An uvsB mutant of Aspergillus nidulans with high variable spontaneous mutation and intergenic mitotic recombination frequencies

Mutation Research, 199 (1988) 167-173

167

Elsevier MTR 04592

An uvsB mutant of Aspergillus nidulans with high variable spontaneous mutation and intergenic mitotic recombination frequencies Nora Babudri and Giorgio Morpurgo Department of Plant Biology, 'La Sapienza' University, Rome (Italy) (Received 15 July 1987) (Revision received 9 November 1987) (Accepted 18 November 1987)

Keywords: Aspergillus nidulans; DNA repair; Spontaneous mutation frequency; Spontaneous frequency of intergenic mitotic crossing-over.

Summary An UV-sensitive mutant has been isolated with a new technique which allows isolation of UV-sensitive and UV-non-mutable mutants in Aspergillus nidulans. This mutant is an allele of the known uvsB gene but shows some features not previously described in the alleles so far isolated. Its more important characteristics are: (1) Frequency of mitotic intergenic recombination is strongly increased in uvs/uvs diploids and it is highly variable in different clones: it varies from a minimum of 40-fold to a maximum of about 1000-fold in comparison with uvs+/uvs + strains. (2) The frequency of mitotic intergenic recombination is increased also in the heterozygous diploids. (3) The frequency of spontaneous mutation is higher and highly variable in different subclones: it may be increased up to 1000-fold.

The pathways that control D N A repair in Aspergillus nidulans have not yet been well characterized, although several mutants which affect D N A repair have been isolated and grouped in 3 epistatic groups, with different features. The characteristics of all the mutants isolated up to this time in A. nidulans have been recently reviewed by K~ifer and Mayor (1986). In the present paper a novel technique for detecting UV-sensitive mutants as well as UVnon-mutable mutants in A. nidulans is described.

Correspondence: Nora Babudri, Dipartimento di Biologia Vegetale, Universith 'La Sapienza', P. le Aldo Moro, 00100 Rome (Italy).

By this technique, a mutant has been isolated which is an allele of the uvsB gene; this mutant has some interesting characteristics: i.e., extremely high and variable spontaneous intergenic mitotic recombination and mutation frequencies.

Materials and methods

Strains Haploid and diploid strains of A. nidulans are listed in Table 1. All are descendants of the A. nidulans isolated by Pontecorvo et al. (1953). The I and IV chromosomes of the uvs+/uosB diploid strain are shown in Fig. 1. For the symbols used throughout this paper see Clutterbuck (1974).

0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

168 TABLE 1 G E N O T Y P E A N D O R I G I N OF H A P L O I D A N D D I P L O I D S T R A I N S Strain number

Genotype

Origin

35 35 F P A R 18

Our collection A spontaneous FPA g m u t a n t

R/1 R / 1 FPA g R/3 R/5 328 333 335 389

pabaA1, anal, yA2," methG; nicA2," nicB8; s12 as 35 but fpaA1 suAladE20, riboA1, proA1, adE20," biA1; pyroA4 as 35 but uvsB as R / 1 but fpaA1 anaA1, yA2, proA1; s12; pyroA4 anaA1, yA2, biA1; methG, uvsB; nicB8 adE20, biA1, uvsA1; meth3 pabaAl08, biA1; uvsBllO proA1, pabaA1, biA1, pyroA4; uvsD153 uvsF201, pabaA1

RD/1 RD/2 RD/3

uvs +/uvs + uvs +/uvsB uusB/ uvsB

C o m p o n e n t haploids: 18 and 35 F P A R C o m p o n e n t haploids: 18 and R / 1 F P A R C o m p o n e n t haploids: 18 and R / 1 FPAR; homozygous for the IV chromosome

Media

The Czapek-Dox minimal medium (CD) and the complete medium (CM) used in this work have been previously described (Sermonti, 1957). Isolation mutants

of

UV-sensitive and

UV-non-mutable

Our collection UV induced in 35 A spontaneous FPA R m u t a n t A recombinant from the cross 18 × R / 1 A recombinant from the cross 18 × R / 1 Fungal Genetics Stock Center (FGSC)-Kansas City FGSC FGSC FGSC

and the plates were then incubated at 3 7 ° C as long as approximately 20-30 colonies per plate developed. At this point, colonies were UV-irradiated and covered with 10 ml of CD medium plus the nutrients required by strain 35, 4% glucose and 3 mM 8-azaguanine (8-aza). Under these condi-

Conidia of strain 35 were plated (200-300 per 100-ram diameter petri dish) on CD medium (10 ml/dish) supplemented with the requirements of the strain and glucose at a strongly reduced concentration (0.01% instead of 2% w/v). On this medium, from each conidium a colony develops which is normal or even increased in size, but with an extremely thin mycelium and with a poor conidiation. Immediately after plating, conidia were mutagenized by UV light at a dose giving 10% survival,

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Fig. 1. I and IV chromosomes of the diploid strain R D / 2 (uvs+/uvsB). The location of uvsB with respect to the centromere is uncertain. The meiotic distances are not shown.

Fig. 2. Colonies of strain 35 on low glucose C D medium. On each colony, the 8-aza-resistant m u t a n t s that develop after 60 sec of UV irradiation and after the addition of the selective agent are visible.

169 tions, small (2-3 mm diameter) compact 8-aza-resistant colonies, each derived from a single conidium, may develop over each expanded colony (see Fig. 2). It is therefore possible to identify colonies without mutants or with a reduced number of mutants and to test them for UV sensitivity and UV mutability.

Test for UV sensitivity To test for UV sensitivity, conidia were plated on CD or CM medium and UV-irradiated on the plates with a 30-watt UV germicidal lamp placed 40 cm above the plates. The dose rate was about 55 erg/mm2/sec at the level of the target. A more rapid procedure was used for testing UV sensitivity when qualitative rather than quantitative data were desired: from a rather dilute conidiai suspension, 4 streaks were done per plate, and their growth after 48 h or more was compared to streaks of strain 35 on UV-non-irradiated and UV-irradiated plates. Measurement of spontaneous and induced mutation frequencies Mutation frequency, either spontaneous or induced, was measured by the 8-aza resistance test (Morpurgo, 1962). Mutagenic treatments UV treatments were done with a germicidal lamp on conidia plated on CD agarized medium. Methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) treatments were done for 1 h on a liquid suspension (liquid test) as previously described (Gualandi et al., 1979). Chromosomal localization Mitotic haploidization of diploids induced by p-fluoro-phenylalanine (FPA) (Morpurgo, 1963) was used to determine the chromosomal localization of the uvs mutant. Diploids were obtained by following the standard procedure of Pontecorvo et al. (1953). Isolation of the homozygous uvs / uvs diploid Conidia of the heterozygous strain R D / 2 were seeded on CD medium supplemented with 0.13 mM FPA and methionine (in fact, it has been previously shown that the uvs mutation is linked

to methG). The homozygous diploid was recovered as a methionine-requiring diploid segregant, induced by FPA.

Determination of mitotic interallefic recombination Mitotic recombination was tested with a selective and a visual method. The selective method is that developed by Beccari et al. (1967), i.e., the estimation of the frequency of diploid green FPA-resistant colonies that develop from a diploid strain. In this work the diploid strains R D / 1 , RD2 and R D / 3 (see Table 1) were used; the inocula were always done in minimal medium: this procedure reduces the possible appearance of clones, because FPA R segregants are leaky for the tyrosine requirement, which is absent in the medium. Moreover, most of the segregants (about 90%) require aneurine, which is also absent in the medium. The visual method is a modification of the technique described by K~fer and Mayor (1986). The original technique was not used because a large part of the colonies were irregularly spotted with yellow patches which made it impossible to count well-defined yellow sectors. Therefore, conidia of the strain under test were seeded on CM plates and yellow colonies were counted. Nutritional requirements of yellow colonies were tested, and the aneurine non-requiring colonies were considered as cross-over types. Results

Morphology of the colonies Colonies of the haploid uvs strain were extremely variable in size and shape, with irregular edges. About 17% of the colonies were dwarf, but dwarf colonies nearly always produced sectors with the normal feature of the strain. The same, or even increased, characteristics were present in the homozygous uvs/uvs diploid. Moreover, the colonies were often yellow spotted, showing irregular patches of various sizes. The majority of the colonies of the heterozygous diploid looked normal, but also in this case there was an excess of dwarf colonies that easily reverted to the normal growth feature. Sectors arising from dwarf colonies did not show color or nutritional segregants; therefore, dwarf colonies were not aneuploids.

170

Chromosomal localization and complementation tests As specified in the Materials and methods section, chromosomal localization was determined by mitotic haploidization of the heterozygous diploid R D / 2 . UV sensitivity was always associated with methG; it is therefore localized on the IV chromosome. The results of the complementation tests (Table 1) clearly show that our uvs mutant is an uvsB allele.

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U V survival and UV-induced mutation The UV sensitivity and the UV mutability of the wild-type strain 35 and of its uvsB derivative R / 1 are shown in Fig. 3. It is evident that, while the uvsB strain is more sensitive to UV light than the wild-type strain, the induced mutation frequencies are similar, except at the highest dose, as a function of UV dose. These data are in agreement with those obtained by KMer and Mayor (1986) with the selenate-resistance system. Spontaneous mutation frequency The spontaneous mutation frequency was tested in 47 clones of the uvsB strain and in 24 clones of the wild-type strain. As can be seen in Fig. 4, the mutation frequencies were extremely variable in the different clones of the uvs strain. Therefore, the mutation frequencies were studied in the cell lineage of 3 different clones. The 3 clones were derived from single conidia and were chosen at random. The spontaneous mutation frequency (as 8-aza resistance) was determined on a conidial suspension from each clone. From the same suspension, conidia were also

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Fig. 3. UV survival and mutagenesis of strains 35 and R / I (uvsB). Open symbols indicate survival and closed symbols mutation. Each point represents the mean value of 3 experiments. Vertical bars represent the standard errors of the mean. In these experiments, the median spontaneous frequency of 8-aza-resistant mutants in uvs + strain was 0.8 × 10-6; in the uvsB mutant it was 4.5 × 10 -6.

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TABLE 2 COMPLEMENTATION TEST IN DIPLOIDS 333

328

335

389

(uosB110)

(uvsA1)

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Fig. 4. Histogram showing the distribution of classes of spontaneous mutation frequencies in strains R/1 and 35.

171

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plated o n C D m e d i u m w i t h o u t 8-aza a n d colonies of the second g e n e r a t i o n were chosen at r a n d o m a n d tested for their s p o n t a n e o u s m u t a t i o n frequency. This o p e r a t i o n was repeated several times. The results (Fig. 5) i n d i c a t e that the m u t a tion frequency m a y vary w i t h o u t a n y a p p a r e n t rule; furthermore, the results are i n c o m p a t i b l e with the hypothesis that exceptionally high m u t a t i o n frequencies m a y be due to the r a n d o m occurrence of 8-aza-resistant clones.

MMS

and EMS

sensitivity and mutability

Fig. 6 shows the survival a n d m u t a t i o n curves for the 35 a n d R / 1 strains after M M S treatment. It is evident that the uvs strain was more sensitive t h a n the wild-type strain to MMS, while the frequency of M M S - i n d u c e d m u t a t i o n was higher only at the highest dose used. Fig. 7 shows the d a t a c o n c e r n i n g the E M S t r e a t m e n t : the 35 a n d the R / 1 strains did n o t significantly differ either i n survival or i n m u t a b i l i t y .

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25 EMS

37.5

50

DOSE (mg/ml)

Fig. 7. Ethyl methanesulfonate (EMS) survival and mutation in strains 35 and P,/1. Open symbols indicate survival and closed symbols mutation. Each point represents the mean value of 3 experiments. Vertical bars represent the standard errors of the mean. In these experiments the median spontaneous frequency of 8-aza-resistant mutant in uvs + strain was 0.5 X 10-6; in the uvsB mutant it was 7x10 -6.

172 RDI2 (U.VS~" / 14VS B ) A (23.7/104 )

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=(lr511o') ct1(34tlO ~1 (Ctl =5OOllO )

10 (> SO0#IO 4) 1~1( d 0 0 1 1 0 ~ 1 '

C (331104 I

(191104 )

Fig. 8. Spontaneous frequency of mitotic crossing-over in the diploid strains R D / 2 and RD/3. The cell lineages from 3 different clones are reported. Numbers in parentheses indicate the frequency of diploid green p-fluoro-phenylalanine-resistant colonies that develop from each clone.

Spontaneous frequency of mitotic crossing-over The spontaneous frequency of mitotic crossingover was determined in RD/1, R D / 2 and R D / 3 by selecting FPA segregants. R D / 2 (uvs+/uvsB) and R D / 3 (uvsB/uvsB) always showed a spontaneous crossing-over frequency higher than that of R D / 1 (uvs+/uvs+); in fact the values obtained for R D / 1 never exceeded 0.5 FPA-resistant colonies/104 survivors (data not shown) while R D / 2 and especially R D / 3 showed very high values, although extremely variable in single clones (see Fig. 8). In the homozygous strain RD/3, the spontaneous frequency of mitotic crossing-over was also determined by the non-selective technique (see Materials and methods). Yellow cross-over segregants appeared at a frequency similar to that of FPA segregants: of a sample of 1257 colonies, 17 were yellow and not requiring aneurine (i.e., the crossing-over frequency was 135 x 10-4). Discussion

In this paper a new efficient technique, which allows the isolation of UV-sensitive and UVnon-mutable mutants, is described. The first mutant isolated is an allele of the uvsB gene already described by Jansen (1970). The same mutant was also isolated by Parag (1977) by selecting for the hyperrec phenotype, and in 2 other laboratories (Wright and Pateman, 1970; Kafer and Mayor, 1986). Therefore, up to this time 9 uvsB alleles have been isolated in different laboratories.

Our allele has some of the characteristics already described but also presents some new features. The most peculiar one is its extremely variable spontaneous mutation frequency. Although the most frequent class is that showing a mutation frequency of 4-6 X 10 - 6 (see Fig. 4), which is in agreement with that observed by Jansen (1972) and K~fer and Mayor (1986), it may vary from 0.5 x 10 - 6 to about 500 x 10 - 6 (i.e., 1000-fold or even more in different clones). The analysis of the cell lineage conclusively shows that this extreme variability is real and is not an artefact due to the sporadic occurrence of 8-aza-resistant clones. The same analysis also shows that the hypermutability is not hereditable: in fact, hypermutable clones may generate clones with low mutation frequencies and vice versa. Another relevant feature of this uvsB allele is the very high frequency of spontaneous mitotic crossing-over. For this parameter, the lowest value observed in the homozygous diploid strain R D / 3 was 19.5 x 10 -4, which is about 40-fold that observed in the wild-type diploid strain RD/1; it must be noted, however, that the spontaneous frequency of crossing-over is also variable among subclones and may be 1000-fold that of the control strain (RD/1). The spontaneous frequency of mitotic crossingover is also higher in the heterozygous diploid R D / 2 ; this means that the hyperrec phenotype is semidominant, a feature which has not been observed in other uvsB alleles (K~ifer and Mayor, 1986). The high variability in the frequencies of spontaneous mutation and spontaneous mitotic crossing-over obviously needs an explanation, but at present only a preliminary hypothesis can be proposed: the expression of the uvs genotype may be sensitive to some microenvironmental conditions, different in the various colonies, finally resulting in an altered mutation (or crossing-over) frequency. The same difficulties exist in explaining the extreme morphological variability observed in the haploid or in the homozygous diploid uvsB strains and, to a lesser extent, also in the heterozygous diploid. The degree of conidiation and the shape of the colonies are not hereditable: platings of small, poorly conidiated colonies produce large

173

colonies and vice versa. As suggested by K~ifer and Mayor (1986), it is possible that the extreme variability in morphology may be related to the hyperrec phenotype, but it is difficult to imagine how hyperrecombination may be related to nonhereditable damages in haploid strains. The different behavior of uvs ÷ and uvsB strains with UV light, MMS and EMS must also be discussed. It is known that UV light, MMS and EMS cause different damages to DNA; in fact, in Escherichia coli MMS and UV damages may in part be repaired by an error-prone mechanism while EMS damage is not (Walker, 1984). Apparently in the uvsB mutant the repair process dealing with the damages caused by either UV light or MMS is altered while the repair process dealing with EMS damage is unaffected. At any rate, the pleiotropic effects of the uvsB mutation suggest, as Kafer and Mayor (1986) pointed out in their paper, that the uvsB gene is very important in the life of A. nidulans and presumably not only in the repair of UV damage.

Acknowledgement This work was supported by the Ministero della Pubblica Istruzione.

References Beccari, E., P. Modigliani and G. Morpurgo (1967) Induction of inter- and intragenic mitotic recombination by fluorodeoxyuridine and fluorouracil in Aspergillus nidulans, Genetics, 56, 7-12.

Clutterbuck, A.J. (1974) Aspergillus nidulans, in: R.C. King (Ed.), Handbook of Genetics, Vol. 1, Plenum, New York, pp. 447-510. Gualandi, G., D. Bellincampi and S. Puppo (1979) MMS induction of different types of genetic damage in Aspergillus nidulans: a comparative analysis in mutagenesis, Mutation Res., 62, 255-266. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-836. K~ifer, E., and O. Mayor (1986) Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMSsensitive hyperrec mutants, Mutation Res., 161, 119-134. Jansen, G.J.O. (1970) Survival of uvsB and uvsC mutants of Aspergillus nidulans after UV-irradiation, Mutation Res., 10, 21-31. Jansen, G.J.O. (1972) Mutator activity in uvs mutants of Aspergillus nidulans, Molec. Gen. Genet., 116, 47-50. Morpurgo, G. (1962) A new method of estimating forward mutation in fungi: resistance to 8-azaguanine and p-fluorophenylalanine, Sci. Repts. Ist. Sup. Sanith, 2, 9-12. Morpurgo, G. (1963) Somatic segregation induced by pfluoro-phenylalanine, Aspergillus Newsl., 4, 4-7. Parag, Y. (1977) Genetic analysis of mutations for low (rec) and very high (pop) mitotic-recombination frequency in Aspergillus nidulans, Molec. Gen. Genet., 155, 319-327. Pontecorvo, G., J.A. Roper, L.M. Hemmons, K.D. MacDonald and A.W.J. Bufton (1953) The genetics of Aspergillus nidulans, Adv. Genet., 5, 141-238. Sermonti, G. (1957) Analysis of vegetative segregation and recombination in Penicillium chrysogenum, Genetics, 42, 433-443. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Wright, P.J., and J.A. Pateman (1970) Ultraviolet-light sensitive mutants of Aspergillus nidulans, Mutation Res., 9, 579-587.