Mutation Research, 263 (1991) 151-158
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© 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100058B MUTLET 0504
Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells Li-Hua Zhang and Dag Jenssen Department of Genetic and Cellular Toxicology, WallenbergLaboratory, Stockholm University, S-106 91 Stockholm (Sweden)
(Received 30 November 1990) (Revision received 26 February 1991) (Accepted 28 February 1991)
Keywords: HPRT mutants; V79 Chinese hamster; HAT resistance; HAsT resistance
Summary H P R T mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., H A T or H A s T medium). However, it has been shown that some H P R T - clones are resistant to both H A T and H A s T medium. The present study was undertaken to investigate whether any c o m m o n structural gene alteration exists for such 6TGr-HATr-HAsT r clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of M N U and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the H P R T gene, these 4 investigated 6TGr-HATr-HAsT r clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and e-azaserine suggesting that these clones carry mutations in the structural part of the H P R T gene. Sequence analysis of PCR-amplified H P R T c D N A from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic D N A after digestion with BgIII, E c o R I and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic D N A using exon 4-specific primers showed that all these 3 mutants had an A T to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the H P R T protein.
Correspondence: Dr. L.-H. Zhang, Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, S-10691 Stockholm (Sweden).
The enzyme hypoxanthine-guanine phosphoribosyltransferase ( H P R T ) converts 5-phosphorylribose-l-pyrophosphate ( P R P P ) and hypoxanthine or guanine to inosine monophosphate (IMP) and guanine monophosphate (GMP), respectively, in
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the salvage pathway of purine biosynthesis. Deficiency of the H P R T enzyme in humans is associated with 2 clinical syndromes, Lesch-Nyhan syndrome and gout. The H P R T gene has been used extensively in mutational studies of cultured m a m malian cells because of its location on the X-chrom o s o m e and because simple selective media allow for growth of H P R T + and H P R T - cells. 6-Thioguanine (6TG) and 8-azaguanine (8AG) have been used as selective agents for the isolation of H P R T mutant clones in the forward mutation assay and H A T (hypoxanthine, aminopterin and thymidine) or H A s T (hypoxanthine, L-azaserine and thymidine) has been applied for selection of H P R T + revertant clones. L-Azaserine, a glutamine analogue, inhibits mainly the conversion of formyl-glycinamide ribonucleoside to formyl-glycinamide ribonucleotide in de novo purine biosynthesis, while aminopterin, an antagonist of folic acid, blocks both de novo purine and pyrimidine biosynthesis by blocking tetrahydrofolate reductase and CH3 transfer. L-Azaserine has been suggested to be a more efficient selective agent when used in the reverse mutation assay (Fujimoto et al., 1971; Fuscoe et al., 1982). In a previous study (Zhang and Jenssen, 1989) we have characterized spontaneously occurring mutations at the H P R T locus of V79 Chinese hamster cells by reverse mutation analysis in an attempt to trace unusual mutational events for further mechanistic studies. We found that 2 H P R T mutant clones (6TG~), one of spontaneous origin and the other EMS-induced, were resistant to both H A T and H A s T medium. The nature of H A T a n d / o r H A s T mutants has not been considered for investigation other than with regard to their enzyme properties (Fenwick et al., 1977; Epstein et al., 1977; Sharp et al., 1973; Taylor et al., 1978). Gillen et al. (1972) reported that about 50°7o of hamster cell lines resistant to 8AG will grow in H A T medium and incorporate radioactive hypoxanthine into nucleic acids. A number of these cell lines had an altered affinity for either of the substrates hypoxanthine or P R P P suggesting that they are true mutations in the structural gene (Fenwick et al., 1977). The present study was under-
taken to investigate whether any c o m m o n structural gene alteration exists for the 6 T G r - H A T rH A s T r clones. Materials and methods Cell culture and isolation o f 6TG r H P R T mutants
Chinese hamster lung V79 cells were used as the parent cell line in this study. Cell cultures of V79 cells were held according to the standard procedure described earlier (Jenssen, 1984). A modified test protocol for forward mutations previously described by Zhang and Jenssen (1989) was followed for isolation of independent H P R T mutants. The induction of mutations was performed by growing wild-type V79 cells in H A T selective medium prior to treatment with 10 mM EMS in HBSS ++ solution for 30 min or 0.05 mM MNU in HBSS ++ solution for 15 rain. Mutants were selected on petri dishes according to the protocol by Jenssen (1984) using 5 /~g/ml 6TG. Only 1 mutant clone was isolated from each petri dish to avoid selection of sister colonies. The ability of these 6 T G ' mutant cells to grow in H A T or H A s T medium was investigated by using 5 x 10 -5 M hypoxanthine, 5 x 10 - 6 M thymidine and 4 × 1 0 -7 M aminopterin or l x 1 0 -5 M Lazaserine. H P R T e n z y m e activity
The H P R T activity of 6TG r mutant clones was measured in growing cells according to a modified protocol previously described by Zhang and Jenssen (1989), using incorporation of 3H-hypoxanthine (Amersham, 5.5 Ci/mmole) in the presence or absence of aminopterin or L-azaserine. H P R T enzyme activity was also measured in cytosolic preparations by the conversion of 3H-hypoxanthine (3H-hx) to I M P according to a modification of the method by Fenwick and Caskey (1975). Cytosolic cell extracts were prepared from 1-2 × 10 v cells which were harvested, washed twice in cold PBS and lysed in 10 mM Tris-HC1 (pH 7.4), 5 mM mercaptoethanol, 0.5% Triton X-100. The lysate was centrifuged and the supernatants which contained 0.8-2.1 m g / m l protein (measured by the method of Petersson, 1977) were stored in aliquots
153 at - 7 0 ° C . An assay mixture of 50 /zl contained 18.2 /xM 3H-hx, 2 mM P R P P , 5 mM MgSO4, 50 mM Tris-HC1 (pH 7.4) and 7.6-13.0/~g protein. In the case of wild-type and EMS3 cells the protein content was 1 /zg per 50 #1 assay mixture. Enzyme reactions were incubated at 37°C, and 10-t~l samples were withdrawn after 0, 15 and 30 rain. The samples were spotted onto DE81 Whatman filters which had previously been spotted with 25 #1 of 0.1 M E D T A (pH 7). After drying the filters were washed with 3 x 10 ml of MeOH:HzO (1:1) to remove unreacted purine bases and dried again. The amount of radioactive IMP on the filters was determined by liquid scintillation counting.
42°C in 40% deionized formamide, 5 x Denhardt's solution (1 × = 0 . 0 2 % (w/v) each of BSA, Ficoll and polyvinyl pyrrolidone), 5 x SSC, 1% SDS and 100/zg/ml sonicated denatured herring sperm DNA solution. Hybridization was carried out for at least 24 h at 42°C under the same conditions apart from the addition of 5% dextran sulfate and the 32p-labeled probe ( > l x l 0 6 cpm/ml). The filter was washed twice with 2 × SSC, 1% SDS and 1 × SSC, 1% SDS for 30 min each at 65°C. After washing with 2 x SSC at room temperature, the filter was exposed to Cronex Xray film in the presence of an intensifying screen for 3-7 days at - 7 0 ° C .
D N A isolation and blot hybridization Cultured cells (20 x 106) were harvested, washed, lysed in 10 ml of 10 mM Tris-HC1 (pH 8), 10 mM NaC1, 10 mM EDTA, 1°70 SDS and incubated at 37°C overnight in the presence of 0.2 mg/ml proteinase K. 1.5 ml of saturated NaC1 was added, the D N A was precipitated with ethanol, dissolved in 0.5-1.0 ml TE, adjusted to 2.5 M NHaAc and precipitated with 50°70 isopropanol. The DNA was then collected with a glass rod, washed in 70°7o ethanol and dissolved in TE solution. Chromosomal DNA (15 #g) was digested with restriction enzymes under conditions recommended by the manufacturer (Boehringer, Mannheim) for 5 h at 37°C. The DNA fragments were electrophoresed on 0.8°70 (w/v) agarose in Tris-bor a t e - E D T A buffer. Before vacuum transfer of DNA to Hybond-N nylon membrane (Amersham) in 2 0 x S S C , the gel was photographed, equilibrated in 0.25 M HCI, denatured in 0.5 M NaOH, 1.5 M NaC1 and finally neutralized in 0.5 M Tris-HC1 (pH 7.0), 1.5 M NaC1. After blotting DNA was fixed onto the nylon membrane by UV light. For hybridization a 1.3-kb PstI fragment of p H P T 5 (Konecki et al., 1982) containing a fulllength cDNA of mouse H P R T was used. 100 ng cDNA fragments were nick-translated in the presence of 20 t~Ci 3zp-dCTP (3000 Ci/mmole) in 15/xl reaction mix to a level of > 108 cpm//~g DNA according to the supplier's protocol (Amersham). The nylon membrane was prehybridized for 4 h at
R N A isolation and blot hybridization Preparation of cytoplasmic RNA was performed according to the protocol described by Vrieling et al. (1988b). Cytoplasmic RNA (20 /zg) was separated by electrophoresis through 1% agarose gel and transferred onto Hybond-N nylon membrane under conditions specified by the supplier. Pre- and hybridization were carried out at 42°C in 5°70 dextran sulfate, 1°70 SDS, 50o7o formamide, 50 /~g/ml sonicated and denatured herring sperm DNA, 1 M NaC1 solution for 4 h and overnight respectively. A 1.3-kb mouse H P R T cDNA insert from p H P T 5 (Konecki et al., 1982) and a l . l - k b mouse 7-actin cDNA fragment (Vrieling et al., 1988a) were used as probes. Before rehybridization the label on the filter was removed by pouring a boiling solution of 0.1% (w/v) SDS on the membrane and allowing it to cool down to room temperature. RNA blots were washed and autoradiographed in the same manner as the DNA blots. Densitometric scans of autoradiogram were performed using a Pharmacia laser densitometer. Sequence analysis o f H P R T cDNA Preparation and amplification of H P R T cDNA by the polymerase chain reaction (PCR) procedure (Saiki et al., 1985) were performed according to the protocol of Vrieling et al. (1989). Amplified DNA was purified and digested with ClaI and EcoRI and cloned into Accl + EcoRI-restricted m p l 9 sequencing vector. Cloning of H P R T cDNA containing
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M13 clones and sequence analysis applying Sanger's dideoxy chain termination technique (Sanger et al., 1977) were performed according to Vrieling et al. (1988b). Oligonucleotide primers for DNA amplification and DNA sequencing were kindly provided by Drs. van Zeeland and Vrieling.
Sequence analysis of H P R T genomic DNA Preparation, PCR amplification of genomic DNA using exon 4-specific primers and direct sequencing of double-stranded PCR-amplified DNA were performed according to the protocol developed by Drs. M. Giphart-Gassler and H. den Dulk (personal communication). Results
SP9 and SP 13 were spontaneously occurring mutant clones and EM$3 was isolated after treatment with 10 mM EMS; MNU4 and MNU8 were independent mutants induced by 0.05 mM MNU. The result of the phenotypical characterization of these mutant clones is summarized in Table 1. Cloning efficiency of the mutants ranged from 34% to 61% giving similar results using MEM with or without 6TG. SP13, a deletion mutant, and wild-type V79 cells were used as positive controls in
this study. Mutants resistant to 6TG are expected to be deficient in their H P R T enzyme activity, unable to utilize hypoxanthine for nucleotide synthesis and unable to survive in medium containing H A T or HAST. The 4 selected 6TG-resistant mutant clones SP9, EMS3, MNU4 and MNU8 have the common feature of being resistant to H A T and HAST. In contrast to wild-type cells and SP 13 cells, analysis of their H P R T enzyme activity by cellular incorporation of exogenous 3H-hx indicated a 2-5-fold enhanced incorporation in the presence of aminopterin and L-azaserine (Table 1). This result is consistent with the result of Gillin et al. (1972) and Caskey et al. (1975) who found that in the case of 8AGr-HAT ~ cells, the utilization of exogenous hypoxanthine or other purine substrates of H P R T is dependent upon the presence of aminopterin in the medium. Although cellular incorporation of 3H-hx for SP9 varied between experiments, the level of enhancement in the presence of aminopterin or L-azaserine was always about the same. As expected, H P R T enzyme activity in a cytosolic preparation of SP13 was below the detection level, while SP9, MNU4 and MNU8 showed about the same low activity. Unexpectedly, EMS3 showed the same activity as wild-type cells.
TABLE 1 C L O N I N G E F F I C I E N C Y A N D A N A L Y S I S O F H P R T E N Z Y M E A C T I V I T Y IN 5 H P R T M U T A N T C L O N E S O F V79 C H I N E S E HAMSTER CELLS Mutant
C l o n i n g efficiency a (°7o) . . . . MEM 6TG HAT
. HAsT b
SP9
47
44
EMS3
42
41
15
+
MNU4 MNU8 SP13 V79 w i l d - t y p e
35 53 61 80
34 51 55 0
10 47 0 44
+ + + + + +
a
25
.
+ +
3 H - H y p o x a n t h i n e i n c o r p o r a t i o n c (%) . . 10 #M 10/~M L-As aminopterin
preparation'
(%)
24.9 16.7 5.1 4.0
69.3 29.7 15.2
70.7 70.9 28.2 8.9
1 ! 8.2
8.9 3.4 4.2 100
25.3 14.5 0.5 125.3
21.9 13.1 1.1 129.8
1.4 2.0 0.0 100
070 c l o n i n g efficiency in M E M (complete), 6 T G - M E M , or H A T - M E M (500 c e l l s / 9 - c m dish). - , no g r o w t h ; + p a r t i a l g r o w t h ; + + , c o m p l e t e g r o w t h in H A s T - M E M (106 c e l l s / 9 - c m dish). °/0 of wild type.
H P R T activity in cytosolic
6.0
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TABLE 2 R E S U L T OF cDNA S E Q U E N C I N G A N D m R N A ANALYSIS OF 5 H P R T M U T A N T CLONES OF V79 C H I N E S E H A M S T E R CELLS Mutant
SP9 EMS3 MNU4 MNU8 SP13 V79 wild type
m R N A level
Target sequence alteration
Remarks
(OD H P R T / O D -y-actin)a
°70 of wild type
cDNA (5' to 3 ' )
genomic D N A (5' to 3 ' )
0.919 1.135 0.566 1.829 0 1.399
66 81 40 131 0 100
exon 4 missing C-416 b, G C > A T exon 4 missing exon 4 missing H P R T gene missing no change
A-338 b, A T - , G C C-331 b, G C - , A T C-331 b, G C - , A T -
splice Thr to Asn splice splice deletion normal
a Ratio of integrated area of optical density for H P R T m R N A and -r-actin m R N A . The range of the measured OD values of the different clones was 1.336-3.606 except for SP13 giving O D = 0 for H P R T m R N A . b Position within the H P R T coding region.
To investigate the molecular nature of the common feature of these mutants, we isolated cytoplasmic RNA for analysis of mRNA levels by Northern blotting using a full-length mouse H P R T cDNA probe. As a reference for the H P R T mRNA level, the blots were reprobed for -y-actin mRNA. The level of H P R T mRNA in the different mutants ranges from 40°70 to 131070 of the wild-type level (Table 2). No H P R T mRNA could be detected in SP13, carrying a complete H P R T deletion. Genomic DNA purified from each mutant clone was analyzed by Southern blotting after digestion with BgllI, EcoRI and PstI. None of the 4 mutant clones SP9, EMS3, MNU4 and MNU8 showed any change in fragment patterns as compared to the wild type. As expected, the fragment pattern of control clone SP13 showed only the bands corresponding to the pseudogene of the hamster H P R T gene. Fig. 1 shows the fragment patterns of DNA from all clones compared to wild-type DNA digested by PstI. The probe identified 5 restriction fragments of 8.2, 6.5, 5.6, 4.0 and 0.96 kb which is comparable with the results of others (Fuscoe et al., 1983; Rossiter et al., 1990). The results from the Southern and Northern blot analyses indicated that the 4 6TGr-HATr-HAsT ~ clones contained no large alterations in H P R T gene suggesting that a point mutation caused the 6TG phenotype. Sequence analysis of the PCR-
A
B
C
D
E
F
23.1 m 9.4-6.6-4.4--
2°3~
2.0--
1.0--
Fig. 1. Autoradiograph of V79 Chinese hamster H P R T gene digested by restriction enzyme PstI. D N A fragments were separated on 0.8% agarose gel and hybridized with a 1.3-kb fulllength mouse H P R T cDNA probe. Lanes A - F correspond to SP13, SP9, EMS3, MNU4, M N U 8 and wild-type V79 cells, respectively.
156
amplified H P R T coding region of these mutants was performed to investigate the precise alterations in the H P R T gene. The result in Table 2 shows that c D N A of 3 of the 4 clones lacked exon 4. EMS3, however, contained a GC to AT transition at position 416 in exon 6. The 3 clones lacking exon 4 from c D N A had normal PstI restriction fragment patterns, which includes a fragment of 6.5 kb containing exon 4 (Rossiter et al., 1990). Further sequence analysis of PCR-amplified genomic DNA, using exon 4-specific primers, showed that SP9 contained an A T to GC transition at position 338 in exon 4, while MNU4 and MNU8 both had a GC to A T transition at position 331 in exon 4. These data indicate that the mutation in all 3 clones probably involves the splicing mechanism of exon 4. Discussion Studies on 8AG-resistant cell lines demonstrated a c o m m o n feature for this type of cells which utilize hypoxanthine poorly during normal growth conditions, but well during inhibition of the de novo purine biosynthesis by aminopterin (Chu et al., 1969; Gillen et al., 1972). Fenwick et al. (1977) found that each resistant clone studied produced H P R T with a reduced catalytic capacity at low concentrations of P R P P . The authors concluded that these cell lines carried mutants in the structural part of the gene. In the present paper we investigated 4 6 T G r - H A T r - H A s T r V79 cell lines which have similar properties as 8AGr-HAT r cell lines with regard to the enhanced incorporation of hypoxanthine in the presence of aminopterin or L-azaserine. Because aminopterin or t-azaserine inhibits de novo purine synthesis leading to an elevated level of P R P P available for the H P R T enzyme, these types of mutants are able to grow in H A T or H A s T medium. Investigation of about 100 H P R T mutant clones showed that about 15°70 of them were resistant to H A T and only the 4 mutants described in this paper were also resistant to H A s T (data not shown). H P R T is one in a group of 8 phosphoribosyltransferases which are functionally related meaning that they have similar physical and catalytic
properties (Musick, 1981) and all use P R P P as the first substrate. The H P R T protein contains 3 proposed domains: the amino acids encoded in exons 1-3, 4-6 and 7-9 correspond to the substratebinding domain, the catalytic domain and a functional unknown domain, respectively. The substrate-binding domain corresponds to the Nterminal 120 residues of the h u m a n H P R T protein and the catalytic domain to about 20 amino acids beginning at residue 126 of the H P R T protein (Agros et al., 1983; Dush et al., 1985). A human mutation (HPRT-Munich) with an amino acid change at residue 103 in a region adjacent to the proposed hypoxanthine binding site showed a strongly reduced affinity for hypoxanthine (Wilson et al., 1983). However, mutant H P R T - L o n d o n with a substitution of leucine for serine at residue 109 had a slightly reduced affinity for hypoxanthine, probably because the mutation was located in a region with a predicted turn that connects 2/5strands in the proposed dinucleotide fold for the putative binding sites of this substrate. Based on their properties of hypoxanthine incorporation (Table 1) the present mutant clones also seem to fit the proposed model for the functional domains in H P R T protein. The amino acid residue sequences encoded in exon 4 are located between the substrate-binding domain and the catalytic domain. The mutations SP9, MNU4 and MNU8 caused by a base substitution giving a defect splicing of exon 4 are suggested to affect the affinity for hypoxanthine and the rate of substrate conversion. According to our data, a deletion of exon 4 (66 bp) has limited effect on the H P R T protein and it still maintains a certain degree of activity. Mutant EMS3 had an amino acid change by a G C - A T transition at residue 139, localized in the proposed catalytic domain, which theoretically should reduce the rate of conversion of P R P P and hypoxanthine or guanine to I M P or G M P . Surprisingly, the enzyme activity in cytosolic preparations was similar to wild-type cells but low using exogenous incorporation of 3H-hx, suggesting a more complicated picture of this clone possibly involving other mechanisms interfering with purine metabolism.
157
We have isolated and characterized about 100 H P R T mutant clones of independent origin, one half occurring spontaneously and the other half induced by EMS or MNU. Only 4 of these mutant clones showed the ability to growh in both H A T and HAsT medium and 3 out of these 4 clones were caused by a splice mutation. Interestingly all 3 mutant clones were caused by a new type of splicing defects: a base change in the coding region resulted in splicing out the exon 4 sequence of the H P R T gene, which is consistent with a recent report by Dorado et al. (1990). The most common/3-globin splicing defects are due to single-base substitutions that prevent splicing at a particular site (reviewed by Collins and Weissman, 1984; Orkin and Kazazian, 1984). Mitchell et al. (1986) analyzed 3 spontaneously occurring mutants at the DHFR locus of Chinese hamster ovary cells and all 3 exhibited exon 5 splicing mutations, suggesting that these splice sites were hot spots for spontaneous mutations. In the H P R T locus, spontaneously occurring mutations are frequently caused by splicing defects (Zhang et al., 1991; Rossi et al., 1990). Since MNU4 and MNU8 were mutants induced by a low dose of MNU exhibiting a mutation frequency as low as 2.6/105 cells compared to a spontaneous mutation frequency of 0.4/105 cells, we cannot exclude the possibility that both these mutants are of spontaneous origin.
Acknowledgements We thank Dr. Harry Vrieling for critically reading the manuscript and Dr. Belinda J.F. Rossiter for providing her manuscript in advance o f publication.
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enzymes in drug-resistant variants of mammalian tissue culture cells, Proc. Natl. Acad. Sci. (U.S.A.), 70, 3145-3149. Taylor, M.W., M.K. Tokito, K.C, Gupta and J. Pipkorn (1978) Regulation of de novo purine biosynthesis in normal and 8-azaguanine-resistant Chinese hamster cells, Biochim. Biophys. Acta, 517, 1-13. Vrieling, H., M.J. Niericker, J.W.I.M. Simons and A.A. van Zeeland (1988a) Molecular analysis of mutations induced by N-ethyl-N-nitrosourea at the HPRT locus in mouse lymphoma cells, Mutation Res., 198, 99-106. Vrieling, H., J.W.I.M. Simons and A.A. van Zeeland (1988b) Nucleotide sequence determination of point mutations at the mouse HPRT locus using in vitro amplification of hprt mRNA sequences, Mutation Res., 198, 107-113. Vrieling, H., M.L. van Rooijen, N.A. Groen, M.Z. Zdzienicka, J.W.I.M. Simons, P.H.M. Lohman and A.A. van Zeeland (1989) DNA strand specificity for UV-induced mutations in mammalian ceils, Mol. Cell. Biol., 9, 1277-1283. Wilson, J.M., A.B. Young and W.N. Kelley (1983) Hypoxanthine-guanine phosphoribosyltransferase deficiency. The molecular basis of the clinical syndromes, N. Engl. J. Med., 309, 900-910. Zhang, L.-H., and D. Jenssen (1989) Isolation and characterization of spontaneously occurring mutations at the HPRT locus in V79 Chinese hamster cells, Mutation Res., 212, 253-262. Zhang, L.-H., H. Vrieling, A.A. van Zeeland and D. Jenssen (1991) Spectrum of spontaneous mutations in the HPRT genc of V79 Chinese hamster cells, in preparation. Communicated by F.H. Sobels
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© 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100058B MUTLET 0504
Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells Li-Hua Zhang and Dag Jenssen Department of Genetic and Cellular Toxicology, WallenbergLaboratory, Stockholm University, S-106 91 Stockholm (Sweden)
(Received 30 November 1990) (Revision received 26 February 1991) (Accepted 28 February 1991)
Keywords: HPRT mutants; V79 Chinese hamster; HAT resistance; HAsT resistance
Summary H P R T mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., H A T or H A s T medium). However, it has been shown that some H P R T - clones are resistant to both H A T and H A s T medium. The present study was undertaken to investigate whether any c o m m o n structural gene alteration exists for such 6TGr-HATr-HAsT r clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of M N U and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the H P R T gene, these 4 investigated 6TGr-HATr-HAsT r clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and e-azaserine suggesting that these clones carry mutations in the structural part of the H P R T gene. Sequence analysis of PCR-amplified H P R T c D N A from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic D N A after digestion with BgIII, E c o R I and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic D N A using exon 4-specific primers showed that all these 3 mutants had an A T to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the H P R T protein.
Correspondence: Dr. L.-H. Zhang, Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, S-10691 Stockholm (Sweden).
The enzyme hypoxanthine-guanine phosphoribosyltransferase ( H P R T ) converts 5-phosphorylribose-l-pyrophosphate ( P R P P ) and hypoxanthine or guanine to inosine monophosphate (IMP) and guanine monophosphate (GMP), respectively, in
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the salvage pathway of purine biosynthesis. Deficiency of the H P R T enzyme in humans is associated with 2 clinical syndromes, Lesch-Nyhan syndrome and gout. The H P R T gene has been used extensively in mutational studies of cultured m a m malian cells because of its location on the X-chrom o s o m e and because simple selective media allow for growth of H P R T + and H P R T - cells. 6-Thioguanine (6TG) and 8-azaguanine (8AG) have been used as selective agents for the isolation of H P R T mutant clones in the forward mutation assay and H A T (hypoxanthine, aminopterin and thymidine) or H A s T (hypoxanthine, L-azaserine and thymidine) has been applied for selection of H P R T + revertant clones. L-Azaserine, a glutamine analogue, inhibits mainly the conversion of formyl-glycinamide ribonucleoside to formyl-glycinamide ribonucleotide in de novo purine biosynthesis, while aminopterin, an antagonist of folic acid, blocks both de novo purine and pyrimidine biosynthesis by blocking tetrahydrofolate reductase and CH3 transfer. L-Azaserine has been suggested to be a more efficient selective agent when used in the reverse mutation assay (Fujimoto et al., 1971; Fuscoe et al., 1982). In a previous study (Zhang and Jenssen, 1989) we have characterized spontaneously occurring mutations at the H P R T locus of V79 Chinese hamster cells by reverse mutation analysis in an attempt to trace unusual mutational events for further mechanistic studies. We found that 2 H P R T mutant clones (6TG~), one of spontaneous origin and the other EMS-induced, were resistant to both H A T and H A s T medium. The nature of H A T a n d / o r H A s T mutants has not been considered for investigation other than with regard to their enzyme properties (Fenwick et al., 1977; Epstein et al., 1977; Sharp et al., 1973; Taylor et al., 1978). Gillen et al. (1972) reported that about 50°7o of hamster cell lines resistant to 8AG will grow in H A T medium and incorporate radioactive hypoxanthine into nucleic acids. A number of these cell lines had an altered affinity for either of the substrates hypoxanthine or P R P P suggesting that they are true mutations in the structural gene (Fenwick et al., 1977). The present study was under-
taken to investigate whether any c o m m o n structural gene alteration exists for the 6 T G r - H A T rH A s T r clones. Materials and methods Cell culture and isolation o f 6TG r H P R T mutants
Chinese hamster lung V79 cells were used as the parent cell line in this study. Cell cultures of V79 cells were held according to the standard procedure described earlier (Jenssen, 1984). A modified test protocol for forward mutations previously described by Zhang and Jenssen (1989) was followed for isolation of independent H P R T mutants. The induction of mutations was performed by growing wild-type V79 cells in H A T selective medium prior to treatment with 10 mM EMS in HBSS ++ solution for 30 min or 0.05 mM MNU in HBSS ++ solution for 15 rain. Mutants were selected on petri dishes according to the protocol by Jenssen (1984) using 5 /~g/ml 6TG. Only 1 mutant clone was isolated from each petri dish to avoid selection of sister colonies. The ability of these 6 T G ' mutant cells to grow in H A T or H A s T medium was investigated by using 5 x 10 -5 M hypoxanthine, 5 x 10 - 6 M thymidine and 4 × 1 0 -7 M aminopterin or l x 1 0 -5 M Lazaserine. H P R T e n z y m e activity
The H P R T activity of 6TG r mutant clones was measured in growing cells according to a modified protocol previously described by Zhang and Jenssen (1989), using incorporation of 3H-hypoxanthine (Amersham, 5.5 Ci/mmole) in the presence or absence of aminopterin or L-azaserine. H P R T enzyme activity was also measured in cytosolic preparations by the conversion of 3H-hypoxanthine (3H-hx) to I M P according to a modification of the method by Fenwick and Caskey (1975). Cytosolic cell extracts were prepared from 1-2 × 10 v cells which were harvested, washed twice in cold PBS and lysed in 10 mM Tris-HC1 (pH 7.4), 5 mM mercaptoethanol, 0.5% Triton X-100. The lysate was centrifuged and the supernatants which contained 0.8-2.1 m g / m l protein (measured by the method of Petersson, 1977) were stored in aliquots
153 at - 7 0 ° C . An assay mixture of 50 /zl contained 18.2 /xM 3H-hx, 2 mM P R P P , 5 mM MgSO4, 50 mM Tris-HC1 (pH 7.4) and 7.6-13.0/~g protein. In the case of wild-type and EMS3 cells the protein content was 1 /zg per 50 #1 assay mixture. Enzyme reactions were incubated at 37°C, and 10-t~l samples were withdrawn after 0, 15 and 30 rain. The samples were spotted onto DE81 Whatman filters which had previously been spotted with 25 #1 of 0.1 M E D T A (pH 7). After drying the filters were washed with 3 x 10 ml of MeOH:HzO (1:1) to remove unreacted purine bases and dried again. The amount of radioactive IMP on the filters was determined by liquid scintillation counting.
42°C in 40% deionized formamide, 5 x Denhardt's solution (1 × = 0 . 0 2 % (w/v) each of BSA, Ficoll and polyvinyl pyrrolidone), 5 x SSC, 1% SDS and 100/zg/ml sonicated denatured herring sperm DNA solution. Hybridization was carried out for at least 24 h at 42°C under the same conditions apart from the addition of 5% dextran sulfate and the 32p-labeled probe ( > l x l 0 6 cpm/ml). The filter was washed twice with 2 × SSC, 1% SDS and 1 × SSC, 1% SDS for 30 min each at 65°C. After washing with 2 x SSC at room temperature, the filter was exposed to Cronex Xray film in the presence of an intensifying screen for 3-7 days at - 7 0 ° C .
D N A isolation and blot hybridization Cultured cells (20 x 106) were harvested, washed, lysed in 10 ml of 10 mM Tris-HC1 (pH 8), 10 mM NaC1, 10 mM EDTA, 1°70 SDS and incubated at 37°C overnight in the presence of 0.2 mg/ml proteinase K. 1.5 ml of saturated NaC1 was added, the D N A was precipitated with ethanol, dissolved in 0.5-1.0 ml TE, adjusted to 2.5 M NHaAc and precipitated with 50°70 isopropanol. The DNA was then collected with a glass rod, washed in 70°7o ethanol and dissolved in TE solution. Chromosomal DNA (15 #g) was digested with restriction enzymes under conditions recommended by the manufacturer (Boehringer, Mannheim) for 5 h at 37°C. The DNA fragments were electrophoresed on 0.8°70 (w/v) agarose in Tris-bor a t e - E D T A buffer. Before vacuum transfer of DNA to Hybond-N nylon membrane (Amersham) in 2 0 x S S C , the gel was photographed, equilibrated in 0.25 M HCI, denatured in 0.5 M NaOH, 1.5 M NaC1 and finally neutralized in 0.5 M Tris-HC1 (pH 7.0), 1.5 M NaC1. After blotting DNA was fixed onto the nylon membrane by UV light. For hybridization a 1.3-kb PstI fragment of p H P T 5 (Konecki et al., 1982) containing a fulllength cDNA of mouse H P R T was used. 100 ng cDNA fragments were nick-translated in the presence of 20 t~Ci 3zp-dCTP (3000 Ci/mmole) in 15/xl reaction mix to a level of > 108 cpm//~g DNA according to the supplier's protocol (Amersham). The nylon membrane was prehybridized for 4 h at
R N A isolation and blot hybridization Preparation of cytoplasmic RNA was performed according to the protocol described by Vrieling et al. (1988b). Cytoplasmic RNA (20 /zg) was separated by electrophoresis through 1% agarose gel and transferred onto Hybond-N nylon membrane under conditions specified by the supplier. Pre- and hybridization were carried out at 42°C in 5°70 dextran sulfate, 1°70 SDS, 50o7o formamide, 50 /~g/ml sonicated and denatured herring sperm DNA, 1 M NaC1 solution for 4 h and overnight respectively. A 1.3-kb mouse H P R T cDNA insert from p H P T 5 (Konecki et al., 1982) and a l . l - k b mouse 7-actin cDNA fragment (Vrieling et al., 1988a) were used as probes. Before rehybridization the label on the filter was removed by pouring a boiling solution of 0.1% (w/v) SDS on the membrane and allowing it to cool down to room temperature. RNA blots were washed and autoradiographed in the same manner as the DNA blots. Densitometric scans of autoradiogram were performed using a Pharmacia laser densitometer. Sequence analysis o f H P R T cDNA Preparation and amplification of H P R T cDNA by the polymerase chain reaction (PCR) procedure (Saiki et al., 1985) were performed according to the protocol of Vrieling et al. (1989). Amplified DNA was purified and digested with ClaI and EcoRI and cloned into Accl + EcoRI-restricted m p l 9 sequencing vector. Cloning of H P R T cDNA containing
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M13 clones and sequence analysis applying Sanger's dideoxy chain termination technique (Sanger et al., 1977) were performed according to Vrieling et al. (1988b). Oligonucleotide primers for DNA amplification and DNA sequencing were kindly provided by Drs. van Zeeland and Vrieling.
Sequence analysis of H P R T genomic DNA Preparation, PCR amplification of genomic DNA using exon 4-specific primers and direct sequencing of double-stranded PCR-amplified DNA were performed according to the protocol developed by Drs. M. Giphart-Gassler and H. den Dulk (personal communication). Results
SP9 and SP 13 were spontaneously occurring mutant clones and EM$3 was isolated after treatment with 10 mM EMS; MNU4 and MNU8 were independent mutants induced by 0.05 mM MNU. The result of the phenotypical characterization of these mutant clones is summarized in Table 1. Cloning efficiency of the mutants ranged from 34% to 61% giving similar results using MEM with or without 6TG. SP13, a deletion mutant, and wild-type V79 cells were used as positive controls in
this study. Mutants resistant to 6TG are expected to be deficient in their H P R T enzyme activity, unable to utilize hypoxanthine for nucleotide synthesis and unable to survive in medium containing H A T or HAST. The 4 selected 6TG-resistant mutant clones SP9, EMS3, MNU4 and MNU8 have the common feature of being resistant to H A T and HAST. In contrast to wild-type cells and SP 13 cells, analysis of their H P R T enzyme activity by cellular incorporation of exogenous 3H-hx indicated a 2-5-fold enhanced incorporation in the presence of aminopterin and L-azaserine (Table 1). This result is consistent with the result of Gillin et al. (1972) and Caskey et al. (1975) who found that in the case of 8AGr-HAT ~ cells, the utilization of exogenous hypoxanthine or other purine substrates of H P R T is dependent upon the presence of aminopterin in the medium. Although cellular incorporation of 3H-hx for SP9 varied between experiments, the level of enhancement in the presence of aminopterin or L-azaserine was always about the same. As expected, H P R T enzyme activity in a cytosolic preparation of SP13 was below the detection level, while SP9, MNU4 and MNU8 showed about the same low activity. Unexpectedly, EMS3 showed the same activity as wild-type cells.
TABLE 1 C L O N I N G E F F I C I E N C Y A N D A N A L Y S I S O F H P R T E N Z Y M E A C T I V I T Y IN 5 H P R T M U T A N T C L O N E S O F V79 C H I N E S E HAMSTER CELLS Mutant
C l o n i n g efficiency a (°7o) . . . . MEM 6TG HAT
. HAsT b
SP9
47
44
EMS3
42
41
15
+
MNU4 MNU8 SP13 V79 w i l d - t y p e
35 53 61 80
34 51 55 0
10 47 0 44
+ + + + + +
a
25
.
+ +
3 H - H y p o x a n t h i n e i n c o r p o r a t i o n c (%) . . 10 #M 10/~M L-As aminopterin
preparation'
(%)
24.9 16.7 5.1 4.0
69.3 29.7 15.2
70.7 70.9 28.2 8.9
1 ! 8.2
8.9 3.4 4.2 100
25.3 14.5 0.5 125.3
21.9 13.1 1.1 129.8
1.4 2.0 0.0 100
070 c l o n i n g efficiency in M E M (complete), 6 T G - M E M , or H A T - M E M (500 c e l l s / 9 - c m dish). - , no g r o w t h ; + p a r t i a l g r o w t h ; + + , c o m p l e t e g r o w t h in H A s T - M E M (106 c e l l s / 9 - c m dish). °/0 of wild type.
H P R T activity in cytosolic
6.0
155
TABLE 2 R E S U L T OF cDNA S E Q U E N C I N G A N D m R N A ANALYSIS OF 5 H P R T M U T A N T CLONES OF V79 C H I N E S E H A M S T E R CELLS Mutant
SP9 EMS3 MNU4 MNU8 SP13 V79 wild type
m R N A level
Target sequence alteration
Remarks
(OD H P R T / O D -y-actin)a
°70 of wild type
cDNA (5' to 3 ' )
genomic D N A (5' to 3 ' )
0.919 1.135 0.566 1.829 0 1.399
66 81 40 131 0 100
exon 4 missing C-416 b, G C > A T exon 4 missing exon 4 missing H P R T gene missing no change
A-338 b, A T - , G C C-331 b, G C - , A T C-331 b, G C - , A T -
splice Thr to Asn splice splice deletion normal
a Ratio of integrated area of optical density for H P R T m R N A and -r-actin m R N A . The range of the measured OD values of the different clones was 1.336-3.606 except for SP13 giving O D = 0 for H P R T m R N A . b Position within the H P R T coding region.
To investigate the molecular nature of the common feature of these mutants, we isolated cytoplasmic RNA for analysis of mRNA levels by Northern blotting using a full-length mouse H P R T cDNA probe. As a reference for the H P R T mRNA level, the blots were reprobed for -y-actin mRNA. The level of H P R T mRNA in the different mutants ranges from 40°70 to 131070 of the wild-type level (Table 2). No H P R T mRNA could be detected in SP13, carrying a complete H P R T deletion. Genomic DNA purified from each mutant clone was analyzed by Southern blotting after digestion with BgllI, EcoRI and PstI. None of the 4 mutant clones SP9, EMS3, MNU4 and MNU8 showed any change in fragment patterns as compared to the wild type. As expected, the fragment pattern of control clone SP13 showed only the bands corresponding to the pseudogene of the hamster H P R T gene. Fig. 1 shows the fragment patterns of DNA from all clones compared to wild-type DNA digested by PstI. The probe identified 5 restriction fragments of 8.2, 6.5, 5.6, 4.0 and 0.96 kb which is comparable with the results of others (Fuscoe et al., 1983; Rossiter et al., 1990). The results from the Southern and Northern blot analyses indicated that the 4 6TGr-HATr-HAsT ~ clones contained no large alterations in H P R T gene suggesting that a point mutation caused the 6TG phenotype. Sequence analysis of the PCR-
A
B
C
D
E
F
23.1 m 9.4-6.6-4.4--
2°3~
2.0--
1.0--
Fig. 1. Autoradiograph of V79 Chinese hamster H P R T gene digested by restriction enzyme PstI. D N A fragments were separated on 0.8% agarose gel and hybridized with a 1.3-kb fulllength mouse H P R T cDNA probe. Lanes A - F correspond to SP13, SP9, EMS3, MNU4, M N U 8 and wild-type V79 cells, respectively.
156
amplified H P R T coding region of these mutants was performed to investigate the precise alterations in the H P R T gene. The result in Table 2 shows that c D N A of 3 of the 4 clones lacked exon 4. EMS3, however, contained a GC to AT transition at position 416 in exon 6. The 3 clones lacking exon 4 from c D N A had normal PstI restriction fragment patterns, which includes a fragment of 6.5 kb containing exon 4 (Rossiter et al., 1990). Further sequence analysis of PCR-amplified genomic DNA, using exon 4-specific primers, showed that SP9 contained an A T to GC transition at position 338 in exon 4, while MNU4 and MNU8 both had a GC to A T transition at position 331 in exon 4. These data indicate that the mutation in all 3 clones probably involves the splicing mechanism of exon 4. Discussion Studies on 8AG-resistant cell lines demonstrated a c o m m o n feature for this type of cells which utilize hypoxanthine poorly during normal growth conditions, but well during inhibition of the de novo purine biosynthesis by aminopterin (Chu et al., 1969; Gillen et al., 1972). Fenwick et al. (1977) found that each resistant clone studied produced H P R T with a reduced catalytic capacity at low concentrations of P R P P . The authors concluded that these cell lines carried mutants in the structural part of the gene. In the present paper we investigated 4 6 T G r - H A T r - H A s T r V79 cell lines which have similar properties as 8AGr-HAT r cell lines with regard to the enhanced incorporation of hypoxanthine in the presence of aminopterin or L-azaserine. Because aminopterin or t-azaserine inhibits de novo purine synthesis leading to an elevated level of P R P P available for the H P R T enzyme, these types of mutants are able to grow in H A T or H A s T medium. Investigation of about 100 H P R T mutant clones showed that about 15°70 of them were resistant to H A T and only the 4 mutants described in this paper were also resistant to H A s T (data not shown). H P R T is one in a group of 8 phosphoribosyltransferases which are functionally related meaning that they have similar physical and catalytic
properties (Musick, 1981) and all use P R P P as the first substrate. The H P R T protein contains 3 proposed domains: the amino acids encoded in exons 1-3, 4-6 and 7-9 correspond to the substratebinding domain, the catalytic domain and a functional unknown domain, respectively. The substrate-binding domain corresponds to the Nterminal 120 residues of the h u m a n H P R T protein and the catalytic domain to about 20 amino acids beginning at residue 126 of the H P R T protein (Agros et al., 1983; Dush et al., 1985). A human mutation (HPRT-Munich) with an amino acid change at residue 103 in a region adjacent to the proposed hypoxanthine binding site showed a strongly reduced affinity for hypoxanthine (Wilson et al., 1983). However, mutant H P R T - L o n d o n with a substitution of leucine for serine at residue 109 had a slightly reduced affinity for hypoxanthine, probably because the mutation was located in a region with a predicted turn that connects 2/5strands in the proposed dinucleotide fold for the putative binding sites of this substrate. Based on their properties of hypoxanthine incorporation (Table 1) the present mutant clones also seem to fit the proposed model for the functional domains in H P R T protein. The amino acid residue sequences encoded in exon 4 are located between the substrate-binding domain and the catalytic domain. The mutations SP9, MNU4 and MNU8 caused by a base substitution giving a defect splicing of exon 4 are suggested to affect the affinity for hypoxanthine and the rate of substrate conversion. According to our data, a deletion of exon 4 (66 bp) has limited effect on the H P R T protein and it still maintains a certain degree of activity. Mutant EMS3 had an amino acid change by a G C - A T transition at residue 139, localized in the proposed catalytic domain, which theoretically should reduce the rate of conversion of P R P P and hypoxanthine or guanine to I M P or G M P . Surprisingly, the enzyme activity in cytosolic preparations was similar to wild-type cells but low using exogenous incorporation of 3H-hx, suggesting a more complicated picture of this clone possibly involving other mechanisms interfering with purine metabolism.
157
We have isolated and characterized about 100 H P R T mutant clones of independent origin, one half occurring spontaneously and the other half induced by EMS or MNU. Only 4 of these mutant clones showed the ability to growh in both H A T and HAsT medium and 3 out of these 4 clones were caused by a splice mutation. Interestingly all 3 mutant clones were caused by a new type of splicing defects: a base change in the coding region resulted in splicing out the exon 4 sequence of the H P R T gene, which is consistent with a recent report by Dorado et al. (1990). The most common/3-globin splicing defects are due to single-base substitutions that prevent splicing at a particular site (reviewed by Collins and Weissman, 1984; Orkin and Kazazian, 1984). Mitchell et al. (1986) analyzed 3 spontaneously occurring mutants at the DHFR locus of Chinese hamster ovary cells and all 3 exhibited exon 5 splicing mutations, suggesting that these splice sites were hot spots for spontaneous mutations. In the H P R T locus, spontaneously occurring mutations are frequently caused by splicing defects (Zhang et al., 1991; Rossi et al., 1990). Since MNU4 and MNU8 were mutants induced by a low dose of MNU exhibiting a mutation frequency as low as 2.6/105 cells compared to a spontaneous mutation frequency of 0.4/105 cells, we cannot exclude the possibility that both these mutants are of spontaneous origin.
Acknowledgements We thank Dr. Harry Vrieling for critically reading the manuscript and Dr. Belinda J.F. Rossiter for providing her manuscript in advance o f publication.
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