J. Mol. Biol. (1992) 223, 627-635

Spectrum of Spontaneously Occurring Mutations in the hprt Gene of V79 Chinese Hamster Cells Li-Hua

Zhangl’f,

Harry Vrieling’, Albert and Dag Jenssen’

A van Zeeland2

‘Department of Genetic and Cellular Toxicology, Wallenberg Laboratory Stockholm University, S-106 91 Stockholm, Sweden 2MGC-Department of Radiation Genetics and Chemical Mutagenesis State University of Leiden, 2333 AL Leiden, The Netherlands (Received 17 June 1991; accepted 22 October 1991) A total of 76 independent spontaneous mutants in the hprt gene of V79 Chinese hamster cells have been analyzed. These mutants were obtained in two different laboratories, 17 and 59 mutants in sets 1 and 2, respectively, under different cell culture conditions. Mutation analysis was performed by amplification of hprt cDNA with the polymerase chain reaction and direct sequencing of the products. The data obtained showed similar spectra of spontaneous mutations in both sets of mutants, suggesting that culture does not play a major role in spontaneous mutagenesis. The majority of the mutations were base substitutions (>60%), with twice as many transversions as transitions. Base changes were evenly distributed throughout the structural gene, including the splice junctions. All types of base substitutions appeared in comparable frequencies, except for A.T to T. A transversions, which were almost absent. The fraction of deletion mutations was low (13%). A striking feature of the observed mutation spectra is that one third of the spontaneous mutations analyzed involved aberrant splicing of the hprt primary transcript, with exon 4 being affected most frequently, indicating that splice mutations are a common mechanism of mutation in the hprt gene.

Keywords: hprt gene; V79 cells; mutational

spectra;

The occurrence of spontaneous mutations in mammalian cells has been shown to play a major role in biological processes such as the development of cancer (Bishop, 1991) and the induction of hereditary diseases. In order to understand by which mechanisms spontaneous mutations in mammalian cells arise, it is essential to investigate the nature of DNA sequence alterations. Furthermore, knowledge of the nature of spontaneously occurring background mutations is important for a better understanding of mutagenesis caused by chemical and/or environmental agents. The molecular nature of spontaneous mutational events has been studied in several genes from both prokaryotes and eukaryotes, e.g. the EacI gene of to whom all correspondence

0022-2836/92/0306274l

$03.00/O

mutations;

splicing

bacterial cells (Schaaper et al., 1986), SupQ0 and URA3 genes of yeast cells (Giroux et al., 1988; Lee et al., 1988), aprt, dhfr and hprt genes in mammalian cells (Mitchell et al., 1986; de Jong et al., 1988; Phear et al., 1989; Recio et al., 1990a; Rossi et al., 1990), and gpt and c-hprt genes in retroviral shuttle vectors (Ashman & Davidson 1987; Ikehata et al., 1989). A comparison of the available spectra of spontaneous mutations indicates that mutation specificity differs between prokaryotic and eukaryotic genes, and between different mammalian genes. There are several explanations of the existence of these discrepancies, such as differences in culture conditions and mutant collection protocols, the kind of mutations giving rise to a mutant phenotype, i.e. the mutational window, but the size and location of the target genes and their genomic organization may play a significant role in this respect. This last point may be especially important in view of the fact that large variations have been reported to

1. Introduction

t Author addressed.

spontaneous

should be 627

0 1992 Academic

Press Limited

L.-H.

628

Zhang

occur in the rate and extent of removal of DNA damage between different regions of the mammalian genome (for a review, see Smith & Mellon 1990). In this study we have focused on the hprt gene, which encodes for the salvage pathway enzyme hypoxanthine-guanine phosphoribosyl-transferase, for sequence characterization of spontaneous mutations. Among the various endogenous genes studied in mutagenesis, the hprt gene has several advantages: good selection procedures for both forward and backward mutations, its large size (36,000 to 57,000 base-pairs) allowing detection of relatively large deletions, Its X-chromosomal location requiring only inactivation of one copy per cell of a hprt mutant phenotype and the availability of the sequence of hprt cDNA from different species and from the entire human hprt gene (Konecki et al., 1982; Jolly et al., 1983; Edwards et aE., 1990). Furthermore, the location of the hprt locus is probably not very close to essential genes, since mutations involving large deletions or rearrangements have been detected (Turner et al., 1985). Moreover, hprt is a genetic marker that has been used extensively for mutational studies in a number of mammalian mutation systems (for a review, see Stout & Caskey, 1985). In addition, total and partial deficiency in HPRT enzyme activity in humans are associated with two clinical syndromes, i.e. the Lesch-Wyhan syndrome and gouty arthritis, respectively. Spontaneous mutations in the hprt gene have been investigated in human cells only in viva. Data concerning the mutation specificity in

st al. cultured mammalian cells in vitro are of interest, among other reasons because most of the studies on chemical or radiation-induced mutations in the hprt gene have been performed with cultured cells (Fuscoe et al., 1983; Thacker & Ganesh, 1989; Vrieling et al., 1989, 1991). A total of 76 independent, 6-thioguanine-resistant, spontaneously occurring mutants in the hprt gene of V79 Chinese hamster cells were isolated in two sets under different cell culture conditions. Mutants selected with 6-thioguanine are expected t,o exhibit changes in HPRTase activity, i.e. mutations were expected to be located in the coding region or in intron sequences essential for accurate processing of the transcripts of the hprt gene. The nature of spontaneous mutations in the hprt gene was therefore determined by applying the (polymerase chain reaction (PCRt) technique (Saiki et aE., 1985) either to amplify hprt cDNA or genomic fragments containing hprt exon sequences, followed by direct sequencing of the PCR products.

2. Materials

and Methods

Cell cultures and the isolation

(a)

of HPRT

mutants

Chinese hamster lung V79 cells were cultured in set 1 under standard conditions as described by Zdzienicka et aE. (1988) and in set 2 as described by Jenssen (1984). For t Abbreviations reaction;

TG’,

used: PCR, polymerase

chain

thioguanine-resistant.

Table 1 Mutation

analysis

of I7 spontaneozcs

hprt

mutants

of V79 cells in set I

-Mutant

clone

Position

Amino acid

Exon

cDNA

Change in genomic DNA

Target Base change

sequence 5-3’

Change in amino acid

A. Transitions SP-v20 SP-VlO

404 E9:-2

135

2 134 222 305 486 E8:-2 E8:-I 547

1 45 74 102 162

6 Del ex 9

Spliceavb

A.T A.T

> G.C > 0.C

GAGG A CATA ttgc a gCAT

Asp

A.T

> C,G

CGTA T GGCG GACAGGACT AATT C TTTG AGAC T GAAG CAAG C TTGC ttac a gTTG taca g TTGT TGAA A TTCC

Met Arg Phe Leu Ser

> Gly

B. Tramversions

SP-V18 SP-V23 SP-VI2 SP-Vl SP-v4 SP-v2 SP-V8 SP-v30

183

1 2 3 3 7 Del ex 8 Del ex 8 8

G,C>T.h

Splice”,b Splice”*b

G.C>T,A z4.T > C.G G-C>C.G A.T>C.G G.C>C.G A.T > T.A

> > > > >

Ile > Phe

C. Deletions SP-v7 SP-v3 D.

-

Del ex 3 Del ex 5

Del 3 kbb Del 4 kbb

Others

SP-VH SP-VI 1 SP-v19 SP-V26 SP-V32 “Identified ‘Identified

Del Del Del Del Del

ex ex ex ex ex

4 4 4 4 4

by PCR amplification of genomic DNA using by digestion of genomic DNA using restriction

Arg Met Leu Arg Arg

Spliceb Spliceb Spliceb Spliceb Spliceb exon specific primers for exons 2, 4 and 7 to 9. kb: lo3 base-pairs. enzymes BgZII, EcoRI, Hind111 and PstI in different, combinations.

1779 Chinese

Hamster

the isolation of hprt mutants in set 2, V79 cells were grown in HAT selective medium (5 x 10m5 Mhypoxanthine, 4 X 10m7 M-aminopterin, 5 X 1o-6 Mthymidine) to reduce the numbers of pre-existing mutant clones. Cell cultures were started at cell densities of lo3 or lo4 V79 cells/Petri dish, in sets 1 and 2; respectively, grown to confluency, harvested separately and plated in 6-thioguanine-containing (5 pg/ml) medium at a density of lo5 cells/Petri dish (94 mm) with 20 dishes/culture. Only 1 6-thioguanine-resistant colony/culture was isolated following 7 days of growth. We have isolated 17 independent 6-TG’ mutants from 30 cultures in set 1 and 59 independcent mutants from more than 100 cultures in set 2.

(b) DNA

isolation

and

Southern

hybridization

High molecular weight DNA was isolated and 10 to 15 pg of DNA was digested with restriction enzymes, separated on a 0+3 o/0 (w/v) agarose gel and after Southern transfer of the DNA to a nylon membrane, hybridized under conditions described elsewhere (Zhang & Jenssen 1991a).

(c) PCR and sequence anaEysis of hprt cDNA Cytoplasmid RNA was used to specifically synthesize hprt cDNA using avian myeloblastosis virus reverse transcriptase (Promega) and primer vrl-16. About 10% of this cDNA product was then amplified by PCR in 30 cycles with the primers zee-1 and vrl-lo-M13, as described (Vrieling et al., 1989). To obtain single-stranded cDNA for direct sequencing; a small proportion of the amplified hprt cDNA was used in a 2nd PCR of 30 cycles with primer vrl-lo-Ml3 and a 5’-biotinylated primer zee-1. The procedure used for direct sequencing of amplified hprt cDNA has been described (Zhang & Jenssen 1991b). Sequencing reactions applying Sanger’s dideoxy chain termination procedure (Sanger et al., 1977) were performed directly on single-stranded DNA bound to the magnetic beads (Dynabeads MZ80 Streptavidin, Dynal AS, Norway). The oligonucleotide primers used in cDNA synthesis and PCR were: vrl-16: 5’-GCAGATTCAACTTGAATTCTCATCGG-3’ vrl-10.M13: S’TCAACTTGAATTCTCATCGGGTAAAACGACGGCCAGT-3’ zee-1: 5’.GGCTTCCTCCTCAGACCGCT-3’

(d) PCR and sequence analysis

of genomic

hprt DNA

A crude cell lysate was prepared as source of genomic DNA for PCR amplification of exons 2, 4 and 7-9 of the hprt gene using exon-specific primers, partly designated as by Rossiter et al. (1991). Sequencing of double-stranded DNA using a modification of the procedure developed by M. Giphart-Gassler and H. den Dulk has been described (Zhang & Jenssen 1991b). The oligonucleotide primers used in PCR and sequencing were: exon 2

Cell hprt

Mutations

629

3. Results (a) Isolation

and analysis

of mutants

Independent, spontaneously arisen 6-thioguanine-resistant mutants from V79 Chinese hamster cells were collected in two different laboratories. The V79 Chinese hamster cell lines used for mutant isolation in these studies were derived from the same cell clone. To study the mutational spectrum of these mutant collections, a cytoplasmic RNA extract was prepared from each mutant clone to allow hprt cDNA synthesis, amplification of the hprt coding region and direct sequencing of the PCR-amplified cDNA. Chromosomal DNA from mutant clones with no detectable hprt cDNA product after PCR, probably because of large deletions, was isolated for Southern analysis. Mutants, which upon cDNA sequence analysis showed a deletion of exon sequences, were further analyzed by amplification of genomic DNA using primers specific to exons 2, 4 and 7-9 and/or Southern analysis. Some of these mutant clones displaying defective splicing of the hprt transcript have been studied at the genomic DNA sequence level to determine the precise nature of the alterations in the hprt gene. (b) Mutation

spectrum

Table 1 shows the mutational specificities in the sequence of the hprt coding region (654 base-pairs) of 76 independent mutants isolated in the two different laboratories. (i) Base substitutions Base changes are the predominant type of spontaneous mutations in the hprt gene, i.e. 59% and 66% in sets 1 and 2, respectively. Only a few of the base alterations were nonsense mutations, the majority were of the missense type. Transversions (42 to 47%) appeared more frequently than transitions (12 to 24%) among the spontaneous hprt mutants analyzed (Tables 1 and 2). Comparison of the different types of base changes presented in Table 3 indicated that occurrence of the A. T to T. A transversion was a rare spontaneous mutational event, while the other base changes were present at comparable frequencies. Tables 1 and 2 demonstrate that base substitutions were spread throughout the coding region and splice sites, with a sight preference for some sites. Four out of 76 mutants studied has an A. T to G. C transition at position 404 in the coding region, whereas mutations at position 190 also occurred at a somewhat higher frequency (3/76).

ham-23: 5’-AGCTTATGCTCTGATTTGAAATCAGCTG-3’ ham-25 5’-ATTAAGATCTTACTTACCTGTCCATAATC-3’ exon 4 ham-43: 5’.GTGTATTCAAGAATATGCATGTAAATGATG-3’ ham-45: 5’XAAGTGAGTGATTGAAAGCACAGTTAC3’ exon 7 + 8 wies-2: &CATCTGATCCAGGTTCCAGGTGG-3’ wies-3: 5’.TTATAGTCAAGGGCATATCC-3’ exon 7-9 wies-2, vrl-16 or vrl-10 (see above).

Mutation

Mutant

clone

Position

analysis

Amino acid

Exon

Table 2 hprt mutants

of 59 spontaneous

cDNA

Change in genomic DNA

of V79 cells in set 2 Target

Base change

sequence 5-3’

Change in amino acid

A. Transitions SP23 SP9 SPl SP30 SP43 SP57 SP52 SP28 SP36 SP59 SP29 SP34 SPll SP15 B. Transversions SP40 SP58 SP50 SP51 SP27 SP56 SP64 SP14 SP47 SP7 SP61 SP65 SP35 SP45 SP19 SP12 SP31 SP60 SP37 SP24 SP38 SPZO SP26 SP32 SP42

134 338 400 404 404 404 415 508 551 568 569 569 602 602 122 190 190 190 191 191 208 222 284 286 E4:-1 358 368 395 395 459 473 473 E7:-1 I7:l 578 580 589 595 606

45 134 135 135 135 139 170 184 190 190 190 201 201 41 64 64 64 64 64 70 74 95 96

123 132 132 153 158 158

193 194 197 199 202

2/del ex 2: 3 Del ex 4 5/del ex 5 6 6 6 6 7 8 8 8 8 8 8 2 3 3 3 3 3 3 3 3 3 Del ex 4 Del ex 4 4/del ex 2, 3, 4 5 5 6 6 6 Del ex 7 Del ex 7 8 8 8 8 8

Splice Splicea*b Splice

G.C>A.T A.T > G,C>A.T A.T > A.T>G.C A.T > A.T > G.C > G.C > G.C > G.C > G.C> A.T>G.C A.T > A.T > G-C>C.G 6.C > G.C>C.G G.C>T.A G.C > G.C>T.A G.C>T,A A.T>C.G A.T > G.C>C.G G.C>T.A G.C>C.G A.T>C.G A.T>C.G G.C>C~G A.T>C.G A.T>C.G G.C>T.A G.C>C.G A.T > G.C > G.C>C.G A,T > G.C! >

Splice” Splice” Splice

Splice” Splice”

G.C 0.C G.C G.C A.T A.T A.T A.T A.T G.C C.G C.G

T.A

C.G

C-G T.A C.G T.A

GACA G GACT GGGG A CATA TGTT G AGGA GAGG A CATA GAGG A CBTA GAGG A CATA TGAC A CTGG CTCT C GAAG ATTC C AGAC TGTT G GATA GTTG G ATAT GTTG G ATAT AGGG A TTTG AGGG A TTTG

Arg t Glu > Asp > Asp > Asp > Thr > Arg > Pro > GIy > Gly > Gly > Asp > Asp >

Lys

GGAG T GATT TGTG G CCCT TGTG G CCCT TGTG G CCCT GTGG C CCTC GTGG C CCTC GAAG G GGGG AATT C TTTG CCCA T GACT CATG A CTGT acta g AATG TGGG G ATGA CTCT C AACT TTGA T TGTT TTGA T TGTT GGTA C AACC ATGG T TAAG ATGG T TAAG aaca g CTTG GACT g taag GCCC T TGAC CCTT 0 ACTA TAAT G AGTA GTAC T TCAG ATTT G AATC

Val Ala Ala Ala Ala Ala Gly Phe Met Thr

Gly Pro Pro Pro Asp Asp Trp Len Arg Pro

Ser Ile Ile Tyr Val Val

Leu Asp Glu Phe Len

> > > > > > > > ;z >

> stop 1 Ser > Ser > stop > Gly > Gly

> > > > >

C. Frame-shift8 SP46 SP21 SP33 SP62

145 207 207 503

3 3/del ex 2, 3 3/del ex 2, 3 7

Splice Splice

Del Del Del Del Del No No No

Del Del Del Del Del Del Del Del

-c +G 4-G -C

AGA GAA GAA GGA

(C) (6) (Gj (C)

TTG GGG GGG CTC

D. Deletions SP48 SP63 SP44 SP17 SP22 SP13 SP49 SPl8

ex 2, 312, 3, 4 ex 2, 3. 4 ex 3/2, 3 ex 4 ex 4 cDNA cDNA cDNA

ex ex ex ex ex ex ex ex

2”/2, 3b 2, 4a 3b 4”~~ qasb llgb 2, 4: 7-9” 3-gash

E. Duplications SP5 SP39

Dupl Dupl

ex 2 ex 2, 3

Del Del Del Del Del No

ex 2’2, 3 ex 3/2, 3 ex 2, 3 ex 5 17 bp ex 9 cDNA

Dupl Dupl

ex 2b ex 2, 3b

P. Others SP16 SP25 SP55 SP54 SP41 SP53 “Identified ‘Identified

by PCR amplification of genomic DNA using by digestion of genomic DNA using restriction

Splice”,b Spliceb Splice”,b Spliceb Spliceb Ex 2, 4, 7 to 9 presenta exon specific primers for exons 2, 4 and 7 to 9. enzymes BglII, EcoRI, Hind111 and PstI in different

Lys Gly Gly Gly Ala stop Leu Arg Gin Glu Giy Gly

combinations.

Arg Tyr Gln Val Phe

V79 Chinese

Hamster

Cell hprt

Mutations

631

Table 3 Comparison Mutation

type

Total no. of mutants Base changes Transitions G.C>A.T A.T>G.C Transversions G.C > T.A G,C>C.G A.T > T.A A.T>C.G Frame-shifts Deletions Duplications Compounds Others Splice

LN and Gout (hpW 42 71 33 26 7 38 12 10 12 5 10 17 0 2 0 12

of spectra

of spontaneous

mutations

in

different

systems

T-cells hpd

31 39 16 13 3 23 10 6 3 3 16 6 0 0 0 39

hprt aprtc

apd

89 62 35 25 10 27 12 10 2 2 7 20 6 1 4 4

30 90 73 73 0 17 13 0 3 0 3 3 0 0 3 7

The values are presented in percentage of the total number of mutants analyzed. aLesch-Nyhan syndrome. Pooled data from Wilson et al. (1983a,b), Wilson & Kelley Fujimori et al. (1988, 1989, 1990), Cariello et al. (1988), Gibbs et al. (1989, 1990). ‘Data from Rossi et al. (1990). “Data from Phear et al. (1989). dData from de Jong et al. (1988). ‘Data from Ashman & Davidson (1987). fData from Ikehata et al. (1989). Vlummarized data from sets 1 and 2 taken from Tables 1 and 2.

(1983,

gpt”

cDNA’

43 26 12 7 5 14 0 7 5 2 2 67 0 0 5

52 23 2 0 2 21 8 0 10 4 38 19 8 2 8

1984), Davidson

et al. (1988a,b,c,

v79 hprtg

76 64 21 9 12 43 13 13 1 16 5 13 3 0 1 33

1989a,b),

(ii) Deletions

(v) Splice

Mutations caused by partial or total deletion of the hprt gene represented only 12 to 14% of the mutants investigated (Tables 1 and 2). Of three deletion mutants that had an undetectable level of hprt CDNA, two exhibited total deletion of the hprt gene, whereas the third one contained only exons 1 and 2 of the hprt gene. All of the partial deletion mutants were located in the 5’ part of the gene.

In sets 1 and 2, 47% and 29% of the mutations, respectively, involved aberrant splicing of the hprt mRNA, in particular of exon 4 (about 20 %). The sequences of some of the splice mutants, missing exon cDNA sequences, have been analyzed at the genomic DNA level using exon-specific primers. The results indicate that splicing of the hprt mRNA can be affected by changes at positions in the DNA other than at the splice sites. Mutants SP9 and SP65 (Table 2) showed a base substitution at positions 338 and 358 in exon 4, respectively, which resulted in splicing out of that exon sequences. The other splice mutants in our collection were either caused by a base substitution (SPl, SP23 and SP35), a frame-shift (SP21 and SP33) or a deletion (SP44 and SP48). One splice mutant, SP41 (Table 2), had a deletion of 17 bases in the beginning of exon 9, which was identical with that in a spontaneous hprt mutant of human T-lymphocytes characterized by Rossi et al. (1990). The last two nucleotides at the 3’ site of the deletion are 6’-AG-3’, which aparently act as a cryptic splice acceptor site, resulting in deletion of 17 base-pairs from the mature hprt mRNA.

(iii) Duplications Duplication of part of the hprt gene is a rare event among spontaneous mutations (0 to 3%). Two mutants had a duplication of exon 2 and of exons 2 including flanking regions and 3, respectively, (Table 2), which may occur after intrachromosomal gene conversion of the hprt gene. The size of one of these duplications (in SP5) is about 2000 base-pairs and contains exon 2 (Zhang & Jenssen 1991c). This mutant clone has an extremely high spontaneous reversion frequency, which is probably caused by intrachromosomal recombination (Zhang & Jenssen, 1989). (iv) Frame-shifts Frame-shift mutations appears only in 0 to 7 y0 of the total cases and involved exclusively G. C basepairs (Table 2). Three of the four mutations found were located in exon 3. Moreover, two of these three mutants had a G-insertion in a run of six G nucleotides at positions 207 to 212, which affected splicing of the hprt transcript and resulted in splicing out of exons 2 and 3.

mutations

(vi) Others Among the 76 mutants isolated and analyzed in this work, we failed to classify only one mutant (SP53, Table 2). In this case, the product obtained from PCR amplification of the cDNA was below the level of detection. However, exons 2, 4 and 7-9 were presented in the genomic DNA from this

L.-H.

632

Zhang

mutant. Southern analysis of genomic DNA from this mutant was also found to be normal, compared to the wild-type. On the basis of these results, this mutant is suggested to have a mutation in the promoter region of the hprt gene. 4. Discussion In this study we have analyzed the sequenceof 76 independent, spontaneously occurring mutants in the hprt locus of V79 Chinese hamster cells isolated in two different’ laboratories under different cell culture conditions. The results indicate that the spectra of mutations in the 654 base-pairs coding region of the hprt gene from these two sets of mutants are similar. Therefore, they are regarded as a single group in the following discussion. The spectrum of spontaneous mutations in V79 cells found here is characterized by a few major features (Table 3). The majority of the mutations (>60%) were base subst,itutions, with twice as many transversions as transitions. The base changes were distributed evenly throughout the structural gene, including the splice junctions, but with somepreference for a few sites. All types of base changes appeared with similar frequencies, except for A’ T to T. A transversion mutations, which were very few. One-third of t.he mutants involved aberrant splicing of the hprt mRNA, with splicing of exon 4 being affected most frequently. The frequency of deletion mutations was low (13 %). It is of interest’ to compare the present results with other studies on spontaneous mutation involving other genesin different systems, as well as the hprt gene in different cells and in different species (Table 3). In a study by Phear et al. (1989), involving 89 spontaneous mutations in the aprt gene in Chinese hamster ovary cells, also a mutation spectrum consisting of a variety of different types of changes was found. However, base substitutions in the aprt gene involved slightly more transitions (35 %) than transversions (27 y/o). Here, G. C t’o A. T was the most frequent (25%) among transitions, whereas A. T to C.G and A.T to T. A were the least common of the transversions (2 %). Studies on spontaneous mutations in the aprt gene have been reported by de Jong et al. ( 1988). They detected a predominance of G *C to A. T transitions, including a “hot spot”; which suggested tha,t deamination of cytosine played a major role in spontaneous mutagenesisin Chinesehamst,er ovary cells. A number of possible explanations for the discrepancy between these two studies have been suggested, such as certain subtle differences in culture conditions, mutant collection protocols or analysis (Phear et al., 1989). However, our similar mutation spectra at the hprt gene in two sets of mutant clones as discussed above lend no support for these explanations. Studies on spontaneous mutations using the hprt gene as a target in human cells in civo either from healthy donors (Recio et al., 1990a,b; Rossi et al., 1990) and from patients having Lesch-Nyhan syndrome or gouty arthritis (Wilson et al., 1983a,b;

et

al.

Wilson $ Kelley 1983, 1984; Cariello et at., 4988; Davidson et al., 1988a,b,c, 1989a,b; Fujimori et al., 1988, 1989, 1990; Gibbs et aZ., 1989, 1990) have been performed. The most striking feature of the spectra of spontaneous mutations a,t the hprt gene is the predominance of base substitution mmations ( > 60 %), with approximate equal frequencies of transitions and transversions in primary cells in vivo, and with a higher frequency of transversions than transitions in cultured cells i?z vitro (Table 3). A possible reason for t,he relatively low frequency (39%) of base change mutations found in healthy human donors might be that a relatively large number of splice mutations, the majority probably of base substitution nature, have not, been characterized. Among the base changes represented, the G. C to A. T transition is a frequent base alteration, while the A. T to G. C transition is the least common in cells in wivo. In contrast, in V79 cells in vitro, the different types of base changes occurred a,t about the same frequency, except for the A. T to T *A transversion, which was almost absent. In contrast to mutational specificities in endogenous cellular genes, the frequency of base substitutions has been reported to be low in retroviral shuttle vector systems. One of these involved a cDNA for t,he human hprt gene (25% base changes, 13/52) and the other the g$ gene from Exherichia eoli (26% base substitutions, 11143)integrated into ehromosomal DNA of mouse cells Ashman $ Davidson, 1987; Ikehata et al., 1989). In the vector with c-hprt, frame-shifts resulting from deletion or addition of one or a few nucleotides were the most frequent mutationa events (38%), and occurred preferentially at sites flanked by short direct repeats. In the study with the integrated gpt gene also, deletions predominated (67%): but here a three base-pair deletion “hot spot” was detect’ed, accounting for about 37O/i of the spontaneous mutations. Deletions represented only a small proportion ( ~20%) of the spontaneous mut,ations in t,he hprt gene (Table 3), which is consistent, with analyses of the aprt gene (de Jong et al., 1988; Phear et al., 1989), as well as with a st,udy by Nicklas et al. (1989), in which gross structural a.lterations in the hprt gene detected by Southern analysis occurred in about 14% (481326) of the mutants derived in human T-lymphocytes in viva. In contrast, Turner et al. (1985) reported that as many as 57% (12/21) of spontaneous hprt mutations in viva in human T-lymphocytes involved substantial gene alterations, including deletions, exon amplifications and novel bands on Southern analysis. Similarly, restrjction mapping of a large number of spontaneous mutations in the hprt gene in a cultured human R-lymphoblastoid cell line showed that 39 y0 (33/85) of these mutants involved a large structural alteration; most of them were deletion mutations (Gennett & Thilly 1988). Studies on spontaneous mutations in the tk gene in the cell line have been reported by Yandel et al. (1990), which indicat,ed that a high percentage of the mutants (SSY;,

V79 Chinese Hamster 151/171) arose by loss of the entire active tk allele. This was also the case for mutants in the aprt gene in Chinese hamster ovary cells (97% of 198 mutants analyzed) reported by Dewyse & Bradley (1989). Deletions were often found to occur at sites clustered with short direct repeats, and such repeat sequences are suggested to be important in the generation of deletions, probably by slippage of the DNA template during replication (Albertine et al., 1982; Ashman & Davidson 1987). Mutations affecting splicing of hprt mRNA have been observed in studies on spontaneous mutations, light chemical-induced on ultraviolet and mutations, as well as in studies on mutations in human patients suffering from Lesch-Nyhan syndrome or gouty arthritis. A large proportion of the spontaneous mutations in our collection were involved in splicing of the hprt mRNA, which is consistent with the studies on spontaneous mutations in human T-lymphocytes (Recio et al., 1990a; Rossi et aE., 1990 and unpublished results), on ultraviolet light-induced mutations in a Xeroderma pigmentosum cell line (Dorado et al,, 1990), as well as the study on 2-cyanoethylene oxide-induced mutations in human lymphoblastoid cells (Recio et al., 1990b) and on N-methyl-Nnitrosourea-induced mutations in V79 cells (Zhang & Jenssen 19916). Reports of mutations in human patients, however, demonstrated a significantly low fraction of splice mutations (Table 3: Wilson et al., 1983a,b; Wilson & Kelley, 1983, 1984; Cariello et al., 1988; Davidson et al., 1988a,b,c, 1989a,b; Pujimori et al., 1988, 1989, 1990; Gibbs et al, 1989, 1990), possibly because of the existence of different criteria for determination of hprt deficiency in genetic diseasesas argued by Rossi et al. (1990). In the case of N-methyl-N-nitrosourea-induced mutations, the G. C to A. T transition predominated and was probably caused by methylation of 06-guanine, occurring preferentially at a consensus sequence 5’purine-G-N-3’ (Richardson et al., 1987; Topal, 1988; Zhang $ Jenssen, 1991b). Interestingly, the consensus sequence of splice junctions is identical to this sequence, which may explain the high frequency of N-methyl-N-nitrosourea-induced mutations that involved aberrant splicing of the hprt mRNA. In contrast, splice mutations among spontaneous mutations in the aprt gene constituted only a small proportion of the total number analyzed (de Jong et aZ., 1988; Phear et al., 1989). Splice mutations in the hprt gene most often involved aberrent splicing of exon 4, suggesting that a larger target sequence regulates correct splicing of that particular exon, which is in agreement with the study by Rossi et al. (unpublished results). A similar observation has been reported by Mitchell et al. (1986), who suggestedthat splice sites for exon 5 in the dhfr gene in Chinese hamster ovary cells are hot spots for spontaneous mutations. According to these results, the occurrence of aberrant splicing seemsto be due to a number of different types of mutations, i.e. mutations at normal splice junctions; mutations creating a new

Cell hprt Mutations

633

splice site or a cryptic splice acceptor site; certain mutations in the form of base substitutions, frameshifts or deletions that are not obviously connected to the splice process and produce a mixture of mRNA products in addition to the expected transcript; certain base substitutions in the coding region, for some unknown reason, result in splicing out of that particular exon to yield a single defective transcript. Our findings support the suggestion by Dorado et al. (1990), that the target size for mutations resulting in aberrant splicing must be quite large and does not solely involve the splice junctions. It seemsthat molecular characterization of hprt splice mutations will provide valuable information about any additional sequences that regulate hprt mRNA splicing. In conclusion, splice mutations are a common mechanism for spontaneous mutations in the hprt gene. Comparison of spontaneous mutation spectra at different target genes shows large differences and suggests that the specificity of spontaneous mutations can be affected by a number of factors. Spontaneous mutations can be caused by replication errors that escape correction mechanisms such as mismatch repair and, to some extent, to the inherent chemical instability of the DNA bases. Endogenous factors, such as oxidative stress, which may cause DNA damage and can give rise to mutations during cell division. In this respect, the etliciency of DNA repair processesmay be important, which has been shown to vary dramatically depending on the activity and genomic organization of the target gene. In addition, “spontaneous” mutations in human cells in viva can be generated by exposure to mutagens both environmentally and occupationally, while mutation frequencies in cultured cells in vitro can be affected by the culture conditions. At present, it is difficult to describe the relative role of these different factors in the specificity of spontaneous mutations in vivo and in vitro. Since new alternative gene products arise from errors in the splicing mechanism, splice mutants might make an important contribution to evolution. We thank MS M.-L. van Rooyen for excellent technical assistance. We are grateful to Mr H. den Dulk for providing the protocol for sequencing of splice mutants and, together with MS J. C. P. Thijssen, for technical assistance in this respect. We also acknowledge Dr D. Hultmark for introducing L.-H. Zhang into molecular biology. This study was supported by the environmental foundation of OK and by the Dutch Cancer Society (contract no. IKW 85-64).

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Edited by J. Karn

Spectrum of spontaneously occurring mutations in the hprt gene of V79 Chinese hamster cells.

A total of 76 independent spontaneous mutants in the hprt gene of V79 Chinese hamster cells have been analyzed. These mutants were obtained in two dif...
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