Mutation Research, 232 (1990) 163-170 Elsevier

163

MUT 04893

Localization of deletion b r e a k p o i n t s in r a d i a t i o n - i n d u c e d m u t a n t s of the hprt gene in h a m s t e r cells John Thacker

a,

Earl W. Fleck b Tracy Morris a, Belinda J.F. Rossiter c and Thomas L. Morgan d

MRC Radiobiology Unit, Chilton, Didcot, Oxon OXll ORD (Great Britain), b Department of Biology, Whitman College, Walla Walla, WA 99362 (U.S.A.), c Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) and d Biology and Chemistry Department, Pacific Northwest Laboratory, Richland, WA 99352 (U.S.A.)

(Received 21 November 1989) (Revision received 22 March 1990) (Accepted 24 April 1990)

Keywords: Hprt gene; Radiation mutagenesis; Deletion breakpoints; Hamster hprt gene mutants

Summary DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiations and others occurring naturally, to identify those with large alterations in part of the gene. DNA from these mutants was restricted further with different endonucleases and probed to establish the patterns of restriction fragments remaining. Of 15 mutants characterized, one showed a duplication of part of the 5' end of the gene, and the remainder showed deletions of various sizes. It was possible to approximately locate the breakpoints of the deletions by comparison of fragment patterns to a recently-established map of the hamster gene. The relatively small number of mutants examined precludes rigorous analysis of the distribution of breakpoints in the hprt gene, but taken with other recent studies of deletion mutagenesis it is suggested that non-random induction or selection of this type of mutation may occur.

Analysis of molecular changes in the DNA of mutant cells is essential for an understanding of the ways in which mutations arise. Recently, it has been possible to analyse the DNA of specific mutant genes in mammahan cells, especially if the gene concerned is relatively small. For example, use of the aprt gene of hamster cells (size < 4 kb) has allowed the cloning and sequencing of a number of mutant forms of the gene (e.g., Nalbantoglu et al., 1987; de Jong et al., 1988). These analyses have identified a variety of types of point muta-

Correspondence: Dr. J. Thacker, MRC Radiobiology Unit, Chilton, Didcot, Oxon OXll 0RD (Great Britain).

tions as well as revealing the mutational spectra of certain DNA-damaging agents in the aprt genetic region. Similarly, it has been possible to describe point mutations in larger genes, such as hprt (size > 30 kb), with the use of techniques such as the polymerase chain reaction where small regions of the gene are amplified for sequence analysis (Vrieling et al., 1988) or by identifying mismatches in hybrid RNA from wild-type and mutant cells (Gibbs and Caskey, 1987). However, in larger genes such as hprt, it has been found that a proportion of the mutants arise through very large genetic changes. In an extended study of mutation in the hprt gene of hamster cells, for example, it was found that large dele-

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

164

tions occur in about 70% of mutants induced by ionising radiations (Brown and Thacker, 1984; Brown et al., 1986; Thacker, 1986; for less extensive studies, see Vrieling et al., 1985; Stankowski et al., 1986, Fuscoe et al., 1986). Very little is known about the molecular nature or mechanism(s) of formation of such mutations. In the present study, those large mutations which resulted in only a part of the hprt gene being altered have been analysed further. This process has required information on the structure of the hamster hprt gene (Rossiter, 1987; H. Vrieling, personal communication) and an interpretation of the hybridization patterns of gene fragments to hprt cDNA probes (Fuscoe et al., 1983). Materials and methods

Cell culture and mutant isolation The Chinese hamster line V79-4 and its culture have been described in detail previously (Thacker, 1981). The isolation of independent mutants resistant to 6-thioguanine was described by Brown and Thacker (1984); the prefix 'S' indicates spon-

EcoRI

taneously-occurring mutants, while 'G' indicates 7-ray induced and ' a ' indicates a-particle induced mutants. The induced mutants were selected from populations irradiated to give approx. 20% survival (5 Gy of y-rays; 1.2 Gy of 6.7 MeV a-particles). The mutant frequencies were: spontaneous 0.62, y-rays 3.88, a-particles 11.3 (mutants per 105 survivors).

DNA isolation and Southern analysis Methods were as described by Thacker (1986), except that in recent analyses 0.6% agarose gels were used to separate large fragments better and the gels were blotted onto GeneScreen Plus membranes (DuPont). Blots were probed with a fulllength Chinese hamster cDNA, isolated from the plasmid pHPT12 (Konecki et al., 1982; kindly supplied by Dr. C.T. Caskey). Results

DNA from the mutants identified by Thacker (1986) as partial deletions/rearrangements was

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hprt gene and pseudogene fragment patterns revealed by Southern analysis of V79-4 cell DNA restricted with various

enzymes and probed with a full-length hamster cDNA. To the left of each pattern is given the size of the fragment in kilobases (kb) and the exons (ex.) contained in that fragment (Rossiter, 1987). ~ = pseudogene fragment (Fuscoe et al., 1983; Thacker, 1986); other

faint fragments were not consistently seen.

165 subjected to further analysis where necessary. Using 4 different enzymes - EcoRI, HindlII, PstI and Bglll - restriction fragment patterns on Southern blots were compared to a map of the hamster hprt gene (Rossiter, 1987; H. Vrieling, personal communication). The map is complete except for the exact location of exon 1; this has been tentatively identified (B.J.F. Rossiter, unpublished) but was not included in the analysis because this information is not essential for the mapping of the majority of the mutations. The fragment hybridization patterns are shown in Fig. 1; it should be noted that, with a cDNA probe, the EcoRI and HindlII digests give two large fragments spanning most of the gene, while the PstI and BgllI digests give several fragments covering smaller areas of the gene (the sizes of these fragments have been reported by Thacker (1986); these are slightly at variance with the accompanying paper probably due to different measurement methods). The PstI and BgllI digests also leave some areas 'unseen' because they lack hybridizing material to the cDNA probe, and the BgllI digest has some fragments which overlap on gels so that it may be non-informative in some analyses. The same fragment patterns and sizes have been seen in all Chinese hamster material examined to date (including V79 and

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CHO cell lines and D N A from primary animal cultures; J. Thacker, unpublished). Examples of the analysis are given in Figs. 2 and 3. In Fig. 2, two examples of internal deletions are shown, G7 and G38. G38 is perhaps the most simple: it is seen that of the two EcoRI fragments spanning the gene, the largest fragment (approx. 17 kb) is slightly smaller than in the non-mutant (V79), indicating a loss of material at the 3' end of the gene. Consistent with this, the smaller HindlII fragment is reduced from 10 kb to about 8 kb. In the PstI digest, all of the fragments are in the same positions in G38 as in V79 except for the 8-kb fragment covering exons 7 and 8 (and probably part of exon 9) which is increased in size (partly obscuring the largest PstI fragment). Since no change is found in the most 3' PstI fragment (0.9 kb) a simple interpretation is that a deletion at the left-hand end of the 8-kb PstI fragment has linked it to another fragment (not normally seen because it lacks material hybridizing to the c D N A probe; Rossiter, 1987) to make it larger. This analysis locates the G38 deletion to the region of exon 5 and sizes it at about 2 kb (see Fig. 5). In mutant G7, we see a slightly different pattern, with the smaller of the two EcoRI fragments increasing in size, suggesting a 5' gene alteration.

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Fig. 2. Parent (V79-4) and mutant hprt gene fragment patterns for two v-ray induced mutants, (a) G7 and (b) G38. Patterns with 3 different restriction enzymesare shown, where arrows point to new band positions in the mutant DNA.

166

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Fig. 3. Parent and mutant hprt gene fragment patterns for two a-particle induced mutants, (a) a16 and (b) a21. Patterns with 4 different restriction enzymes are shown, where arrows point to new band positions in the mutant D N A .

Again, the HindIII pattern is consistent with this, where the larger (5') fragment is decreased in size to run at nearly the same position as the smaller fragment (i.e., loss of about 2.5 kb). The PstI digest again gives a more precise localization, with the only alteration seen in the 6-kb fragment car-

rying exon 4. Since this fragment still hybridizes at its new position (at about 9.5 kb), only part of exon 4 or essential intron material in this region can be lost. This loss fuses the right-hand ends of the altered EcoRI and PstI fragments to fragments which are not normally seen, and locates

167

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Fig. 4. hprt gene fragment patterns for parental cells, mutant G8 and subclones of G8. The enzyme HindIII was used, and the arrow points to the duplicated fragment.

the G7 deletion to the right side of exon 4, as shown in Fig. 5. Examples of hybridization patterns indicating more extensive deletions are given in Fig. 3. A glance at these patterns indicates that mutants a16 and ct21 have somewhat complementary losses of hprt gene material. Taking a16, it is not possible to say which parts of the gene are deleted from the EcoRI or HindIII patterns; the single fragments remaining could come from either of the normal pair. However, the PstI data suggest that only the 3' end of the gene remains (all normal fragments missing except the 0.9-kb fragment carrying part of exon 9), and this is confirmed by the BglII data (presence of 2.1-kb fragment carrying exon 9). Since no trace of the 1.5-kb BglII fragment (exons 7 and 8) is seen the breakpoint must lie between exons 8 and 9 (the new faint fragment seen in the PstI pattern at about 1.8 kb will be the remains of the 8-kb fragment, which normally carries exons

6 - 8 and part of 9). In a similar fashion, it can be shown that a21 has a deletion of the 3' end of the gene, including exons 7, 8 and 9 (loss of PstI and BgllI fragments containing these exons, and size reduction of larger EcoRI or HindlII fragments to the expected size). The only mutant of this series which cannot be described in terms of a simple deletion is G8. This mutant showed additional hybridizing bands without loss of the normal set. To eliminate the possibility that this D N A pattern was derived from two sub-populations of mutants (one with the normal pattern and the other with only the new fragment), a number of subclones were derived from single G8 cells and their D N A screened with the hprt probe. In each case, the extra band was seen (Fig. 4), suggesting that this mutant really has a duplication of part of the gene. The duplicated material does not hybridize to a probe covering exons 7 - 9 (not shown), suggesting that it is a 5' partial-gene duplication. The location of each of the 14 deletion mutants is shown in Fig. 5. It will be noted that deletions occur in all parts of the gene but that the breakpoints occur more frequently in some parts of the gene than others (e.g., 8 out of the 28 breakpoints occur between exons 4 and 5). It is also seen that the majority of the breakpoints are intragenic. However, the number of deletions examined is too small for a useful analysis of distributions. Discussion

We have already shown by molecular and other analyses of a much larger series of mutants, that a wide range of mutation types can be detected using the hprt gene as a target (Thacker, 1981, 1986; Brown and Thacker, 1984; Brown et al., 1986; Thacker and Ganesh, 1989). The present data take us further in understanding one of the types - partial gene deletions - by defining the approximate locations of the breakpoints in a series of 14 mutants. It is of interest that a range of sizes of deletions has been detected, from the relatively small deletions of mutants G22, a20, and S l l to those which encompass much of the gene as well as 3' or 5' flanking sequences. This sample of mutants, therefore, gives no clear indication of any size

168

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Fig. 5. Size and position of partial deletions/rearrangements of the hamster hprt gene in a series of mutants. A scale with arbitrary positioning is shown at the top, and the next line shows the approximate locations of the known exons of the gene (Rossiter, 1987). The mutants analysed are listed below with the approximate sizes of deletions (open boxes) shown where possible; the angles of the vertical lines indicating breakpoints are drawn to include the uncertainties of location. Dashed lines indicate that the deletion extends into flanking DNA.

limitation on mutation formation (the technique of Southern analysis has, for the present, imposed a lower limit on measurement of deletion size). As noted above, the positions of breakpoints are not entirely random, but the sample size is too small for a useful analysis of breakpoint distribution. However, evidence for a non-random distribution of breakpoints has also been found for radiationinduced hprt deletion in C H O cells (accompanying paper: Morgan et al., 1990), and for spontaneous hprt deletions in human T K 6 cells (Gennett and Thilly, 1988). In contrast, spontaneous deletion breakpoints were found to be spread approximately evenly across the gene in adult human lymphocytes, but examples were found of the independent occurrence of 'identical' mutations (at the level of Southern analysis; Nicklas et al., 1989). It may be speculated, therefore, that either some form of site-specificity occurs in deletion muta-

genesis or that non-random selection of mutation types has occurred. The possibility of non-random selection must always be borne in mind, although at present there is no evidence with the hprt gene that a deletion mutation at one site has any phenotypic distinction to (and, therefore, potential selective advantage over) a similar mutation at another site within the gene. However, there are ways in which this might occur. For example, in other systems it has been found that alternative splicing of exons occurs such that different gene products result from one gene (review: Breitbart et al., 1987); a deletion at one site could mimic this process to yield a less extreme phenotype than deletion elsewhere. In a different way, human X-linked muscular dystrophy presents an example of the difficulty of predicting the phenotypic outcome of deletion mutation: the severe (Duchenne's) form

169 a n d the mild (Becker) form of m u s c u l a r d y s t r o p h y can b o t h arise through deletion of the same parts of the very large d y s t r o p h i n gene. While it has b e e n suggested that the mild disease results from deletions which c a n keep the reading frame of the gene in phase - so that a shortened b u t active gene p r o d u c t is formed - a n d that similar deletions giving the severe form are frameshift ( n o n sense) m u t a t i o n s ( M o n a c o et al., 1988), this seems to be an oversimplification ( M a l h o t r a et al., 1988). A n alternative possibility is that site-specificity occurs through m e c h a n i s m s which favour deletion f o r m a t i o n at certain sequences or structures in D N A . This possibility has been explored i n the a c c o m p a n y i n g paper with respect to scaffold att a c h m e n t sites in particular, b u t could involve a variety of other sites such as q u a s i - p a l i n d r o m i c regions or highly repetitive sequences. These sequences could have r e c o m b i n a t i o n - l i k e functions or m a y form regions which affect the repair of d a m a g e d D N A . It has already b e e n shown that very small s p o n t a n e o u s or r a d i a t i o n - i n d u c e d deletions (up to a b o u t 20 bp) i n m a m m a l i a n genes are often located between sites of short repeat sequences, suggesting some form of n o n - h o m o l o gous r e c o m b i n a t i o n process ( N a l b a n t o g l u et al., 1986, Breimer et al., 1986; de Jong et al., 1988; Grosovsky et al., 1988). However, the m e c h a n ism(s) of f o r m a t i o n of larger deletions, such as those described here, r e m a i n s to be demonstrated.

Acknowledgements This study was supported i n part b y the Commission of the E u r o p e a n C o m m u n i t i e s contract B16-E-144-UK, the Office of H e a l t h a n d E n v i r o n m e n t a l Research ( O H E R ) U.S. D e p a r t m e n t of Energy u n d e r contract D E - A M 0 6 - 7 6 R L O 1830 to Pacific Northwest Laboratory, a n d contract DEA M 0 6 - 7 6 - R L O 2 2 2 5 to the Northwest College a n d U n i v e r s i t y Association for Science (University of Washington).

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170 Nalbantoglu, J., D. Hartley, G. Phear, G. Tear and M. Meuth (1986) Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini, EMBO J., 5, 1199-1204. Nicklas, J.A., T.C Hunter, J.P. O'Neill and R.J. Albertini (1989) Molecular analyses of in vivo hprt mutations in human T-lymphocytes, III. Longitudinal study of hprt gene structural alterations and T-cell clonal origins, Mutation Res., 215, 147-160. Rossiter, B.J.F. (1987) Structure and mutation of the Chinese hamster HPRT gene, Ph.D. Thesis, University of Manchester, England. Stankowski, L.F., K.R. Tindall and A.W. Hsie (1986) Quantitative and molecular analyses of EMS- and ICR191-induced mutation in AS52 cells, Mutation Res., 160, 133-147. Thacker, J. (1981) The chromosomes of a V79 Chinese hamster line and a mutant subline lacking HPRT activity, Cytogenet. Cell. Genet., 29, 16-25.

Thacker, J. (1986) The nature of mutants induced by ionising radiation in cultured hamster cells, III. Molecular characterization of HPRT-deficient mutants induced by y-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene, Mutation Res., 160, 267-275. Thacker, J., and A. Ganesh (1989) Molecular analysis of spontaneous and EMS-induced mutations of the hprt gene in hamster cells, Mutation Res., 210, 103-112. Vrieling, H., J.W.I.M. Simons, F. Arwert, A.T. Natarajan and A.A. van Zeeland (1985) Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions, Mutation Res., 144, 281-286. Vrieling, H., J.W.1.M. Simons and A.A. van Zeeland (1988) Nucleotide sequence determination of point mutations at the mouse HPRT locus using in vitro amplification of HPRT m R N A sequences, Mutation Res., 198, 107 113.