine, 1972), a sensitiveindex to cellular contamination by mycoplasma, did not show any correlation with thelevel of specific activity of HGPRT in these cells. In some subclones of A9, in whichthe uracil conversion index at 180 min was less than 25 %, indicating no or very lowPPLO contamination (Levine, 1972), the heat-labile HGPRT activity was stillpresent. On the other hand, subclones of L929 or B82 in which mycoplasma contami-nation could be demonstrated by the conventional agar plate method of Hayflick(i960) contained no atypical HGPRT activity characteristic of the enzyme in the A9cell. Though it is not possible to rule out absolutely a transient or low-level contami-nation of A9 by mycoplasma, the above results suggest strongly that the residualHGPRT activity in A9 is an intrinsic property of the A9 itself and not due to extrinsicgenetic elements.

In Table 6, the important distinguishing parameters of HGPRT in the wild-typeL929, the mutant A9, and the phenotypically normal revertants of A9 are summarizedfor comparison.


- 4 - 2 0 2 41/[Hypoxanthine],

10

Fig. 3. Lineweaver-Burk plot for HGPRT activity of L929 (•) and A9 (CO-[PRPP], 5 x 10-1 M.

Table 6. Properties of the HGPRT activity in normal,mutant and revertant mouse cell lines

Specific activityKm for hypoxanthine

( X io4, M/L)Catalytic product from

hypoxanthinePrecipitable fraction by

anti-HGPRT antiaerumThermolabile fraction after

1 min, 70 °C

• Data from 3 independently derived,

L929

i-oo

0 2 2

IMP

100%

0 %

cloned revertants

A9

001—006i - o

IMP

0 %

100%

isolated from

A9-revertants#

o-5o-i-i70-25-0-30

IMP

100%

0 %

A9 (Shin et al. 1973)

DISCUSSION

Cellular resistance to 8-azaguanine or thioguanine is usually due to reduced orabsent activity of HGPRT. Cells deficient in this enzyme activity cannot convert theabnormal purine into its corresponding ribonucleotide, thus preventing its lethalincorporation into nucleic acids. In man, a severe sex-linked neurological disorder,first described by Lesch & Nyhan (1964), has been shown to be associated with adeficiency of this enzyme (Seegmiller, Rosenbloom & Kelley, 1967). Fibroblasts orlymphocyte cultures derived from patients with this disease have little or no detectableHGPRT activity (Migeon et al. 1968; Choi & Bloom, 1970), and are resistant tosynthetic purine analogues.

246 S. Shin

In principle, a de novo stable shift from the HGPRT+ phenotype to the HGPRT-phenotype in a cell could result from any of the following events: (1) Gene deletionsinvolving part or all of the hgprt locus; (2) mutations within the hgprt locus (i.e., thelocus coding for HGPRT), such as base-pair substitutions or frame-shift mutations,or suppressible tRNA mutations, which give rise to inactive enzyme molecules orcatalytically incompetent fragments of the enzyme; and (3) mutations of a regulatorygene, or any other epigenetic changes, which result in a partial or complete repressionof enzyme synthesis. Gene deletions would be ruled out for any mutant cell in which agenuine reversion to the HGPRT+ phenotype could be shown to occur. On the otherhand, the presence of immunologically cross-reacting material (CRM) to specificantibody directed against the wild-type HGPRT would suggest that the given mutantarose through a mutation within the structural gene for HGPRT. Furthermore, if aseries of HGPRT+ revertants were to be recovered from HGPRT" cells, only mutantsof the second class would occasionally produce colonies with HGPRT which hasnormal activity but is physically distinguishable from the original wild-type enzyme.Variants of the third class would contain unaltered normal HGPRT molecules directlyproportional to the extent of repression. Operationally, any cellular variant caused bya 'stable phenotypic shift' as suggested by Harris (1971) would also fall into this lastcategory.

In fact, erythrocytes from Lesch-Nyhan patients have been shown to contain analtered HGPRT molecule, which migrates differently from the normal enzyme inelectrophoresis (Bakay & Nyhan, 1972) and cross-reacts with an antiserum against theHGPRT of normal human erythrocytes (Rubin et al. 1971). These results indicate thata base-substitution mutation in the X-linked gene is responsible for the cellular defect.After the experiments described in this paper had been completed, Beaudet, Roufa &Caskey (1973) reported the results of a study in which 8-azaguanine-resistant mutantstrains from a Chinese hamster cell line were analysed by a closely analogous approach.

As mentioned earlier, we have shown that both A9 and RAG do in fact revert to theHGPRT+ phenotype with apparent frequency of i-8 x io~8 and 9 x io~8, respectively(Shin et al. 1973). It was clear therefore that the mutant phenotype (HGPRT~) inneither A9 nor RAG was caused by a gene deletion. Data presented here were obtainedin our further attempts to distinguish between the second and the third alternativesdescribed above.

Mutation in Aq

Data summarized in Table 6 indicate that the tertiary structure of the enzyme inthe mutant is quite different from the catalytically normal enzymes found in the wild-type or in the revertant lines. It is possible that the Ag-type HGPRT is the productof a second hgprt locus which is expressed only when the major locus is not. Consideringthe large amount of work on this enzyme in mammals, it is extremely unlikely thatanother, undiscovered locus for this enzyme exists. HGPRT" mutants can be recoveredfrom untreated L929 cells at a frequency of about io~6 (Littlefield, 1963), while thereversion index for A9 has been found to be of the order of io~8 (Shin et al. 1973). Itseems very probable therefore that A9 is the product of a single point mutation at the


hgprt locus, and that the revertants of A9 resulted from either an exact reverse mutationor a second mutation that restores the original conformation of the active site ofHGPRT.

The ligprt locus is X-linked in man (Lesch & Nyhan, 1964; Migeon et al. 1968),horse (Deys, 1972), and almost certainly in hamster (Westerveld, Visser, Freeke &Bootsma, 1972). In view of the evolutionary conservation of homology in the mam-malian sex chromosome (Ohno, 1967), as well as recent experimental evidence (Epstein,1972), this locus is most probably X-linked in mouse also. In euploid. mammaliancells in which more than one X-chromosome is present, gene-dosage compensation isachieved for the X-linked genes by genetic inactivation of all but one X-chromosome(Lyon, 1961; Russell, 1961), thereby maintaining the effective hemizygosity of theX-borne loci. The mouse L-cell (Earle, 1943) and sublines derived from it are grosslyheteroploid, with a modal number of chromosomes greater than 50. Even though ithas been shown by somatic cell fusion that more than one X-chromosome can begenetically functional in a proliferating cell (Siniscalco et al. 1969), it is not knownwhether the usual process of heteroploidization which accompanies the cellularadaptation to long-term culture in vitro permits the acquisition and maintenance ofmore than one functional equivalent of X-chromosomes. The biochemical and geneticdata presented above suggest that at least in the L-cell derivatives analysed here, thehgprt locus is likely to be present in a single functional dose. If, for instance, the wild-type mouse L cell had contained at least 2 functionally active copies of the hgprt locus,having the genotype hgprt+jhgprt+, the genotype of its mutant derivative A9 would behgprt~/hgprt~, and the immediate result of a single-step reversion should be a hetero-zygote of the genotype hgprt+jhgprt~. In the absence of genetic inactivation (Lyon,1971), such an heterozygote revertant should express an intermediate level of HGPRTactivity. Gene-dosage effect in cells from heterozygous carriers of recessive autosomaltraits is well established in man (see Nitowsky, 1972), as well as for sex-linked enzymesin mouse oocytes in which the X-chromosome inactivation has not yet occurred(Epstein, 1969, 1972). Littlefield has actually attempted to interpret the occurrence ofintermediate levels of resistance to 8-azaguanine in L cells in terms of multiple genecopies for HGPRT (Littlefield, 19646). However, all of the revertants which weisolated from A9 and RAG regained the normal range of specific activity of HGPRTtogether with complete sensitivity to 8-azaguanine (Shin et al. 1973), and in the A9revertants studied here, no residual activity of the mutant-type HGPRT was detectable.If A9 had the genotype hgprtmjhgprtm, produced perhaps as a result of a mutationinvolving one of the two hgprt+ cistrons in the wild-type cell followed by mitoticrecombination and segregation, the one-step revertant would have the genotypehgprt+jhgprtm, and contain both the normal and the mutant-type HGPRT in equi-molar concentration. The actual experimental data, therefore, would be most easilyexplained if it is assumed that L-cell sublines contained only one functional equivalentof the X-chromosome.

In an immunologically responsive host, a foreign protein antigen will in generaldisplay a collection of overlapping but distinctive antigenic determinants, giving rise inturn to a population of heterogeneous antibody molecules with a range of specificities.

248 S. Shin

This of course is the basic rationale for the CRM test performed above. The completelack of cross-reactivity of the mutant HGPRT with the antiserum against the normalHGPRT was therefore surprising. However, other examples of this kind have beendescribed. Shaltiel (1968) has demonstrated that a precipitating antiserum against apurified glycogen phosphorylase b did not cross-react significantly with the apoenzymefrom which the prosthetic group pyridoxal phosphate had been removed. In this case,pyridoxal phosphate itself was not a major antigenic determinant, and the apoenzymeregained the full antigenicity upon re-addition of the prosthetic group. Antigenicdifferences between metmyoglobin and apomyoglobin (Crumpton & Poison, 1965),and between the normal haemoglobin and the mutant haemoglobin-S (Reichlin, Hay &Levine, 1964), have been well described. This last example shows clearly that a singleamino acid substitution in a globular protein can result in a dramatic change in itsantigenicity. Anti-enzyme antibodies are often directed against a restricted number ofmajor conformational determinants (Arnon, 1971). In fact, one of the two rabbitswhich have been immunized with the mouse HGPRT in this study did not produceany detectable antibody to the enzyme. This suggests that mouse HGPRT is a poorimmunogen in the rabbit and contains a very limited number of antigenic deter-minants.

Mutation in RAG

The absence of any detectable HGPRT activity or CRM in RAG is compatiblewith the view that A regulatory mutation may be involved for this mutant strain.Whether RAG represents a novel class of cellular variants not involving gene mutationson the DNA level, as proposed by Harris (1971), cannot be decided with the presentlyavailable data. However, if the variant phenotype in RAG is due to a mutation of aregulator gene, this could be demonstrated by the fusion of RAG and A9. Therecovery of HAT-resistant hybrids from the fusion of RAG and LM (TK~) mousecell strain (Klebe et al. 1970 a) rules out the presence of dominant regulator gene muta-tion in RAG. Even though a direct proof of the kind obtained from the A9-humancell hybrids (Shin et al. 1971) has not been provided, available data from the inter-species cell hybrids with RAG (Klebe, Chen & Ruddle, 19706; Ruddle et al. 1971)indicate that the regulator gene, if it existed at all, would be species-specific. Theisolation of HGPRT+ hybrids from a fusion of RAG and A9 would therefore indicatethat a species-specific, recessive regulator gene mutation is involved in RAG. Inaddition, the complementation of A9 and RAG should result in the production in thehybrid cell of both the heat-resistant, cross-reacting normal HGPRT (contributed bythe de-repressed hgprt+ of RAG) as well as the mutant-type HGPRT (coded by thehgprtm of A9). The occurrence of such hybrids would be further evidence that drug-resistant cellular variants can arise through genuine gene mutations in cultured animalcells.

I thank Dr C. Steinberg for helpful discussions, suggestions and critical reading of themanuscript, Drs B. A. Askonas and D. Braun for help with immunization, and Dr A. Kelus forthe sheep anti-rabbit IgG antiserum. I also thank Ann Langeveld and Ruth Steward-Silber-schmidt for technical assistance. This work was supported in part by a Genetics Center Grantto the Albert Einstein College of Medicine from the National Institutes of Health (GM-19100-01).


REFERENCES

ARNON, R. (1971). Antibodies to enzymes - A tool in the study of antigenic specificity deter-minants. In Current Topics in Microbiology "and Immunology 54, 47-93. Berlin, Heidelberg,New York: Springer Verlag.

ARNON, R. & NEURATH, H. (1969). An immuHological approach to the study of evolution oftrypsins. Proc. natn. Acad. Sci. U.S.A. 64, 1323-1328.

AUGUSTI-TOCCO, G. & SATO, G. H. (1969). Establishment of functional clonal lines of neuronsfrom mouse neuroblastoma. Proc. natn. Acad. Sci. U.S.A. 64, 311-315.

BAKAY, B. & NYHAN, W. L. (1972). Electrophoretic properties of •hypoxanthine-guaninephosphoribosyltransferase in erythrocytes of subjects with Lesch-Nyhan syndrome. Bio-chem. Genet. 6, 139-146.

BEAUDET, A. L., ROUFA, D. J. & CASKEY, C. T. (1973). Mutations affecting the structure ofhypoxanthine: guanine phosphoriboayltransferase in cultured Chinese hamster cells. Proc.natn. Acad. Sci. U.S.A. 70, 320-324.

BRAY, G. A. (i960). A simple efficient liquid scintillator for counting aqueous solutions in aliquid scintillation counter. Analyt. Biochem. 1, 279—285.

CHOI, K. W. & BLOOM, A. D. (1970). Biochemically marked lymphocyte lines: establishmentof Lesch-Nyhan cells. Science, N. Y. 170, 89-90.

CHU, G. H. Y. & MALLING, H. V. (1968). Mammalian cell genetics. II. Chemical induction ofspecific locus mutations in Chinese hamster cells in vitro. Proc. natn. Acad. Sci. U.S.A. 61,1306-1312.

COFFINO, P. & SCHARFF, M. D. (1971). Rate of somatic mutation in immunoglobulin productionby mouse myeloma cells. Proc. natn. Acad. Sci. U.S.A. 68, 219-223.

CRUMPTON, M. J. & POLSON, A. (1965). A comparison of the conformation of sperm whalemetmyoglobin with that of apomyoglobin. J. molec. Biol. i t , 722-729.

DEYS, B. F. (1972). Demonstration of X-Linkage of GRPD, HGPRT and PGK in the Horse byMeans of Mule-Mouse Somatic Cell Hybridization. Ph.D. Thesis, tjniversity of Leiden,Leiden, The Netherlands.

EARLE, W. R. (1943). Production of malignancy in vitro. IV. The mouse fibroblast cultures andchanges seen in the living cells. J. natn. Cancer Inst. 4, 165-212.

EPSTEIN, C. J. (1969). Mammalian oocytes: X chromosome activity. Science, N.Y. 163,1078-1079.

EPSTEIN, C. J. (1972). Expression of the mammalian X-chromosome before and after fertiliza-tion. Science, N.Y. 175, 1467-1468.

GRZESCHIK, K.-H., ALLDERDICE, P. W., GRZESCHIK, A., OPTIZ, J. M., MILLER, O. J. & SINIS-CALCO, M. (1972). Cytological mapping of human x-linked genes by use of somatic cellhybrids involving an x-autosome translocation. Proc. natn. Acad. Sci. U.S.A. 69, 69—73.

HARRIS, H. (1970). Cell Fusion: The Dunham Lectures. Oxford: Clarendon Press.HARRIS, H. & COOK, P. R. (1969). Synthesis of an enzyme determined by an erythrocyte

nucleus in a hybrid cell. J. Cell Sci. 5, 121-134.HARRIS, M. (1971). Mutation rates in cells at different ploidy levels. J. cell. Physiol. 78,

177-184.HAYFLICK, L. (i960). Decontamination of tissue cultures infected with pleuropneumonia-like

organisms. Nature, Land. 185, 783-784.KLEBE, R. J., CHEN, T. R. & RUDDLE, F. H. (1970a). Controlled production of proliferating

somatic cell hybrids. J. Cell Biol. 45, 74-82.KLEBE, R. J., CHEN, T. & RUDDLE, F. H. (19706). Mapping of human genetic regulator element

by somatic cell genetic analysis. Proc. natn. Acad. Sci. U.S.A. 66, 1220-1227.KLEIN, G., BREGULA, U., WIENER, F. & HARRIS, H.(i97i). The analysis of malignancy by cell

fusion. I. Hybrids between tumour cells and L cell derivatives. J. Cell Sci. 8, 659-672.KRENITSKY, T. A., PAPAIOANNOU, R. & ELION, G. B. (1969). Human hypoxanthine-guanine

phosphoribosyltransferase, I. Purification, properties and specificity. J. biol. Chem. 244,1263-1270.

LESCH, M. & NYHAN, W. L. (1964). A familial disorder of uric acid metabolism and centralnervous system function. Am. J. Med. 36, 561-570.

250 5. Shin

LEVINE, E. (1972). Mycoplasma contamination of animal cell cultures: a simple rapid detectionmethod. Expl Cell Res. 74, 99-109.

LITTLEFIELD, J. W. (1963). The inosinic acid pyrophosphorylase activity of mouse fibroblastspartially resistant to 8-azaguanine. Proc. natn. Acad. Sd. U.S.A. 50, 568-576.

LITTLEFIELD, J. W. (1964a). Selection of hybrids from mating of fibroblasts in vitro and theirpresumed recombinants. Science, N.Y. 145, 709-710.

LITTLEFIELD, J. W. (19646). Three degrees of guanylic acid-inosinic acid pyrophosphorylasedeficiency in mouse fibroblasts. Nature, Loud. 203, 1142-1144.

LITTLEFIELD, J. W. (1966). The use of drug resistant markers to study the hybridization ofmouse fibroblasts. Expl Cell Res. 41, 190-196.

LYON, M. (1961). Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature,Lond. 190, 372-373-

LYON, M. (1971). Possible mechanisms of X-chromosome inactivation. Nature, New Biol. 232,229-232.

MEZGER-FREED, L. (1972). Effect of ploidy and mutagens on bromodeoxyuridine resistance inhaploid and diploid frog cells. Nature, New Biol. 235, Z45-246.

MIGEON, B. R., DER KALOUSTIAN, V. M., NYHAN, W. L., YOUNG, W. J. & CHILDS, B. (1968).X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: heterozygote hastwo clonal populations. Science, N. Y. 160, 425-427.

MILLER, O. J., COOK, P. R., MEERA KHAN, P., SHIN, S. & SINISCALCO, S. (1971). Mitoticseparation of two human x-linked genes in man-mouse somatic cell hybrids. Proc. natn. Acad.Sd. U.S.A. 68, n6-120.

NITOWSKY, H. (1972). Use of cell culture techniques in diagnosis and studies of inheriteddisease. Seminars in Hematology g, 403-429.

OHNO, S. (1967). Sex Chromosome and Sex Linked Genes. Berlin, Heidelberg, New York:Springer Verlag.

REICHLIN, M., HAY, M. & LEVINE, L. (1964). Antibodies to human Aj hemoglobin and theirreaction with A21S, C, and H hemoglobins. Immunochemistry 1, 21-30.

RUBIN, C. S., DANCIS, J., YIP, L. C, NOWINSKI, R. C. & BALIS, M. E. (1971). Purification ofIMP: pyrophosphate phosphoribosyltransferases, catalytically incompetent enzymes inLesch-Nyhan disease. Proc. natn. Acad. Sd. U.S.A. 68, 1461-1464.

RUDDLE, F. H. (1972). Linkage analysis using somatic cell hybrids. In Advances in HumanGenetics, vol. 3 (ed. H. Harris & K. Hirschhorn), pp. 173-236. New York and London:Plenum Press.

RUDDLE, F. H., CHAPMAN, V. M., RICCIUTI, F., MURNANE, M., KLEBE, R. & MEERA KHAN, P.(1971). Linkage relationships of seventeen human gene loci as determined by man-mousesomatic cell hybrids. Nature, New Biol. 232, 69—73.

RUSSELL, L. B. (1961). Genetics of mammalian sex chromosomes. Science, N. Y. 133, 1795-1803.SCHWARTZ, A. G., COOK, P. R. & HARRIS, H. (1971). Correction of a genetic defect in a mam-

malian cell. Nature, New Biol. 230, 5-8.SEEGMILLER, J. E., ROSENBLOOM, F. M. & KELLEY, W. N. (1967). Enzyme defect associated

with a sex-linked human neurological disorder and excessive purine synthesis. Science, N. Y.155, 1682-1684.

SHALTIEL, S. (1968). An immunochemical comparison between phosphorylase b and its apo-enzyme. Israel J. Chem. 6, 104P.

SHIN, S., CANEVA, R., SCHILDKRAUT, C, KLINGER, H. & SINISCALCO, S. (1973). Cells withphosphoribosyl transferase activity recovered from mouse cells resistant to 8-azaguanine.Nature, New Biol. 341, 194-196.

SHIN, S., MEERA KHAN, P. & COOK, P. R. (1971). Characterization of hypoxanthine-guaninephosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electro-phoretic method. Biochem. Genet. 5, 91-99.

SINISCALCO, S., KLINGER, H., EAGLE, H., KOPROWSKI, H., FUJIMOTO, W. Y. & SEEGMILLER,J. E. (1969). Evidence for intergenic complementation in hybrids derived from two humandiploid strains each carrying an X-linked mutation. Proc. natn. Acad. Sd. U.S.A. 62,793-799.

SZYBALSKA, E. H. & SzYBALSKi, W. (1962). Genetics of human cell lines. IV. DNA-mediatedheritable transformation of a biochemical trait. Proc. natn. Acad. Sd. U.S.A. 48, 2026-2034.


SZYBALSKI, W., SZYBALSKA, E. H. & RAGNI, G. (1962). Genetic studies with human cell lines.Natn. Cancer Inst. Monogr. 7, 75-89.

THOMPSON, L. H., MANKOVITZ, R., BAKER, R. M., WRIGHT, J. A., TILL, J. E., SIMINOVITCH, I..& WHITMORE, G. F. (1971). Selective and non-selective isolation of temperature sensitivemutants of mouse L-cells and their characterization. J. cell. Physiol. 78, 431-440.

VAN ZEELAND, A. A., VAN DIGGELEN, M. C. E. & SIMONS, J. W. I. M. (1972). The role ofmetabolic cooperation in selection of hypoxanthine-guanine-phosphoribosyl-transferase(HG-PRT)-deficient mutants from diploid mammalian cell strains. Mutation Res. 14,355-363-

WESTERVELD, A., VISSER, R. P. L. S., FREEKE, M. A. & BOOTSMA, D. (1972). Evidence for link-age of 3-phosphoglycerate kinase, hypoxanthine-guanine phosphoribosyl transferase, andglucose-6-phosphate dehydrogenase loci in Chinese hamster cells studied by using arelationship between gene multiplicity and enzyme activity. Biochem. Genet. 7, 33-40.

{Received 27 June 1973)

I 7 CEL 14

nature of mutations conferring resistance to 8 …tissue hgprt was prepared to test a9 and rag for...

Documents