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Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans
Mutatton Research, 230 (1990) 187-195
187
Elsexaer MUT 04863
Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans Nora Babudri 1 and Giorgio Morpurgo 2 I Department of Plant Btology, Unwerstty of Rome 'La Sapwnza" Rome (Italy) and 2 Instttute of Cellular Bwlogy, Unwerstty of Perugla, Perugta (Italy)
(Recewed 17 July 1989) (Rexasion recewed 18 October 1989) (Accepted 11 January 1990) Keywords" Aspergdlus mdulans; Diploid strain; RecessivemutaUon frequency
Summary The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on either resting or germinating conidia. Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the haploid strain as well as the mitotic recombination frequencies. We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.
The observable frequency of recessive mutations in diploid cells should be the square of the mutation frequency in the hemizygous condition, i.e., the square of the mutation frequencies at X-linked loci. Mutation rates higher than expected have been observed by several authors in diploid cells in culture. The following mechanisms have been advocated to explain this phenomenon: (1) functional hemizygosity of cell lines for certain loci; (2) high incidence of mitotic recombination or other mechanisms of somatic segregation; (3) epigenetic inactivation of genes (for a review see Siminovitch, 1976).
Correspondence: Nora Babudn, Istituto dl BiologdaCellulare, Via Elce Da Sotto, 06100 Perugda(Italy).
In spite of the nlzmerous efforts, the problem is so far at least partially unsolved. In this work we measure the frequency of recessive mutations in diploid cells of Aspergzllus ntdulans, with the aim of understanding the nature of the phenomenon described above. Actually this mould represents a model organism where genetlcal analysis is feasible; hence it allows the study of possible mechanisms that could bring a mutauon in homozygosis. The data reported in this paper show that in a diploid strain of A. mdulans the frequency of recessive mutations at the fpaA locus (p-fluorophenylalanine (FPA) resistance) (Morpurgo, 1962a; Srivastava and Sihna, 1975) is in the expected order of magnitude. Comparable data were obtained by the 8-azaguanine (8-AZA) resistance test.
0027-5107/90/$03.50 © 1990 ElsevierSctencePubhshers B.V (Biome&calDivision)
188
We review some papers on mammalian cell lines and conclude that in some cases a really higher mutation rate has been observed (Gupta, 1978; Tischfield et al., 1982; Turker et al., 1984) while m others the frequency of recesswe mutations at autosomal loci was not different from that expected (Chasin, 1974; Eves and Farber, 1981; Bradley and Letovanec, 1982; Simon and Taylor, 1983).
the haploid was deternuned by the FPA resistance test (Morpurgo, 1962a). The method for the evaluauon of mitotic recombination leading the fpaA locus to homozygosis has been described in Beccari et al. (1967). This method allows the selection of fpaA/fpaA colomes arising from fpaA+/fpaA diploid conidia either by crossing over or by gene conversion. fpaA mutation mapping and characterization are described in Morpurgo (1962a) and Srlvastava and Sinha (1975). In UV-irradiation experiments a 30-W germicidal lamp placed 40 cm above the plates was used. The dose rate at ttus distance was about 40 erg/mmZ/s at the level of the target.
Materials and methods
Strains Haploid and diploid strains of Aspergtllus mdulans are listed in Table 1. All are descendants of A. mdulans isolated by Pontecorvo et al. (1953). Chromosome I of the 18/35 fpaA strain is shown in Fig. 1. For the symbols used throughout this paper see Clutterbuck (1974).
Determmatton of the number of UV-mduced fpaA / fpoA d~plo~ds Two experimental protocols were devised for the determination of the number of UV-lnduced fpaA/fpaA diploids: (1) resting comdla: conidia (400000/plate) were plated on CD-NH~--FPA medmm supplemented only with biotin and immediately UV-irradiated; (2) germinating conidla: conidia (4 × 106/plate) were plated on 12.5 ml of CD-NH~- medium supplemented with thiamine, p-aminobenzoic acid, proline and riboflavine (CDNH~--APPBR medium); on this medium f p a A / fpaA diploids could be recovered also when they resulted from mitotic recombination or non-disjunction events leading UV-induced fpaA mutations to homozygos~s. After plating, conidia were allowed to germinate (4.5 h) and then UV-lrradiated. After 3 h at 37 °C a second layer of CD-NH~-APPBR medium plus FPA was added. This procedure was adopted in order to have the conditions as similar to those of mammalian cell culture as possible. It must be noted that with germinating comdia the survival of the 18/35 strain at 90 sec of UV
Medta The Czapek-Dox minimal medium (CD) and the AF medium containing a mixture of 3-aminotyrosine and phenylanthranilic acid (6 mg/mi and 0.15 mg/ml respectively, final concentrations) have been previously described (Hopwood and Sermonti, 1962; Calvori and Morpurgo, 1966). For the isolation of FPA-resistant (FPAR) dlplolds, sodium nitrate was replaced by ammonium sulfate (equlmolar) in the CD medmm and the pH was adjusted to 7 instead of 6.2 (CD-NH~ medmm); this medium allows better conidiation of FPAR colonies. FPA was added to the CD-NH~medium at a final concentration of 0.15 mg/ml (CD-NH~--FPA medium).
Evaluatton of the mutatton frequency at the fpaA locus and of mitotic recombmation tn the 18/35 fpaA stram The mutation frequency at the fpaA locus in 1
+
+
fpaA
anAl
SUAladE20
rlboA1
+
+
Fig 1. Chromosome I of the diploid straan 18/35
fpaA
+ proA1
pabaA1
yA2
+
4-
+ adeE20
+ blA2
Meiotic distances are not shown Only markers relevant to this paper are reported
189 TABLE 1 GENOTYPE AND ORIGIN OF HAPLOID AND DIPLOID STRAINS Strata number 35 18
35fpaA 35azgP1 18/35 18/35fpaA
Genotype pabaAl, anAl,yA2, methG, incA2;sC suAladE20,nboA1 proA1, adE20,blA1, pyroA4 as 35 but fpaA as 35 but AZAR
18/ 35(fpaA/ fpaA) 18/35 azgP1
Origin Our collection Our collection A spontaneous fpaA mutant A UV-induced AZAR strain Component haploids' 18 and 35 Component haplolds. 18 and 35fpaA Component haploids: 18 and 35fpaA a Component haploids: 18 and 35 azgP1
a 18/35(fpaA/fpaA) was obtained by point-moculatmgCD plates supplementedwith thiamane, p-anunobenzolcacid and FPA and by isolating wgorous slightlygreen &piold emergingsectors, which were shown by haploidmation to be homozygousfor the fpaA mutation irradiation was lower than with resting conidia (10% instead of 27%); moreover the colonies were very small compared to normal colonies. This phenomenon is well known in Aspergdlus nidulans and it has been extensively studied by Kiifer (1969). For this reason the number of conidia plated was 4 x 106/plate instead of 4 × 105/plate. In both treatments the surviving fraction was determined on the same medium without FPA. After 7 days conidiating colonies were isolated and tested on CD-FPA and AF medium. On A F medium the growth of fpaA mutants is inhibited (Calvori and Morpurgo, 1966). When slight growth developed on A F medium and A F resistance was not completely clear, mutants were tested on CD medium with and without L-phenylalanine. In fact, the great majority of fpaA mutants requires Lphenylalanine for optimal growth (Calvori and Morpurgo, 1966). 18/35 and 18/35 (fpaA/fpaA) were used as controls.
Expected frequency estimation In resting conidia plated on CD-NH~--FPA medium plus biotin the expected frequency of fpaA/fpaA diploids is the square of the mutation frequency in the haploid strain. In germinating conidia the expected frequency of fpaA/fpaA diploids is given by the frequency in resting conidia plus 2 x (frequency of fpaA
mutants in the h a p l o i d ) x (frequency of mitotic recombination). The frequency of mutation is doubled because 2 loci can undergo recombination. The expected frequencies are calculated using data reported in Figs. 2 and 3. The frequency of non-disjunction is not considered in calculations because the frequency of non-disjunctional haploid or diploid segregants for chromosome I is only 1 x 10-5 (Morpurgo, 1962b) and this frequency is not increased by UV irradiation (Morpurgo, 1962b; K~ifer, 1969). Actually, the spontaneous frequency of non-disjunction is much higher, about 0.1-1 x 10 -3 (Morpurgo et al., 1979) but many slowly growing, aneuploid colonies are completely eliminated on FPA medium (Morpurgo et al., 1979). Finally it must be noted that we define 'mutation frequency' as the frequency of fpaA/fpaA diploids derived from + / + conidia, although these resistant cells could derive from true mutational events as well as from mitotic recombination.
8-Azaguanme reststance in haploid and diploM cells of A. nidulans The spontaneous and induced frequencies of 8-azaguanine-resistant (8-AZA R) mutants in the haploid and diploid conidia of the strain 35 were determined according to the test described by Morpurgo (1962a) on CD medium plus biotin,
190
nicotinic acid and 8-AZA. The selective agent (8-AZA) concentration was 0.2 m g / m l (final concentration in the medium). For the determination of spontaneous and induced mitotic recombination frequencies, conidia of the 18/35 azgP1 strain were plated (104/plate) on CD medium supplemented with biotin, nicotinic acid and 8-AZA (0.2 mg/ml, final concentration in the medium). After 72 h at 37 ° C 8-AZA R colonies were counted. The surviving fraction was determined on the same medium without 8-AZA. The 35 azgP1 mutant was obtained in our laboratory by UV-irradmting conidia of the 35 strain. By the standard genetical procedures (Pontecorvo et al., 1953) the azgP1 allele was assigned to the V chromosome (it is linked to the incA2 locus of the 35 strain). This mutant was used to obtain the 18/35 azgP1 strata. It must be noted that it is not known exactly how many loci conferring 8-AZA resistance can be selected by the mutagenesis method used in this work, although it is likely that one locus is involved (Darlington and Scazzocchio, 1967).
"11
350
C~
100
200 <: 5C
150
O U~
100
- 50
is
3o
,s UV
go
is
9o
0
DOSE (see)
Results
Fig. 2 UV survival and frequency of F P A R colonies in the haploid strain 35 and in the diploid strata 18/35 Open symbols indicate survwal and closed symbols mutagenesls strata 35 (resting corn&a), o O; strain 18/35 (resting comdla), Q II; strata 18/35 (gerrmnatlng corn&a), zx The spontaneous frequency of F P A R (fpaA) m u t a n t s m haploid strata 35 was 0 2 × 1 0 - 6 in resung comdla The spontaneous frequency of F P A R (not fpaA) m u t a n t s m strain 18/35 was 1 5 x 1 0 -8 (resting corn&a). Each point represents the mean value of at least 3 experiments
Frequency of mutation at the fpaA locus and frequency of mltottc recombinatmn m the 18/35 fpaA strain The frequency of UV-induced FPA R (fpaA)
order to detect a small increase in mutation frequency compared to that expected, a far higher number of viable conidia should have been plated.
mutants in the 35 haploid strain (resting conidia) is shown in Fig. 2. The frequency of fpaA mutants in germinating conidia is similar to that observed for resting conidia (Gualandi et al., 1978). The spontaneous frequency of mitotic recombination leading the fpaA locus to homozygosls was about 1 × 10-4; It did not significantly change upon UV irradiation (Fig. 3).
Spontaneous frequency of fpaA / fpaA diplotds from + / + comdta The spontaneous frequency of fpaA/fpaA mutants was determined in resting and germinating conidia (see Materials and methods for the protocols used). The expected frequency of fpaA/ fpaA mutants is 4 × 1 0 - 1 4 in resting conidia and 5.6 × 10-la in germinating ones. In 6 experiments we plated about 3 × 108 viable cortidia and did not find any fpaA/fpaA mutants (Table 2). In
UV-mduced frequency of fpaA /fpaA dtplotds from + / + conidia Table 2 shows the data obtained in UV-irradiation experiments on resting and germinatmg conidia, Unexpectedly, a considerable number of FPA R mutants were recovered in these experiments. They turned out to be dominant, grew on A F medium and did not require tyrosine. In most cases they were lethal in hemizygosis and did not map on the I chromosome; therefore they were not fpaA/fpaA mutants (Babudri and Morpurgo, Genetics, in press). In Fig. 2 the survival and the frequency of FPA R mutants after UV irradiation of the 18/35 strain are reported. At 90 sec the expected frequency of fpaA/fpaA mutants with resting conidia was 1.22 × 1 0 - 7 while with germinating comdia it was 3.32 × 10 -7. No fpaA/ fpaA mutant was recovered from 565 FPA R col-
191 TABLE 2 SPONTANEOUS AND UV-INDUCED FREQUENCY OF fpaA/fpaA DIPLOIDS Irradiation time (nun)
Survtval (%)
Number of viable comdia plated
0 90
100 27
2 × 108 8 × 106
0 90
100 10
1 × 108 7.8 x 106
lO0
o
-3o
Expected frequency of
colomes
fpaA/fpaAdlplo]ds
30 565
0 0
4 ×10 -14 1.22 × 10- 7
43 1492
0 0
56 ×10 -1] 3.32×10 -7
onies tested on A F medium. Hence there was no increase in the frequency of fpaA/fpaA mutants over that expected in diploid conidia of A. nidu-
-40
>
fpaA/fpaA
FPAa colomes recovered
_m
lans.
z
-20 lOO0
loo
-10
,
i
J
UV DOSE
,
,
,
?
o
(see)
Fig. 3. Recombination frequency in the 18/35 fpaA and 18/35 azgP1 strains. Open symbols indicate survival and dosed symbols recombmation frequencaes: swain 18/35 (resting eonidla), <3; strain 18/35 (germinating conidia, zx; strain 18/35fpaA (resting comcha), v; strain 18/35fpaA (germinating conidla), *; strata 18/35azgPl (resting comdia), e. The survival for the 18/35fpad and 18/35azgP1 strains was similar to that reported for strata 18/35. The spontaneous frequency of recombinants in the 18/35 azgP1 strain (49×10 -4) has been subtracted. For the 18/35 fpaA strain the spontaneous frequency of recombinants has not been subtracted. In fact the presence of recombinant clones could be excluded in the 18/35fpaA comd]al populauon used in these experiments because it was derived from a streak grown on CD medium supplemented only with biotin. On thts medium the growth of fpaA/fpad recombinants is strongly reduced (Morpurgo, 1962a). Each point represents the mean value of at least 2 experiments.
.~ 5c
500
~
%
m
6
15
3'0
4'S
6'0
7'S
9'0
UV DOSE ( s e c )
Fig. 4, UV survival and frequency of 8-AZAg mutants in haploid stram 35 and m diploid strata 18/35. Open symbols indicate survwal and closed symbols indicate mutagenesis: strain 35 (resting comdia), o e; strata 18/35 (resting conidia), • D. The spontaneous mutation frequency for the 35 strain was 0.9 × 10 -6 (1-ustoncal mean). The frequency of mutation for the 18/35 strain was 3.5 × 10 -6 at 60 sec of UV irradiauon and 10 x 10 -6 at 90 sec. Each pomt represents the mean value of at least 3 experiments.
TABLE 3 FREQUENCY OF 8-AZAR DIPLOID MUTANTS IN RESTING CONIDIA Irradiation ume (ram)
Surwval (%)
Frequency of 8-AZAR Frequency of 8-AZAR diploid mutants from mutants in the haploid wild-type conidia strain 35 a
Frequency of rmtotic recombination a
Expected frequencies of AZAR mutants from wild-type comdia b
60 90
58 27
3.5 × 10 -6 1 x l 0 -5
1.5 X 10-3 3.8X10 -3
1 ×10 -6 0.76 × 10- 5
3.3 X 10 -4 1 x l 0 -3
a Controls have been subtracted. b The expected frequency of 8-AZAR diploids is given by the square of the frequency m the haploid plus 2 × (frequency of 8-AZAR mutants in the haploid)× (frequency of rmtotic recombination).
192
Frequency of 8-AZA R mutants The UV survival and the frequency of 8-AZAa mutants in haploid strain 35 are shown in Fig. 4. The recombination frequencies in the 18/35 azgP1 strain are reported in Fig. 3. Clearly the frequency of mitotic recombination leading the azgP1 locus to homozygosis increased upon UV irradiation. The frequency of 8-AZAa diploid conldia derived from + / + ones after UV irradiation is shown in Table 3: it was not different from that expected.
Reconstruction experiments Since in UV-irradiation experiments a large number of FPAa diploid colonies developed, we wondered whether they might hamper the growth of fpaA/fpaA mutants. 100-400 conidia of the 18/35 (fpaA/fpaA) diploid strain, homozygous for the yA2 marker, were mixed with 4 × 106 conidia of the 18/35 wild-type (pale green) strain. UV irradiation was carried out as described for germinating conidia. Almost complete recovery of the fpaA/fpaA yellow conidia was obtained (data not shown); therefore the growth of fpaA/fpaA colonies was not limited by other FPAa diploid colonies. Discussion
The data reported in this paper demonstrate that the frequency of UV-induced recessive mutations in diploid cells of A. mdulans is in the expected order of magnitude. Both the mutation frequency in the haploid and diploid strains and the mitotic recombination have been assessed in resting as well as germinating conidia with the FPA and 8-AZA resistance tests. It must be noted that the UV treatment induced mitotic recombination leading the azgP1 locus to homozygosis while it did not significantly increase the homozygosity at the fpaA locus. The reason of the different recombinogemc effects of UV light in the 2 systems is unknown. Possibly a less stringent selection with 8-AZA allowing more residual growth or an interference of drugs with recombinogenic processes could account for this difference. Actually Wood and K~ifer (1969) obtained a strong increase in somatic recombination frequencies upon UV irradiation using a non-
selective technique, Le., scoring for yellow sectors in a y~ + diploid. Concerning mammalian cell lines, several authors state that the frequency of recessive mutations at some autosomal loo is higher than expected (Siminovitch, 1976; Gupta et al., 1978; Eves and Farber, 1981; Bradley and Letovanec, 1982; Tischfield et al., 1982; Simon and Taylor, 1983; Turker et al., 1984). Their conclusions can be explamed by assuming that: (1) genetic or epigenetic phenomena peculiar to mammahan cells m culture lead to high frequencies of recessive mutations at autosomal loci; (2) some data have been masinterpreted and standard genetical mechanisms alone could explain the observed frequencies. Let us now review some papers about this topic. Gupta et al. (1978; Gupta, 1984) extensively studied this problem in Chinese hamster ovary (CHO) cells in culture. They found that the mutation frequency at the autosomal Emt R lOCUS(conferring emetine resistance) was almost identical to that observed at X-linked loci, Le., at hemizygous loci. They demonstrated (Gupta et al., 1978) that the CHO cell line studied was hemizygous for the Emt a lOCUS,thus explaining the phenomenon. The frequency of mutation at the same locus in other CHO cell lines examined by Gupta (1984) was much lower and in the order of magnitude of that expected taking into account a reasonable frequency of mitotic recombination. This problem has been studied also by other authors, often with a molecular approach. The systems used in these works are selection for diaminopurine resistance (adenine phosphoribosyltransferase (APRT) deficiency), selection for bromodeoxyuridine resistance (thymidine kinase (TK) deficiency) and selection for toyocamycln resistance (adenosine kinase (ADK) deficiency). These systems utihze autosomal recessive mutations and allow the selection from the wild-type condition ( + / + , fully sensitive) to the heterozygous condition ( + / - , intermediate sensitivity) and from this to the - / (fully resistant) condition. Hence each step from the + / + condition to the fully resistant one ( - / - ) can be independently studied. Eves and Farber (1981) state in their paper 'the observed frequencies of recessive mutation in cul-
193 TABLE 4 M U T A T I O N F R E Q U E N C I E S A T T H E A D K , H P R T A N D A P R T LOCI Locus
Cell line
ADK
CAK-BR c
HPRT
CS1 (CHO)
HPRT
CS1 (CHO)
HPRT
V79 (CHO)
HPRT
V79 (CHO)
APRT
D-416(CHO)
Dormnant and Xhnked genes m man
Mutatton frequency
Expression a
Survival
Expected b
Spontaneous
EMS-induced
time
(Sg)
frequencies
0.5-1 × 1 0 - 6 d
10
50
1.39 × 1 0 - 6
4
100
Farber (1981) T h o m p s o n et
4
66
al. (1980) T h o m p s o n et
5
100
2-8 X 10- 6 7.5 x 10-4 3 × 10-6 1.18 × 10-3 5.9 × 1 0 - 7 e _ . ~ 8.8 X 10-5 0.18 x 10 - 6 ~ 1.05 x 10 - 4
5 4
Eves and
al. (1980) Abbondandolo et al. (1980) Abbondandolo
40 9 100
References
3 × 10- 6
et al. (1980) Bradley and Letovanec (1982) Vogel and Motulsky (1988)
a Days of subculture m non-selective conditions before plating m non-selecuve m e d m m . b The expected frequency is the square of the mutation frequency at X-linked loci. For the calculatmn the data of Abbondandolo et al. (1979) have been used. ¢ Mouse embryonic fibroblast line. d Frequency of fully resistant from fully sensiUve cells ( A D K + / A D K + to A D K - / A D K - ). e Frequency of A P R T - / A P R T - from A P R T + / A P R T - cells
tured mammalian cells are generally much higher than would be predicted by comparison with the frequencies of dominant mutations in the same cell lines or with recessive mutations in haploid organisms'. In Table 4 the frequency obtained by Eves and Farber at the autosomal ADK locus in a near-diploid mouse cell line after treatment with ethyl methanesulfonate (EMS) and the frequencies obtained by other authors at the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in CHO cells are reported. On the basis of these data the expected frequency of fully toyocamycinresistant cells obtained from fully sensitive ones should be about 1 × 10-6; somatic segregation events, if any, should increase this value. Actually, the value obtained by Eves and Farber (0.5-1 × 10 -6 ) is not dissimilar from that expected. Therefore these authors have studied the problem of high frequencies of recessive mutations in cultured mammalian cells in a system where mutation frequencies were not different from those expected. Simon and Taylor (1983) stated that 'one of the most puzzling findings in somatic cell genetics has
been the high frequency occurrence of recessive mutations at autosomal loci (Siminovitch, 1976, Cell, 7, 1-11)'. They used the D416 cell line to clarify this problem. The D416 cell line is an A P R T - / A P R T ÷ heterozygous cell line obtained from CHO cells by Bradley and Letovanec (1982) who carefully evaluated the mutation rates in their newly isolated cell line by 2 different methods, one of which is the P0 calculation according to Lea and Coulson (1949). The values, according to P0 calculation, varied from 8.8 x 10 -5 to 5.9 × 10 -7. These figures do not seem exceptionally high; the highest value is not very different from that obtained for some dominant or X-linked genes in humans (Vogel and Motulsky, 1988) (Table 4). However, Bradley and Letovanec (1982) stated that D416 gave fully diaminopurine-resistant colonies at a relatively high frequency and Simon and Taylor (1983) used this cell line for the purpose cited above. Moreover, in these papers (Simon and Taylor, 1983; Bradley and Letovanec, 1982) the word 'mutation' is frequently used to describe a highfrequency event leading from the + / to the
194
-/condition. In the Discussion the authors always correctly suggest that this event could be some kind of somatic rearrangement. We think, however, that the use of the word 'mutation' in their context is misleading and may accredit the unproved assertion that observed frequencies of recessive mutations in diploid cells are higher than expected. Really higher mutation frequencies have been observed in some systems. For example, Tischfield et al. (1982) report that resistance to 2,6-diaminopurine occurred at a rate of about 10-3 per cell per generation in mouse L cells, while mutation rates at other loci were normal. The authors demonstrate that this high frequency was the result of a mutation rather than of an epigenet~c event. They suggest that a mutational hot-spot, a locus-specific mutator gene or a site of integration for an insertion sequence may be the cause of the high mutation rates in their heteroploid cell line where it is likely that only one copy of the A P R T gene is functioning. Turker et al. (1984) in teratocarcinoma cells reported a frequency of the event leading from the A P R T + / A P R T - to the A P R T - / A P R T - con&tlon and vice versa In the order of magnitude of 1 × 10 -2 and probably this phenomenon is related to the peculiarity of teratocarcinoma cells. In conclusion we think that, with the exception of some cases, in mammalian cell lines the spontaneous and induced frequencies of recessive mutations are not parucularly high at autosomal loci. Acknowledgements
This work was supported by the Ministero della Pubblica Istruzione (MPI 40% and 60%) and by the Consiglio Nazionale deUe Ricerche (Progetto Strategico Mutagenesi). References Abbondandolo, A , S. Bonatti, A. Mazzacarro, R Randazzo, L Madam, G Mamscalco and A. D1 Leonardo (1979) m G.E. Magni (Ed.), Mutagenesl Amblentale - Metodlche dl Analisl, Vol. I, CNR, Rome, pp. 195-222. Beccan, E., P. Modtgham and G Morpurgo (1967) Inducuon of rater- and intragemc mitottc recombmatlon by fluorodeoxyundme and fluorouracil m Aspergdlus mdulans, Genetics, 56, 7-12
Bradley, W E C , and D Letovanec (1982) High-frequency nonrandom mutational event at the ademne phosphorlbosyltransferase (aprt) locus of slb-selected vanants heterozygous for aprt, Somat. Cell Genet, 8, 51-66 Calvorl, C , and G. Morpurgo (1966) Analysis of induced mutations m Aspergdlus ntdulans 1 UV- and HNO2-mduced mutations, Mutation Res., 3, 145-151 Chasm, L A (1974) Mutations affecting adenine phosphorlbosyltransferase actlwty in Chinese hamster cells, Cell, 2, 37-41 Clutterbuck, A J (1974) Aspergdlus ntdulans, m R.C Kmg (Ed), Handbook of Genetics, Vol 1, Plenum, New York, pp. 447-510 Darhngton, A.J, and C Scazzocctuo (1967) Use of analogues and the substrate-sensmwty of mutants m analys~s of punne uptake and breakdown in Aspergtllus mdulans, J Bacterlol, 93, 937-940 Eves, E M , and R A Farber (1981) Chromosome segregation is frequently assocmted with the expression of recessive mutauons in mouse cells, Proc Natl Acad. Scl. (U S A ), 78, 1768-1772 Gualandl, G , G Morpurgo and S Puppo (1978) Relative efficiency of mutagens in inducing different types of genetic damage In A mdulans, Atti Ass Genet. Ital, 23, pp 151-153. Gupta, R.S (1984) Genetic markers for quantitative mutagenesis stu&es in Chanese hamster ovary cells, apphcation to mutagen screemng stuches, m' B.J. Kalbey, M. Legator, W Nichols and C. Ramel (Eds), Handbook of Mutagemoty Test Procedures, Elsevaer, Amsterdam, pp 291-319 Gupta, R.S, D.Y H Chan and L Sln'unovltch (1978) Evidence for functional henuzygoslty at the Emt r locus m CHO cells through segregation analysis, Cell, 14, 1007-1013 Hopwood, D A., and G Sermontl (1962) Genetics of Streptomyces coehcolor. Adv Genet, 11,273-342 Kafer, E (1969) Effects of ultraviolet irradiation on heterozygous diplolds of Aspergdlus mdulans II, Recovery from UV-mduced mutation m matotic recombinant sectors, Geneucs, 63, 821-841 Lea, D E, and C A Coulson (1949) The distribution of the numbers of mutants m bacterial populaUons. J Genet., 49, 264-285 Morpurgo, G (1962a) A new method of esttrnatmg forward mutation in fungi: resistance to 8-azaguamne and p-fluorophenylalamne, Scl. Rep. Ist Sup Samth, 2, 9-12. Morpurgo, G (1962b) Quantltauve measurement of reduced somatic segregation in Aspergdlus ntdulans, Scl Rep Ist Sup Samt~, 2, 324-329 Morpurgo, G , D Belhncampi, G. Gualandi, L Baldinelh and O Serlupl (1979) Analysis of rmtotlc non-disjunction with Aspergdlus mdulans, Envtron. Health Perspect., 31, 81-95. Pontecorvo, G., J A. Roper, L.M. Hemmons, K.D MacDonald and A W J Burton (1953) The genetics of Aspergdlus ntdulans, Adv Genet., 5, 141-238 Sirmnovttch, L (1976) On the nature of heredltable variation in cultured somatic cells, Cell, 7, 1-11 SLmon, A E., and M.W. Taylor (1983) High-frequency mutauon at the ademne phosphonbosyltransferase locus in
195 Clunese hamster ovary cells due to deleuon of the gene, Proc. Natl. Acad. Sci. (U S.A.), 80, 810-814. Snvastava, S., and U. Sinha (1975) Six new loci controlling resistance to p-fluorophenylalanme in Aspergdlus mdulans, Genet Res., 25, 29-38. Thompson, L.H., S Fong and K. Brookman (1980) Vahdatlon of condltmns for efficient detecUon of HPRT and APRT mutatmns m suspension-cultured Clunese hamster ovary cells, Mutation Res., 74, 21-36. Tlschfield, J.A, J.J Trill, Y.I. Lee, K Coy and M.W. Taylor (1982) Geneuc mstablhty at the adenine phosphonbo-
syltransferase locus in mouse L cells, Mol. Cell. Biol., 2, 250-257. Turker, M.S., A.C. Smith and G.M. Martin (1984) High frequency "switclung" at the ademne phosphonbosyltransferase locus m mulUpotent mouse teratocarcanoma cells, Somat Cell Mol. Genet., 10, 55-69. Vogel, E.W., and Motulsky (1988) Human Genetics- Problems and Approaches, Spnnger-Verlag, Berhn, p 343. Wood, S., and E K~ifer (1969) Effects of ultravmlet lrradlataon on heterozygous &plolds of Aspergdlus mdulans. I. UV-mduced matOtlC crossmg over, Geneucs, 62, 507-518.
187
Elsexaer MUT 04863
Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans Nora Babudri 1 and Giorgio Morpurgo 2 I Department of Plant Btology, Unwerstty of Rome 'La Sapwnza" Rome (Italy) and 2 Instttute of Cellular Bwlogy, Unwerstty of Perugla, Perugta (Italy)
(Recewed 17 July 1989) (Rexasion recewed 18 October 1989) (Accepted 11 January 1990) Keywords" Aspergdlus mdulans; Diploid strain; RecessivemutaUon frequency
Summary The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on either resting or germinating conidia. Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the haploid strain as well as the mitotic recombination frequencies. We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.
The observable frequency of recessive mutations in diploid cells should be the square of the mutation frequency in the hemizygous condition, i.e., the square of the mutation frequencies at X-linked loci. Mutation rates higher than expected have been observed by several authors in diploid cells in culture. The following mechanisms have been advocated to explain this phenomenon: (1) functional hemizygosity of cell lines for certain loci; (2) high incidence of mitotic recombination or other mechanisms of somatic segregation; (3) epigenetic inactivation of genes (for a review see Siminovitch, 1976).
Correspondence: Nora Babudn, Istituto dl BiologdaCellulare, Via Elce Da Sotto, 06100 Perugda(Italy).
In spite of the nlzmerous efforts, the problem is so far at least partially unsolved. In this work we measure the frequency of recessive mutations in diploid cells of Aspergzllus ntdulans, with the aim of understanding the nature of the phenomenon described above. Actually this mould represents a model organism where genetlcal analysis is feasible; hence it allows the study of possible mechanisms that could bring a mutauon in homozygosis. The data reported in this paper show that in a diploid strain of A. mdulans the frequency of recessive mutations at the fpaA locus (p-fluorophenylalanine (FPA) resistance) (Morpurgo, 1962a; Srivastava and Sihna, 1975) is in the expected order of magnitude. Comparable data were obtained by the 8-azaguanine (8-AZA) resistance test.
0027-5107/90/$03.50 © 1990 ElsevierSctencePubhshers B.V (Biome&calDivision)
188
We review some papers on mammalian cell lines and conclude that in some cases a really higher mutation rate has been observed (Gupta, 1978; Tischfield et al., 1982; Turker et al., 1984) while m others the frequency of recesswe mutations at autosomal loci was not different from that expected (Chasin, 1974; Eves and Farber, 1981; Bradley and Letovanec, 1982; Simon and Taylor, 1983).
the haploid was deternuned by the FPA resistance test (Morpurgo, 1962a). The method for the evaluauon of mitotic recombination leading the fpaA locus to homozygosis has been described in Beccari et al. (1967). This method allows the selection of fpaA/fpaA colomes arising from fpaA+/fpaA diploid conidia either by crossing over or by gene conversion. fpaA mutation mapping and characterization are described in Morpurgo (1962a) and Srlvastava and Sinha (1975). In UV-irradiation experiments a 30-W germicidal lamp placed 40 cm above the plates was used. The dose rate at ttus distance was about 40 erg/mmZ/s at the level of the target.
Materials and methods
Strains Haploid and diploid strains of Aspergtllus mdulans are listed in Table 1. All are descendants of A. mdulans isolated by Pontecorvo et al. (1953). Chromosome I of the 18/35 fpaA strain is shown in Fig. 1. For the symbols used throughout this paper see Clutterbuck (1974).
Determmatton of the number of UV-mduced fpaA / fpoA d~plo~ds Two experimental protocols were devised for the determination of the number of UV-lnduced fpaA/fpaA diploids: (1) resting comdla: conidia (400000/plate) were plated on CD-NH~--FPA medmm supplemented only with biotin and immediately UV-irradiated; (2) germinating conidla: conidia (4 × 106/plate) were plated on 12.5 ml of CD-NH~- medium supplemented with thiamine, p-aminobenzoic acid, proline and riboflavine (CDNH~--APPBR medium); on this medium f p a A / fpaA diploids could be recovered also when they resulted from mitotic recombination or non-disjunction events leading UV-induced fpaA mutations to homozygos~s. After plating, conidia were allowed to germinate (4.5 h) and then UV-lrradiated. After 3 h at 37 °C a second layer of CD-NH~-APPBR medium plus FPA was added. This procedure was adopted in order to have the conditions as similar to those of mammalian cell culture as possible. It must be noted that with germinating comdia the survival of the 18/35 strain at 90 sec of UV
Medta The Czapek-Dox minimal medium (CD) and the AF medium containing a mixture of 3-aminotyrosine and phenylanthranilic acid (6 mg/mi and 0.15 mg/ml respectively, final concentrations) have been previously described (Hopwood and Sermonti, 1962; Calvori and Morpurgo, 1966). For the isolation of FPA-resistant (FPAR) dlplolds, sodium nitrate was replaced by ammonium sulfate (equlmolar) in the CD medmm and the pH was adjusted to 7 instead of 6.2 (CD-NH~ medmm); this medium allows better conidiation of FPAR colonies. FPA was added to the CD-NH~medium at a final concentration of 0.15 mg/ml (CD-NH~--FPA medium).
Evaluatton of the mutatton frequency at the fpaA locus and of mitotic recombmation tn the 18/35 fpaA stram The mutation frequency at the fpaA locus in 1
+
+
fpaA
anAl
SUAladE20
rlboA1
+
+
Fig 1. Chromosome I of the diploid straan 18/35
fpaA
+ proA1
pabaA1
yA2
+
4-
+ adeE20
+ blA2
Meiotic distances are not shown Only markers relevant to this paper are reported
189 TABLE 1 GENOTYPE AND ORIGIN OF HAPLOID AND DIPLOID STRAINS Strata number 35 18
35fpaA 35azgP1 18/35 18/35fpaA
Genotype pabaAl, anAl,yA2, methG, incA2;sC suAladE20,nboA1 proA1, adE20,blA1, pyroA4 as 35 but fpaA as 35 but AZAR
18/ 35(fpaA/ fpaA) 18/35 azgP1
Origin Our collection Our collection A spontaneous fpaA mutant A UV-induced AZAR strain Component haploids' 18 and 35 Component haplolds. 18 and 35fpaA Component haploids: 18 and 35fpaA a Component haploids: 18 and 35 azgP1
a 18/35(fpaA/fpaA) was obtained by point-moculatmgCD plates supplementedwith thiamane, p-anunobenzolcacid and FPA and by isolating wgorous slightlygreen &piold emergingsectors, which were shown by haploidmation to be homozygousfor the fpaA mutation irradiation was lower than with resting conidia (10% instead of 27%); moreover the colonies were very small compared to normal colonies. This phenomenon is well known in Aspergdlus nidulans and it has been extensively studied by Kiifer (1969). For this reason the number of conidia plated was 4 x 106/plate instead of 4 × 105/plate. In both treatments the surviving fraction was determined on the same medium without FPA. After 7 days conidiating colonies were isolated and tested on CD-FPA and AF medium. On A F medium the growth of fpaA mutants is inhibited (Calvori and Morpurgo, 1966). When slight growth developed on A F medium and A F resistance was not completely clear, mutants were tested on CD medium with and without L-phenylalanine. In fact, the great majority of fpaA mutants requires Lphenylalanine for optimal growth (Calvori and Morpurgo, 1966). 18/35 and 18/35 (fpaA/fpaA) were used as controls.
Expected frequency estimation In resting conidia plated on CD-NH~--FPA medium plus biotin the expected frequency of fpaA/fpaA diploids is the square of the mutation frequency in the haploid strain. In germinating conidia the expected frequency of fpaA/fpaA diploids is given by the frequency in resting conidia plus 2 x (frequency of fpaA
mutants in the h a p l o i d ) x (frequency of mitotic recombination). The frequency of mutation is doubled because 2 loci can undergo recombination. The expected frequencies are calculated using data reported in Figs. 2 and 3. The frequency of non-disjunction is not considered in calculations because the frequency of non-disjunctional haploid or diploid segregants for chromosome I is only 1 x 10-5 (Morpurgo, 1962b) and this frequency is not increased by UV irradiation (Morpurgo, 1962b; K~ifer, 1969). Actually, the spontaneous frequency of non-disjunction is much higher, about 0.1-1 x 10 -3 (Morpurgo et al., 1979) but many slowly growing, aneuploid colonies are completely eliminated on FPA medium (Morpurgo et al., 1979). Finally it must be noted that we define 'mutation frequency' as the frequency of fpaA/fpaA diploids derived from + / + conidia, although these resistant cells could derive from true mutational events as well as from mitotic recombination.
8-Azaguanme reststance in haploid and diploM cells of A. nidulans The spontaneous and induced frequencies of 8-azaguanine-resistant (8-AZA R) mutants in the haploid and diploid conidia of the strain 35 were determined according to the test described by Morpurgo (1962a) on CD medium plus biotin,
190
nicotinic acid and 8-AZA. The selective agent (8-AZA) concentration was 0.2 m g / m l (final concentration in the medium). For the determination of spontaneous and induced mitotic recombination frequencies, conidia of the 18/35 azgP1 strain were plated (104/plate) on CD medium supplemented with biotin, nicotinic acid and 8-AZA (0.2 mg/ml, final concentration in the medium). After 72 h at 37 ° C 8-AZA R colonies were counted. The surviving fraction was determined on the same medium without 8-AZA. The 35 azgP1 mutant was obtained in our laboratory by UV-irradmting conidia of the 35 strain. By the standard genetical procedures (Pontecorvo et al., 1953) the azgP1 allele was assigned to the V chromosome (it is linked to the incA2 locus of the 35 strain). This mutant was used to obtain the 18/35 azgP1 strata. It must be noted that it is not known exactly how many loci conferring 8-AZA resistance can be selected by the mutagenesis method used in this work, although it is likely that one locus is involved (Darlington and Scazzocchio, 1967).
"11
350
C~
100
200 <: 5C
150
O U~
100
- 50
is
3o
,s UV
go
is
9o
0
DOSE (see)
Results
Fig. 2 UV survival and frequency of F P A R colonies in the haploid strain 35 and in the diploid strata 18/35 Open symbols indicate survwal and closed symbols mutagenesls strata 35 (resting corn&a), o O; strain 18/35 (resting comdla), Q II; strata 18/35 (gerrmnatlng corn&a), zx The spontaneous frequency of F P A R (fpaA) m u t a n t s m haploid strata 35 was 0 2 × 1 0 - 6 in resung comdla The spontaneous frequency of F P A R (not fpaA) m u t a n t s m strain 18/35 was 1 5 x 1 0 -8 (resting corn&a). Each point represents the mean value of at least 3 experiments
Frequency of mutation at the fpaA locus and frequency of mltottc recombinatmn m the 18/35 fpaA strain The frequency of UV-induced FPA R (fpaA)
order to detect a small increase in mutation frequency compared to that expected, a far higher number of viable conidia should have been plated.
mutants in the 35 haploid strain (resting conidia) is shown in Fig. 2. The frequency of fpaA mutants in germinating conidia is similar to that observed for resting conidia (Gualandi et al., 1978). The spontaneous frequency of mitotic recombination leading the fpaA locus to homozygosls was about 1 × 10-4; It did not significantly change upon UV irradiation (Fig. 3).
Spontaneous frequency of fpaA / fpaA diplotds from + / + comdta The spontaneous frequency of fpaA/fpaA mutants was determined in resting and germinating conidia (see Materials and methods for the protocols used). The expected frequency of fpaA/ fpaA mutants is 4 × 1 0 - 1 4 in resting conidia and 5.6 × 10-la in germinating ones. In 6 experiments we plated about 3 × 108 viable cortidia and did not find any fpaA/fpaA mutants (Table 2). In
UV-mduced frequency of fpaA /fpaA dtplotds from + / + conidia Table 2 shows the data obtained in UV-irradiation experiments on resting and germinatmg conidia, Unexpectedly, a considerable number of FPA R mutants were recovered in these experiments. They turned out to be dominant, grew on A F medium and did not require tyrosine. In most cases they were lethal in hemizygosis and did not map on the I chromosome; therefore they were not fpaA/fpaA mutants (Babudri and Morpurgo, Genetics, in press). In Fig. 2 the survival and the frequency of FPA R mutants after UV irradiation of the 18/35 strain are reported. At 90 sec the expected frequency of fpaA/fpaA mutants with resting conidia was 1.22 × 1 0 - 7 while with germinating comdia it was 3.32 × 10 -7. No fpaA/ fpaA mutant was recovered from 565 FPA R col-
191 TABLE 2 SPONTANEOUS AND UV-INDUCED FREQUENCY OF fpaA/fpaA DIPLOIDS Irradiation time (nun)
Survtval (%)
Number of viable comdia plated
0 90
100 27
2 × 108 8 × 106
0 90
100 10
1 × 108 7.8 x 106
lO0
o
-3o
Expected frequency of
colomes
fpaA/fpaAdlplo]ds
30 565
0 0
4 ×10 -14 1.22 × 10- 7
43 1492
0 0
56 ×10 -1] 3.32×10 -7
onies tested on A F medium. Hence there was no increase in the frequency of fpaA/fpaA mutants over that expected in diploid conidia of A. nidu-
-40
>
fpaA/fpaA
FPAa colomes recovered
_m
lans.
z
-20 lOO0
loo
-10
,
i
J
UV DOSE
,
,
,
?
o
(see)
Fig. 3. Recombination frequency in the 18/35 fpaA and 18/35 azgP1 strains. Open symbols indicate survival and dosed symbols recombmation frequencaes: swain 18/35 (resting eonidla), <3; strain 18/35 (germinating conidia, zx; strain 18/35fpaA (resting comcha), v; strain 18/35fpaA (germinating conidla), *; strata 18/35azgPl (resting comdia), e. The survival for the 18/35fpad and 18/35azgP1 strains was similar to that reported for strata 18/35. The spontaneous frequency of recombinants in the 18/35 azgP1 strain (49×10 -4) has been subtracted. For the 18/35 fpaA strain the spontaneous frequency of recombinants has not been subtracted. In fact the presence of recombinant clones could be excluded in the 18/35fpaA comd]al populauon used in these experiments because it was derived from a streak grown on CD medium supplemented only with biotin. On thts medium the growth of fpaA/fpad recombinants is strongly reduced (Morpurgo, 1962a). Each point represents the mean value of at least 2 experiments.
.~ 5c
500
~
%
m
6
15
3'0
4'S
6'0
7'S
9'0
UV DOSE ( s e c )
Fig. 4, UV survival and frequency of 8-AZAg mutants in haploid stram 35 and m diploid strata 18/35. Open symbols indicate survwal and closed symbols indicate mutagenesis: strain 35 (resting comdia), o e; strata 18/35 (resting conidia), • D. The spontaneous mutation frequency for the 35 strain was 0.9 × 10 -6 (1-ustoncal mean). The frequency of mutation for the 18/35 strain was 3.5 × 10 -6 at 60 sec of UV irradiauon and 10 x 10 -6 at 90 sec. Each pomt represents the mean value of at least 3 experiments.
TABLE 3 FREQUENCY OF 8-AZAR DIPLOID MUTANTS IN RESTING CONIDIA Irradiation ume (ram)
Surwval (%)
Frequency of 8-AZAR Frequency of 8-AZAR diploid mutants from mutants in the haploid wild-type conidia strain 35 a
Frequency of rmtotic recombination a
Expected frequencies of AZAR mutants from wild-type comdia b
60 90
58 27
3.5 × 10 -6 1 x l 0 -5
1.5 X 10-3 3.8X10 -3
1 ×10 -6 0.76 × 10- 5
3.3 X 10 -4 1 x l 0 -3
a Controls have been subtracted. b The expected frequency of 8-AZAR diploids is given by the square of the frequency m the haploid plus 2 × (frequency of 8-AZAR mutants in the haploid)× (frequency of rmtotic recombination).
192
Frequency of 8-AZA R mutants The UV survival and the frequency of 8-AZAa mutants in haploid strain 35 are shown in Fig. 4. The recombination frequencies in the 18/35 azgP1 strain are reported in Fig. 3. Clearly the frequency of mitotic recombination leading the azgP1 locus to homozygosis increased upon UV irradiation. The frequency of 8-AZAa diploid conldia derived from + / + ones after UV irradiation is shown in Table 3: it was not different from that expected.
Reconstruction experiments Since in UV-irradiation experiments a large number of FPAa diploid colonies developed, we wondered whether they might hamper the growth of fpaA/fpaA mutants. 100-400 conidia of the 18/35 (fpaA/fpaA) diploid strain, homozygous for the yA2 marker, were mixed with 4 × 106 conidia of the 18/35 wild-type (pale green) strain. UV irradiation was carried out as described for germinating conidia. Almost complete recovery of the fpaA/fpaA yellow conidia was obtained (data not shown); therefore the growth of fpaA/fpaA colonies was not limited by other FPAa diploid colonies. Discussion
The data reported in this paper demonstrate that the frequency of UV-induced recessive mutations in diploid cells of A. mdulans is in the expected order of magnitude. Both the mutation frequency in the haploid and diploid strains and the mitotic recombination have been assessed in resting as well as germinating conidia with the FPA and 8-AZA resistance tests. It must be noted that the UV treatment induced mitotic recombination leading the azgP1 locus to homozygosis while it did not significantly increase the homozygosity at the fpaA locus. The reason of the different recombinogemc effects of UV light in the 2 systems is unknown. Possibly a less stringent selection with 8-AZA allowing more residual growth or an interference of drugs with recombinogenic processes could account for this difference. Actually Wood and K~ifer (1969) obtained a strong increase in somatic recombination frequencies upon UV irradiation using a non-
selective technique, Le., scoring for yellow sectors in a y~ + diploid. Concerning mammalian cell lines, several authors state that the frequency of recessive mutations at some autosomal loo is higher than expected (Siminovitch, 1976; Gupta et al., 1978; Eves and Farber, 1981; Bradley and Letovanec, 1982; Tischfield et al., 1982; Simon and Taylor, 1983; Turker et al., 1984). Their conclusions can be explamed by assuming that: (1) genetic or epigenetic phenomena peculiar to mammahan cells m culture lead to high frequencies of recessive mutations at autosomal loci; (2) some data have been masinterpreted and standard genetical mechanisms alone could explain the observed frequencies. Let us now review some papers about this topic. Gupta et al. (1978; Gupta, 1984) extensively studied this problem in Chinese hamster ovary (CHO) cells in culture. They found that the mutation frequency at the autosomal Emt R lOCUS(conferring emetine resistance) was almost identical to that observed at X-linked loci, Le., at hemizygous loci. They demonstrated (Gupta et al., 1978) that the CHO cell line studied was hemizygous for the Emt a lOCUS,thus explaining the phenomenon. The frequency of mutation at the same locus in other CHO cell lines examined by Gupta (1984) was much lower and in the order of magnitude of that expected taking into account a reasonable frequency of mitotic recombination. This problem has been studied also by other authors, often with a molecular approach. The systems used in these works are selection for diaminopurine resistance (adenine phosphoribosyltransferase (APRT) deficiency), selection for bromodeoxyuridine resistance (thymidine kinase (TK) deficiency) and selection for toyocamycln resistance (adenosine kinase (ADK) deficiency). These systems utihze autosomal recessive mutations and allow the selection from the wild-type condition ( + / + , fully sensitive) to the heterozygous condition ( + / - , intermediate sensitivity) and from this to the - / (fully resistant) condition. Hence each step from the + / + condition to the fully resistant one ( - / - ) can be independently studied. Eves and Farber (1981) state in their paper 'the observed frequencies of recessive mutation in cul-
193 TABLE 4 M U T A T I O N F R E Q U E N C I E S A T T H E A D K , H P R T A N D A P R T LOCI Locus
Cell line
ADK
CAK-BR c
HPRT
CS1 (CHO)
HPRT
CS1 (CHO)
HPRT
V79 (CHO)
HPRT
V79 (CHO)
APRT
D-416(CHO)
Dormnant and Xhnked genes m man
Mutatton frequency
Expression a
Survival
Expected b
Spontaneous
EMS-induced
time
(Sg)
frequencies
0.5-1 × 1 0 - 6 d
10
50
1.39 × 1 0 - 6
4
100
Farber (1981) T h o m p s o n et
4
66
al. (1980) T h o m p s o n et
5
100
2-8 X 10- 6 7.5 x 10-4 3 × 10-6 1.18 × 10-3 5.9 × 1 0 - 7 e _ . ~ 8.8 X 10-5 0.18 x 10 - 6 ~ 1.05 x 10 - 4
5 4
Eves and
al. (1980) Abbondandolo et al. (1980) Abbondandolo
40 9 100
References
3 × 10- 6
et al. (1980) Bradley and Letovanec (1982) Vogel and Motulsky (1988)
a Days of subculture m non-selective conditions before plating m non-selecuve m e d m m . b The expected frequency is the square of the mutation frequency at X-linked loci. For the calculatmn the data of Abbondandolo et al. (1979) have been used. ¢ Mouse embryonic fibroblast line. d Frequency of fully resistant from fully sensiUve cells ( A D K + / A D K + to A D K - / A D K - ). e Frequency of A P R T - / A P R T - from A P R T + / A P R T - cells
tured mammalian cells are generally much higher than would be predicted by comparison with the frequencies of dominant mutations in the same cell lines or with recessive mutations in haploid organisms'. In Table 4 the frequency obtained by Eves and Farber at the autosomal ADK locus in a near-diploid mouse cell line after treatment with ethyl methanesulfonate (EMS) and the frequencies obtained by other authors at the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in CHO cells are reported. On the basis of these data the expected frequency of fully toyocamycinresistant cells obtained from fully sensitive ones should be about 1 × 10-6; somatic segregation events, if any, should increase this value. Actually, the value obtained by Eves and Farber (0.5-1 × 10 -6 ) is not dissimilar from that expected. Therefore these authors have studied the problem of high frequencies of recessive mutations in cultured mammalian cells in a system where mutation frequencies were not different from those expected. Simon and Taylor (1983) stated that 'one of the most puzzling findings in somatic cell genetics has
been the high frequency occurrence of recessive mutations at autosomal loci (Siminovitch, 1976, Cell, 7, 1-11)'. They used the D416 cell line to clarify this problem. The D416 cell line is an A P R T - / A P R T ÷ heterozygous cell line obtained from CHO cells by Bradley and Letovanec (1982) who carefully evaluated the mutation rates in their newly isolated cell line by 2 different methods, one of which is the P0 calculation according to Lea and Coulson (1949). The values, according to P0 calculation, varied from 8.8 x 10 -5 to 5.9 × 10 -7. These figures do not seem exceptionally high; the highest value is not very different from that obtained for some dominant or X-linked genes in humans (Vogel and Motulsky, 1988) (Table 4). However, Bradley and Letovanec (1982) stated that D416 gave fully diaminopurine-resistant colonies at a relatively high frequency and Simon and Taylor (1983) used this cell line for the purpose cited above. Moreover, in these papers (Simon and Taylor, 1983; Bradley and Letovanec, 1982) the word 'mutation' is frequently used to describe a highfrequency event leading from the + / to the
194
-/condition. In the Discussion the authors always correctly suggest that this event could be some kind of somatic rearrangement. We think, however, that the use of the word 'mutation' in their context is misleading and may accredit the unproved assertion that observed frequencies of recessive mutations in diploid cells are higher than expected. Really higher mutation frequencies have been observed in some systems. For example, Tischfield et al. (1982) report that resistance to 2,6-diaminopurine occurred at a rate of about 10-3 per cell per generation in mouse L cells, while mutation rates at other loci were normal. The authors demonstrate that this high frequency was the result of a mutation rather than of an epigenet~c event. They suggest that a mutational hot-spot, a locus-specific mutator gene or a site of integration for an insertion sequence may be the cause of the high mutation rates in their heteroploid cell line where it is likely that only one copy of the A P R T gene is functioning. Turker et al. (1984) in teratocarcinoma cells reported a frequency of the event leading from the A P R T + / A P R T - to the A P R T - / A P R T - con&tlon and vice versa In the order of magnitude of 1 × 10 -2 and probably this phenomenon is related to the peculiarity of teratocarcinoma cells. In conclusion we think that, with the exception of some cases, in mammalian cell lines the spontaneous and induced frequencies of recessive mutations are not parucularly high at autosomal loci. Acknowledgements
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