Copyright 0 1992 by the Genetics Society of America
Effects of Mutagen-Sensitivemus Mutations on Spontaneous Mitotic Recombination in Aspergillus
Ping Zhao and Etta Kafer Department of Biology, McGill University, Montreal, Canada H3A 1Bl Manuscript received September 26, 1990 Accepted for publication December3, 199 1
ABSTRACT Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to muss. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations frommus+ controls in both tests. Two mutations,musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels onlyfor intragenic recombination. Effects were relatively small, but averages betweenhypo- and hyperrec mus differed 15-20-fold. In musL diploids, mostof the rare color segregants resulted from mitotic malsegregationrather than intergenic crossing over.This indicates that the musL gene productis required for recombination and thatDNA lesions lead to chromosome loss when it is deficient. In addition, analysis theof genotypes of intragenic (ad’) recombinants showed that themusL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygousfor ad+).Similar analysis revealed differences between the effects of two hyperrec mutations;musN apparently caused high levels solely of mitotic crossingover, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over,while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.
ENETIC analysis of recombination has revealed transactions (e.g., ligase, analyzed in several species; G a large number of different genes which affect or topoisomerase, recently identified as the product recombination and many of these are also important of the HPRl gene in yeast; ACUILERAand KLEIN for DNA replication and/or repair. However, even 1990). In contrast, mutations which reduce recombi- the number of genes which specifically affect recom- nation are more likely to cause defects in proteins bination was foundto be very high, since several specifically requiredfor recombination. The latter different pathways of homologous recombination can was demonstrated, e.g., for the RAD52 gene in yeast occur in a single species (MAHAJAN1988; reviewed by in which disruption abolishes certain types of recom- SMITH1989). So far, few enzymes involved primarily bination (MALONEet al. 1988). By “interspecies com- inrecombination have been characterized. Only in plementation” the RAD52 protein was shown to sub- Escherichia coli and its phages has a variety of products stitute for thefunctions of two phage T4 recombina- from such genes been identified (Cox and LEHMAN tion mutants (CHENand BERNSTEIN1988). Using a 1987). different approach, recombination function was de- In eukaryotes, mutants which increase or reduce duced for “endo-exonuclease” of Neurospora which recombination have also revealed an increasing num- showed antigenic relatedness to the recC component ber of different types of recombination in assays that of the recBCD enzyme in E. coli (FRASER,KOA and use a variety of substrates [as reviewed, e.g., for yeast CHOW 1990). Antibody against this Neurospora en- by ORR-WEAVER andSZOSTAK (1985); or for mam- zyme crossreacts with nucleases from many eukaryotes malian cell systems by BOLLAG,WALDMAN and LISKAY and was used to clone the corresponding genein yeast (1989)~ In some cases, mutants which cause increased (CHOWand RESNICK1988) (reviewed by FRIEDBERC recombinationcould be used to identify enzymes, 1988). It precipitates almost 100% of the correspond- when the corresponding genes were cloned and se- ing nuclease of Aspergillus which shows all the inter- quences comparedto those of prokaryotic gene prod- esting properties of the Neurospora enzyme (KOA, ucts. Such mutants may accumulate recombinogenic FRASERand KAFER 1990). lesions which result in channeling of DNA repair into For the analysis of genetic recombination in Asper- recombination pathways. Theirgene products fre- gillus, we have analyzed alluvs mutants which are quentlyfunction more generally in variousDNA cross-sensitive to methyl methane-sulfonate(MMS) for
Genetics 130: 717-728 (April, 1992) 718 P. Zhao and E. Kafer effects on recombination (KAFER and MAYOR1986). (mus, uvs, or ribo, ad, etc.). Such crosses frequently were We found that a few of them closely resembled the heterozygous for translocations and produced increased fre- quencies of specific disomic types. These aneuploids were well analyzedUV-sensitive mutantsof E. coli and used to map the break points of translocations which caused yeast.Some uvs mutantswere hyperrec types and no mutant effects (KAFER 1977). showedproperties of excision-defectivemutants, Parasexual cycle, mitotic analysis and construction of diploid while others resembled rec- types. However, we also strains: Diploids heterozygous for nutritional and conidial identified “novel” types; e.g., hyperrec mutants which color markers were selected from forced heterokaryons in which rare fusion of vegetative nuclei occurs. Diploids of appear to cause chromosomal aberrations resembling Aspergillus are quite stable, but mitotic recombinants are mus mutations in Drosophila(GATTI 1979). To obtain formed at low frequency and occasionallyhaploids are null mutations in additional recombination genes for found, completing the “parasexual” cycle (PONTECORVOet unambiguoustests of epistaticrelationships, we in- al. 1953). Such haploid products of nondisjunction show ducedfurther MMS-sensitive mutants by y-rays. practically no crossing over. They were increased by treat- ment with the spindle poisons benomyl or chloral hydrate Among such mutants, various types with alteredlevels and used to map mus mutations and translocations to chro- of recombination have been obtained in several spe- mosomes and to analyze diploid genotypes (KAFER 1977). cies. Also many rec- mutations are known to be hy- Diploid tester strains, mostly homozygous for mus muta- persensitive to this alkylatingagent (among themrecC tions or mus+, were constructed from equivalent pairs of of E. coli and rad52 of yeast). It was expected, there- suitably marked haploid strains (Table 1; for gene symbols and genetic map, see CLUTTERBUCK1987). All strains were fore, that atleast some of our mutations would affect heterozygous for the main markers required for tests of recombination andpossibly include a case withaltered inter- and intragenic recombination (especially pabaA yA levels of endo-exonuclease. Of the recovered muta- adE8 / adE20; see last part of “Materials and Methods”). tions in Aspergillus, several were defective in DNA Notall test diploids were translocation free. Three mus repair, cross-sensitive to various mutagens (mus-221 mutations could not be separated from translocations which unavoidably were homozygous in such diploids [muss224 to mus-234). Among these, two new alleles of uvsB T3(III;VII), mus0226 T2 (III;VII) and musP234 TI(II;VII)]. have been identified (uvsB22I and B2??). The others In addition, a widespread spontaneous translocation, complemented and/or recombined freely with all uvs Tl(I;III) with break points on theleft arms of chromosomes mutations that caused MMS sensitivity (KAFER and I and III, was inadvertently retained in strains of musR223 MAYOR1986). and muss224 (diploids 2743 and 2744; Table 1). No effects on recombination were found for any of these translocations We now report results from testsof mitotic recom- in tests which used the yA and adE markers on the right arm bination for eight mus mutations which represent new of chromosome I. DNA repairgenes of Aspergillus nidulans. Five of As “positive” controls, similar diploids were constructed them showed significant differences from wild-type for two UV-sensitive mutations, namely the hyperrec uvs- controls, generallyfor intergenic crossingover as well F201 (SHANFIELDand KAFER 1969) and the rec- mutant uusCII4 UANSEN 1970). as intragenic recombination. Measurements of intergenicrecombination frequen- cies: For each experiment, cultures were started on solid MATERIALS AND METHODS complete medium (CM),using conidia from one of 20 original parallel cultures for inoculation. After incubation Media and special procedures:Standard Aspergillus me- (3 days at 37” and 2-4 days at room temperature) conidia dia and genetic methods were used as developed by PON- were harvested. Suspensions of1 07/ml were prepared which TECORVO et al. (1953). For minor modificationsof the media, served for all recombination assays. To measure survival and for methods specific to the genetic analysis of mitotic and intergenic recombination, samples were plated on CM recombination and DNA repair, detailed procedures were (aiming at 30-40 colonies per plate, based on hemacyto- as described previously (SCOTTand KAFER 1982; KAFERand meter counts). Plates were incubated at 37” for 3 days (4- MAYOR1986). 5 days at room temperature formusO and Q which conidiate Genetic analysis; constructionof strains: In A. nidulans, poorly at 37 ”). Colonies were counted and survival relative methods of genetic mapping include meiotic recombination, to that of wild type was determined. Intergenic recombi- mitotic recombination and mitotic chromosome loss. All nants which express recessive mutations for conidial color three methods were used for mapping of mus mutations and were identified as macroscopically visible color “sectors” in translocations and for constructing isogenic tester strains diploid green colonies (whole colonies ofmutant color were (for a recent review of the life cycle, and especially of the not included so that all counts represent independent development of vegetative conidiospores, see TIMBERLAKEevents). The frequencies of color sectors were calculated in 1990). two ways: (i) as the percent of sectors per colony (i.e., the Sexual cycle and meiotic analysis: Aspergillus is homothallic number of sectors per 100 colonies; KAFER and MAYOR and all strains are derived from a single haploid nucleus 1986) and (ii) as the number of color sectors per 10 plates. (PONTECORVOet al. 1953). Any two haploid strains can be The latter measure is more appropriate when survival and crossed if nutritional markers are used to force fusion of colony size vary between strains as found in a few cases. For hyphal tips. Cleistothecia, formed under conditions of re- both these frequencies, averages and standard errors (SE) stricted aeration, provided abundant material for random were determined, as well as the levels of significance for spore analysis and meiotic mapping of mus mutants and deviations from control. translocations. Selfing was eliminated in most crosses be- Tests to distinguish mitotic crossovers from nondisjunctional tween mus and standard strains whichusually contained segregants: In standard strains, most color sectors are diploid mutations that are unable to produce selfed cleistothecia recombinants and haploid or diploid products of mitotic mus Effect on Mitotic Recombination 719
TABLE 1
Genotype of haploids used to construct adE8/adE20 test diploids
Diploid Haploid strains numbers Genotypes A. Homozygous mus diploids 2742a 3485 musL222pabaA6adE8; yA2AcrAl; choA1; riboB2 chaAl 345 1 musL222anAl adE2O biA1; AcrAl;ActAl; nicA2; sB3;fiuAZ 2742b3485 musL222pabaA6adE8; yA2AcrAI; choA1; riboB2 chaAl 3633 musL222SulAl anAl adE2O biA1;wAZ cnxEI6;ActAl;pyroA4;nicA2;wA2 27433448 musR223SulAlanAl pabaA6adE8;yA2T,(l;lll;Vl); riboB2 chaAl 3354 musR223 riboAI adE2O biA1; T,(I;III);AcrAI; sbA3; choA1;fiuAZ 27443422 musS224T~(III;VII); SulAlyA2 adE8;T,(I;III); lysB5; riboB2 chaAl 3357 musS224T~(lll;Vll); pabaAl adE2O biAI;ActAl; sB3;fiuA2 27563478 mus0226 T2(lll;VlI);riboAlpabaA6 adE8;yA2AcrAI; riboB2 chaAl 3445 mus0226 T~(111;V11);anAl adEZO biAl;AcrAl;ActAl;nicA2;sB3;choAI;&A2 2757a 3063 musN227AcrAl;adE8;sD85fwA2pabaA6yA2 2797 musN227 SulAl adE20;AcrAl; pyroA4 ssbA1; facA303; riboB2 chaAl 2757b3063 musN227AcrAl;adE8;sD85fwAZpabaA6yA2 363 I musN.227 SulAlanAl adE.20 biA1; ActAl; pyroA4; facA303; riboB2 chaAl 27583360 musK228pabaA6adE8; yA2AcrAl; facBlOl riboB2 chaAl 3364 musK228 SulAl anAladE2O biAI; ActAl;nicA2; choA1;hAZ 2760a 3488 musQ.230 pabaA6 yA2 adE8; AcrAl; nicA2;sbA3; riboB2 chaAl 3460 musQ230 anAladE20 biAl; ActAI; sB3; choA1;fiuAZ 2760b3489 musQ230pabaA6 adE8;yA2AcrAl; nicA2; riboB2 chaAl 3851 musQ230 SulAlanAl adE20 biA1; ActAl; pyroA4; choA1;fiuAZ 2754344 1 musP234T,(ll;Vll); pabaA6 adE8;yA2 AcrAl; OliC2 nicB8; chaAl 3443 musP234T,(ll;Vll); SulAlanAl adE2O; OliC2 pantoBlO0 choA1;fiuAZ B. Control diploids: mus+/+, ?nus/+ and uvs/uvs 2752a 2890 musfadE8; pabaA6yA2 riboB2 chaAl 2357 musfanAl adEZO biA1; AcrAl;ActAl; choA1;fiuAZ 275213 2890 mus+adE8; pabaA6yA2 riboB2 chaAl 3629 mus+ SulAlanAl adE20 biA1; wA2cnxE16; ActAI; nicA2; sbA3;fiuAZ 2842 28902842 mus+ pabaA6 yA.2 adE8; riboB2 chaAl 3633 musL222SulAl anAl adE2O biA1; wA2cnxEl6; ActAl; pyroA4; nicA2;fiuAP 2857 28902857 mus+ pabaA6 yA.2 adE8; riboB2 chaAl 3631 musN227 SulAlanAl adEZO biAl;ActAI; pyroA4; facA303; riboB2 chaAl 27483423 uvsF2OI pabaA6adE8;AcrAI;yA2 malA1; riboB2 chaAl 2512 uvsF.201 anAl adE2O biAl;nicA2; sbA3;fiAZ 2749 34562749 uvsCl14 pabaA6adE8;AcrAI;nicA2;yA2ma1Al;fwAP 3455 uvsCll4 adE2O biAI;OliC2 pantoB100; riboB2 nondisjunction are very rare (aneuploids conidiate too KAFER 1969). Similarly, white sectors were analyzed and poorly to show up; KAFER et al. 1982). To check the pro- classified from especially constructed musL and control dip- portion of the three types of euploid segregants in muslmus loids, heterozygous for WA and linked markers (diploids diploids, random samples of yellow sectors from suitably 2742b and 2752b; Table 1; ZHAO 1991). marked diploids were purified and tested for segregation of Intragenic recombination frequencies (adenine proto- linked markers. The important mutations were pabaA, in trophs selected from heteroallelicadE diploids): The adE coupling on the same chromosome arm as yA, andthe system for analysis ofintragenic recombination was designed semidominant mutation SulA (sulfanilamide resistance) in by PRITCHARD(1955, 1960) who identified the very low repulsion on the opposite arm (Table 1).All but a small reversion rates in homozygous diploids of the adE alleles fraction of crossovers are expected to be paba-, but only used here. Conidia from the adE8ladEZO diploids were nondisjunctional yA/yA diploids normally are sensitive, plated to adenine-free medium at two different densities, homozygous for SUI+. Any yA haploids differed in color, yA 1O5 conidia/plate and 106/plate. After 4-5 days, conidiating being modified either by fwA (= fawn) or chaA (= char- ad+ colonies were counted. Their frequencies were deter- treuse), two color mutations located on opposite homologs mined taking into account the survival observed in CM of chromosome VZZZ. The latter markers also served to platings. Averages (~sE)were calculated for both densities detect complementary products of crossing over as fw//cha and P values for differences from control determined sepa- “twin spots,” especially in hyperrec strains (SHANFIELDand rately. However, levels of significance are based on a com- 720 and P. Zhao E. Kafer bined assessment of both values [SOKAL andROHLF (1981), produceddifferent levels of significance (e.g., for 18.1; FISHER(1954), 21.11. musK strains; discussed below). Statistical consideration: In contrast to color sectors ob- served in growing colonies, ad+ recombinants are formed For most strains, namely six of the eight rnus dip- during growthand formation of the conidiawhich are loids, mus+ control and uvsF, average frequencies of plated. Therefore, occasional clones of ad+ recombinants all colorsegregants were used to assess effects on could be found. However, large clones wererare, since each mitotic crossing over. Genetic tests of yellow segre- conidium results froma different cell division and does not gants indicated that in these diploids practically all divide further (TIMBERLAKE1990). Such recombinants therefore do not show a Luria-Delbriick distribution (for colorsectors were diploid crossovers (only 11200 special features and assumptions, see SARKAR1991). Even tested was a yA/yA nondisjunctional type). Control thoughthe frequencies determined do not fit exactly a diploids produced the expectedlow frequency of color normaldistribution, the statisticsused provide a useful sectors (-2% per colony from diploids heterozygous approach andare considered an appropriate approximation for three color markers; KAFERand MAYOR 1986). (out of a total of >I60 cases, three very large values deviated by 50-100 X SE from the corresponding averages; these Similar frequencies were observed for four of the six were excluded, namelyone case eachfor musL222, musN227 mus strains, musR22?, S224, 0226 and P234 (Table and musK.228). As a simplecheck, median values were 2). For the other two, musN227 and musK228, devia- determined forall sets of experiments. tions from control were significant. In musN diploids, Analysis of genotypes of intragenic recombinants: Se- lected ad+ recombinants were analyzed from diploidswhich frequent color sectors wereespecially obvious, similar all had the same arrangement of markers on chromosome to thosefound for uvsF. Reciprocal crossing over I, namely on the right arm, pabaA yA adE8 / adE2O biA between genes and their centromeres was clearly in- (Table 1). The yA mutation is very closely linked to adE, creased and cha//fw twin sectors were regularly found proximal by 0.1 cM, while biA is distal to adE by 6 cM. The (for musN about 1/600 colonies, while none was seen adE alleles were distinguishable because adE2O is slightly leaky; adE8, and double mutant strains, do not grow at all in mus+ platings; >8000 colonies inspected from each on minimal media (MM). To identify genotypes,small sam- diploid). In contrast,decreases in the musK strain were ples of green diploid recombinants from manyexperiments statistically significant only at the 5% level, and only werepurified and haploidized. About 20 haploids were when considered as “number of sectors per plate.” tested from each recombinant. These always showed two However, the smaller decrease evident for “percent majority types correspondingto the two homologs of chro- mosome I. per colony” is very likely the result of the unusually low densities andtherefore larger size of colonies RESULTS obtained in musK platings (average 22 per plate, com- pared to 40 in control diploids). New rnus genesand associated translocations: Forthe other two mus mutations, musQ230 and Tests for allelism and genetic mapping of mus muta- musL222, and also for UVSC,genetic tests revealed tions identified ninenew DNA repair genes ofAsper- increased proportions of nondisjunctional segregants gillus, musK to muss (continuinggene assignments among color sectors. Corrected values are therefore fromthe MMS-sensitive UVSJ;KAFER and MAYOR 1986). One of these, musM, while clearly located on shown in Table 2 which specifically represent cross- chromosome VI, showed aberrant meiotic linkages over frequencies. FormusQ, 25% of yellow segregants and has been excluded from current analyses. Five were nondisjunctional types (10/40 tested). In addi- mus mutations were separated from all translocations tion, some haploid segregants were obtained; the fre- (musL222, R22?, N227, K228and musQ2?0; a map of quency of these segregants was hard to judgebecause the new DNA repair genes, and information on mu- they grew better than the original diploid. However, tagen sensitivities and epistatic relationships of mus the levels of identifiable crossovers were also signifi- mutants, will be published elsewhere; E. KAFER and cantly increased (about 4-fold, Table 2) but no twin S.-K. CHAE, manuscriptin preparation). Three other spots were observed. mutations, musS224,0226 and P234, were inseparable In the case of the hyporec musL mutant, which from induced translocations(Table 1). These will have produced dull-colored conidia so that yellow sectors to be mapped further to identify thechromosome were difficult to distinguish, white sectors from wA/+ containing their wild-type sequence. diploids were tested (ZHAO1991). Very few of these Intergenic mitotic recombination: In Table 2, re- sectors were crossovers (3/54) but nondisjunctional sults from all recombination assays are summarized diploid and haploidsegregants were unusually fre- for eight mus mutations and for control strains (ie., quent. Overall, color sectors were therefore reduced mus+/+ and two uvs/uvs diploids). The average fre- only toabout half thecontrol level, even though quencies of color sectors which result from intergenic recombination was actually reducedat least 6-fold recombination were calculatedas numbers percolony (Table 2). Similar corrected values for uvsC were and also as numbers per plate. The two values agreed based on previous tests of yellow sectors, andon fairly well, especially when plating densitieswere close current results which identified only haploid segre- to those of the control. However, in some cases they gants. on Mitotic RecombinationmusMitotic Effect on 72 1
TABLE 2
Effects of mus mutations on intergenic and intragenic mitotic recombination
Intergenic recombination (color sectors) Intragenic recombination (selected ad+)"
Average frequencies (fSE) No. X No. of of Factor Survival (on No. of Factor of Diploid type expts. No./100 colonies No./lO plates change CM) (%) expts. Average f SE Median change
~~~ ~~ ~ Homozygous mus musL222 1 lb <0.3'** <1'** <1/6X 80 f 6* 16 2.8 f 0.6*** 1.9 <1/4x musR223 11 1.3 f 0.2 4.1 f 0.5 -1x 97 f 21 3 10 f 1.3 9.3 -IX muss224 14 1.5 f 0.2 4.2 f 0.5 -1x 94 f 10 6 11 f 2.1 11 -lx mus0226 13 2.9 f 0.4 5.3 f 0.7 -1x 80 f 5* 10 24 f 4.7** 19 -2.5X musN227 15 4.9 f 0.5** 14.7 f 1.3* 2.5X 105 f 10 16 28 f 3.2*** 25 -3x musK228 1.3 9 f 0.3 2.7 f 0.4* -1/2x 71 f 12* 7 3.5 f 1.0** 3.6 4/3x musQ230 12.2 9 f 4.3(** 12.2 f 1.8'** -4x 68 f 17 10 70 f 20** 61 -6X musP234 7 3.3 f 0.5 7.8 f 0.7 -1x 101 f 5 5 13 f 2.8 13 -1x + Controls - +/+ 1.8 21b f 0.15 6.3 f 0.2 - 100 (f7) 25 11 f 0.9 11 +/mud -b - - - 80 f 20 5 10 f 1.4 11 -1x +/musN - (ND) (ND) - 128 f 15 5 10f 1.5 9 -1x uvs controls, hyperrec and hyporec types uvsF2OI 11.0 8 f 2.3** 21.4 f 3.0** -5x 87 f 10 5 31 f 5.9** 34 -3x uvsCII4 6 <0.4"** Intragenic recombination (selected ad+ recombi- (Figure 1B). In addition, the musO mutant displayed nants from heteroallelic adE8/20 diploids): Results significantly (if only -%fold) higher levelsof ad+ in Table 2 show excellent agreement between aver- recombinants than control strains. ages and median values for the frequencies of intra- Genotypes of ad+ recombinants;classification of genic recombinants from low density platings ( lo5 recombinant types:Only a few genotypes were found conidia/plate). Even closer correspondence was found for intragenicrecombinants from mus+/+ controls for values from high density platings (data notshown). while fromthe three analyzed mus diploids many The former presumably are closer to the real values, different types were obtained. All of these are shown because the numbers perplate were very low (averages and interpretedin Figures 2 and 3. Based on recovery ranged from 0.3 to 7 per plate). Recovery must have and arrangement of adE alleles and linked markers, been excellent even for hyperrec strains. Frequencies ad+ recombinants were grouped into five classes (Fig- were 2-4 times lower in high density platings, dem- ure 2). In all cases the simplest interpretation, i.e., the onstrating the well known Grigg effect. These larger smallest number of exchange events which could have samples were however less variable and contributed produced the observed results, was used for classifi- considerably to the significance for differences from cation as shown in Figures 2 and 3. Since in the mitotic control. As expected, for mutants which showed sig- assay systememployed only recombinants are selected nificant deviations values were clearly correlated when which produce at least one ad' strand, andin addition both sets of results were compared (Figure 1). half of the time only one of the two strands involved The control mus+/+ diploid generated ad+ recom- is recovered, information is incomplete (as in the case binants at a frequencyof about l 0-5 (Table 2). Similar of selected half tetrads, or attached X chromosomes values were obtained for the heterozygous diploids, in Drosophila). Therefore, alternative interpretations musL/+ and musN/+, indicating that these two muta- often are possible and are not meant to be ruled out. tions are recessive for their effect on recombination. Classes I and I1 represent simple conversion and The well known hyporec effects of the uvsC mutation crossover types, respectively; classes I11 and IV the and thehyperrec effects of uvsF were again con- corresponding types but associated with an outside firmed. For the eight muslmus diploids, average fre- marker exchange. Class V includes all more complex quencies varied considerably. Deviations from control and unusual types, which were obtained mainly from were found mainly for those mus mutants in which mus diploids. For classes 111-V, many different sub- the levels of color sectors were altered. Recombina- types were obtainedonly from musN and musQ strains tion was significantly decreased for musK and musL (shown in detail in Figure 3, and grouped in Figure 2 (Figure 1A) and increased for musN and musQ strains with totals in parentheses). 722 P. Zhao and E. Kafer A These crossovers all are expected to show the same recombinant arrangement for outside markers in the 2o A ad+ strand (y ad++ bi in these diploids, based on the map of adE alleles; PRITCHARD1960). This ad+ strand is expected to segregate half the time with the opposite parental strand and show homozygosis for distal mark- ers (bilbi, type Ha); alternatively, when segregating with the opposite recombinant strand, all mutant al- leles remain heterozygous and adE20-8 double mu- tants are found (type IIb; Figure 2). Many recombinants from the mus+ control diploid showed single allele conversions (class I, 84%, Table B 3), mostly of adE8 (type Ia, Figure 2). The remaining cases were mainly of two types which fitted the geno- types predictedfor crossovers between adE alleles (types IIa and IIb). In addition, their proportions(3:6) 80 2are not incompatible with theexpected 1: 1 ratio. However, it cannot be ruled out that some of them represent convertants with adjacent marker exchange 60 I (class 111). Conversion with associated marker exchange (class ZZZ): This conversion is expected to produce foursubtypes resulting from conversion of adE8, or of E20, each segregating either with the parental or the recombi- nantopposite homolog. Such recombinants would 0 show the same type of ad+ strand as class I1 (y ad++ + bi), but no doublemutants, and different proportions of subtypes would be expected. Recombinants with FIGURE1.-Frequencies (X lo-‘) of ad+ recombinants from genotypes like “type IIa” would be the most frequent adEB/adEZO test diploids in platings of conidia at different densities: 0 = 1O‘/plate; 0 = 105/plate (from Table 2). Averages and standard type (at least twice as frequent as those with IIb-like errors (+sE) shown (except where too small to be visible).A, Diploids phenotypes, since conversion of both alleles can pro- with normal or reduced recombination: + =control diploid, normal duce the former, but only conversion of adE20 the for repair; uvsC114 (= rec- control) and musL222, R223, S224, latter). Also, conversion of adE8 (plus adjacent marker K228 and P234. B, mus+/+ control and hyperrec types: uvsF201, exchange) when segregating with the opposite recom- mus0226, N227 and Q230 (note difference in scale). binant strand, would produce ad+ types with “reverse The frequencies resulting from the classification as linkage” (i.e.,y ad++ bi / y+ adE20 bi+).Such types were shown in Figures 2 and 3 are summarized in Table 3. not recovered from mus+ controls and arenot shown. The relative frequencies of the various classes are Double crossovers (class ZV): These arerecombinants shown, as well as the estimated absolute frequencies resulting from two reciprocal exchanges, one between of each class (based on results listed in Table 2). The the adE alleles and the otherin the adjacent interval. latter values more clearly demonstratethe effects Two cases fromthe mus+ control could represent caused by each of the three analyzed mus mutations. such types (IVa; Figure 2). Alternatively, these could However, this summary is an oversimplification in be convertants of adE20 with double crossing over some cases because the fiveclasses each contain at (type Va). least two subtypes each, sometimes in unexpected Multiple events, classV: These refer tocomplex types proportions as shown in Figures 2 and 3 (butnot resulting from more than two events of conversion Table 3). The various classes and types are defined and/or crossing over. Some of them may represent and interpreted for controlrecombinants as follows. conversion of one or the other adE allele, accom- Simple convertants (class I): Simple convertants show panied with exchanges of linked markers on bothsides one adE mutant allele replaced by the wild-type se- of the adE locus (type Va). Others are more unusual quence (adE8 or adE20, subtypes Ia or Ib; Figure 2). (e.g., ad+/+ types) and presumably arise from short They retainthe parental (P type) arrangement for conversion stretches in opposite strands and frequent closely linked outside markers and result from unidi- crossing over (found only for mus diploids; see below). rectional transfer of wild-type information, i.e., gene Recombinant types from musL, musN and musQ conversion. diploids: In mus diploids, simple convertants were a Simple crossovers between the adE alleles (class ZZ): smaller fraction of all recombinants analyzed than in mus Effect on Mitotic Recombination 723 -ad€ + paba y + 8 + Parental diploids SUI + + 20 + bi Class Type Genotypes of selected ad+ No. from each diploid recombinants + musL ~USN~USQ I. Single Conversion (Conv.) + paba y++ + a) Conv. of ad8 43 15 SUI - + + 20+ bi + paba y + 8 + - 13 19 b) Conv. of ad 20 SUI + +++ bi 11. Single Crossing over (CO) + paba y + + bi - 3 21 a) CO and P strand SUI + + 20 + bi + paba y + + bi - 6 0 b) Both CO strands SUI + + 208 + 111. Conversion & CO a) Cow. of ad8 0 0 + pa& y + 8 bi 0 1 b) Conv. of ad 20 SUI - + +++ + IV. Double CO + - paba y + + + a) Both ad CO strands - + + 20 8 bi 2 0 b) ad + CO and P strand 0 0 V. Multiple events SUI - + y+8 bi 0 1 a) Conv. anddouble CO + - paba + + + + (or bi) + - paba y + + + b) Homozygous ad+ SUI + +++ bi 0 5 (or +) + paba + + + bi c) Homozygous Y+ SUI " + + 20 + bi 0 1 or + paba ++8 + sui - + +++ bi "-0 2 Total 67 65 56 FIGURE2,"Genotypes (relevant markers) of ad+ recombinants from musL, musN and musQ diploids compared to mus+ control, and numbers obtained in samples of similar size. Grouping into classes I-V assumes a minimum number of conversion and/or crossing over events (see text). All types identified among mus+ and mud recombinants are shown (double mutants adEZO-8 in crossovers between the two alleles are assumed but have not been demonstrated). For musN and musQ, only recombinants of classes I and I1 are included; those of classes 111-V are grouped and total numbers indicated in parentheses (details in Figure 3). 724 P. Zhao and E. Kafer -ad€ + paba y + 8 + Parental diploid SUl + + 20 + bi -~~ Class - Type Genotypes of selected ad+ No. identified recombinants musN musQ 111. Conversion and CO a) Conv. of ad 8 and proximal CO Sul + v +8+ b) Conv. of ad20 and proximal CO + paba + + + bi + paba y + 8 bi and distal CO SUI - + ++++ + paba y + 8 + - FIGURE3.--Genotypes and classifica- Sui + + +++ tion of complex ad+ recombinants from musN and musQdiploids, classes 111-V, i.e., + - paba y + 8 bi recombinants resulting from more than SUI + +++bi one simple conversion and/or recombina- IV. Double CO tion event (as defined in text; cases shown in detail here are summarized in Figure (or Conv. and double CO) 2). + - paba v + + + a)Both ad+ COstrands Sul + + 20 8 bi and distal CO + - paba y + + b, Sui + + 20 8 bi + - paba y + + + Sui + +208+ b) ad+ CO strand and P strand + paba y + + bi with distal CO Sui + +20++ V. Multiple events SUl + y +8bi Conv. and double CO - ~~ + paba + + + bi SUI + y20++ + paba + + + bi Total 26 13 mus+ control strains, but absolute frequencies varied one of the two expected recombinant types was found considerably between diploids (Table 3). and was much increased in frequency (namely appar- Effects of the musL mutation: Comparedto mus+ ent crossovers segregating with the parental opposite controls, simple convertants (class I) were reduced 7- homolog; type IIa, Figure 2). The other,equally likely fold in musL diploids (Table 3). In contrast, the fre- type, was completely missing (namely ad++ crossover quency of apparent crossovers between alleles (class strands segregating with the reciprocal opposite hom- 11) seemed to be reduced much less. However, only olog; type IIb). The apparent crossovers could there- mus EffectRecombination on Mitotic 725 TABLE 3 Relative and absolute frequenciesof crossover (CO) and coversion (Conv.) types among ad+ recombinantsfrom mud, musN and mu.@ diploids compared to wild type Relative frequencies Absolute fre uencies" (%) (Xlo-9 ) Recombination classes mu@ +musN musL mu@ +musN musL Simple cases Single Conv.I. Single 52 84 25 47 9.2 1.433.3 7.0 11. Single co -13 -32 -29 -22 - 1.58.1 -0.9 - -15.6 Total 5497 84 69 10.7 2.3 15.1 48.9 Double or multiple events 111. Conv. + CO 0 1 18 22 0 0.03 5.0 15.6 IV. Double CO2 3 2 23 0.05 0.3 1.4 6.5 V. Multiple events 0 2 0 5 0 0 1.4 1.4 or homozygous' -0 -12 -0 -4 -0 -0.4 Ob -2.8 Total 3 15 4612.9 300.48 0.3 21.1 Total all types 100 70.0 100 28.0 2.8 10011.0 99 Average values of low density platings used(Table 2). One case homozygous ad+ (but none y+) was obtained from the musN diploid 2757a (which lacks the marker and is not included _I biA here) (detailsin Z~~O'i991).' fore equallylikely represent unusual adE8 conver- of the total (47% rather than 84%) while two other tants, either with unusually frequent crossing over of specific types were disproportionately increased. The outside markers (21 cases compared to 15 without latter had genotypes like the subtypes IIa and IIIb exchange) or possiblywith extended conversion (Figures 2 and 3). They have in common that both of stretches leading to coconversion of adE8+ and biA these groups could represent convertants with associ- (Figure 2). The remaining cases from musL diploids ated distal marker exchange (conversion of adE8 in were unusual types, homozygous for ad+ or for y+. IIa-like types, and conversion of adE20 in IIIb types). These presumably resulted from coincident conver- There arehowever crucialdifferences between them. sion on opposite strands, frequently associated with Only for conversion ofadE8 is biA an adjacent marker distal crossing over (types Vb and Vc; Figure 2). so that, alternatively, IIa-like types could beproduced Recombinant types from hyperrec musN and musQ dip- by coconversion of biA (as suggested above for the loids (results for classes I and 11 in Figure 2; details for similar types from musL diploids). In contrast, in the classes 111-V in Figure 3): The combined results for case of adE20 conversion (IIIb types) the observed musN recombinants demonstrated that mainly cross- genotypes could not be formed by this mechanism. over types were increased compared to mu+. The They must either have resulted from frequent distal identified genotypes and their proportions suggest crossing over or from discontinuousconversion that crossing over between alleles was very frequent stretches including the biA locus as well as adE20. (class 11) and that crossing over of outside markers was also increased (classes 111-V). Such recombinants DISCUSSION represented a large fraction ofall recovered types When the results for the various mus mutants are (75% for musN, compared to 16% in mus+/+) and compared for mitotic inter- and intragenic recombi- showed about a 40-fold increase in absolute terms nation, itcan be seen that effects in general were (Table 3). In contrast, simple convertants (class I) were concordant. Most mus mutations increased or de- apparently not affected. They represented a much creased both typesof recombination or caused no smaller fraction of the total musN sample but, com- change. An interesting exception is the musO mutation pared to the control diploid, their level was actually which only increased intragenic recombination. This unchanged. mutant interacted synergistically with all uvs mutants For musQ, the overall increase ofintragenic recom- tested so far (E. KAFER and S.-K. CHAE, unpublished bination was even larger than found for musN, but no results). In addition it showed reduced conidiation at simple pattern emerged to suggesteffects on one higher temperatures. However,close linkage of a specific type of recombination. Compared to control, second induced mutation responsible for this latter simple convertants (class I) were increased 4-fold and, effect has not been ruled out (the musO mutation, as as in mus+ diploids, conversion ofadE8 predominated well as musK which slightly reduces recombination, (Figure 2). However, among all musQ recombinants, need to be analyzed in more detail to identify their class I types actually represented a reduced fraction specific effects). 726 P. Zhao and E. Kafer The MMS-sensitivemus mutants which did not show strate differences in yeast, have not yet been carried effects on recombination may well be deficient in out. functions specifically required for repair of methyla- More specific effects of a few mus mutations were tion damage. On the other hand, all of the mutants apparent when the genotypes of intragenic ad+ recom- which altered mitotic recombinationfrequencies binants were identified and compared to those from showed pleiotropic effects. They also were defective mus+/+ control diploids. These control results were in some step of the meiotic cycle and produced no surprisingly different from those obtained by PRIT- viable progeny in homozygous crosses (tests of meiotic CHARD (1955, 1960). His extensive analysis indicated recombination were therefore not possible). Further- that the adE region has the following two unusual more, most of them caused reduced viability of coni- features, namely, (i) relatively frequent reciprocal dia. Presumably DNA lesions, accumulating even in crossing over between adE alleles (usually >50% of the absence of mutagenic treatment, were not only the total) which regularly produces adE double mu- recombinogenic but also lethal for a fraction of cells. tant chromosomes and (ii) crossing over between dis- Such multiple effects suggest that these mutants are tant adE alleles at a higher rate than between adE and deficient in genefunctions which are involved in the proximal marker JA. important aspects of DNA metabolism as well as re- In our analysis simple convertants were by far the combination. majority type (>80%). However, also in the current An exception was the musN mutation. It resembles sample most other ad+ recombinants (-1 5% of the uvsF which tentatively was classified as a member of total) best fitted expectations for crossing over be- the excision repair class (OZAand KAFER 1990). Both tween alleles (ratherthan conversion plus marker exchange;but evidence is not compelling without of these mutants showed normal survival of conidia isolation of double mutants). Our interpretation gains and greatly increased mitotic crossing over.They support from very similar results obtained in Asper- presumably cause DNA lesions which are fully re- gillus by FORTUIN (1971). In this case, simple conver- paired; e.g., nicks which provide a substrate for recip- tants also were by far the majority when paba+ recom- rocal recombinational repair that restores the normal binants were selected from diploids heterozygous for sequence for bothhomologs. In contrast, the hyperrec two pabaA mutations. In addition, about 15% of in- mutant musQ did not increase reciprocal events, even tragenic crossing over was clearly demonstrated, since thoughintergenic recombination was greatly in- pabaA double mutants were recovered in half of the creased. In addition, it caused relatively high levels of cases. In most other ascomycetes such types are rare malsegregation. This may account for the observed or absent (e.g.,in yeast, they may be no more frequent poor survival of conidia (which was however so vari- than recombinants homozygous for wild-type alleles able that deviations were not statistically significant). of the originally heteroallelic gene; ESPOSITO1978). Increased levels of “nondisjunctional” segregantswere In a few genes, however, a high proportion of recip- also observed in musL and UVSCdiploids and have rocal crossing over between distant alleles has been been found for rec- mutants in various organisms. In found in other organisms (e.g.,in Sordaria by KITANI all these cases a large fraction of malsegregants were 1978). haploid and presumably resulted from chromosome The difference between the “historical control” of loss. Nondisjunctional diploid segregants were how- PRITCHARDand the current results in Aspergillus is ever also increased. Inthe hyperrec musQ strains, surprising, considering the origin of all A. nidulans some of the latter could have resulted from double strainsfrom a single haploid nucleus. Possibly, the crossing over across the centromere. Alternatively, drasticmutagenic treatments used toinduce most they could represent secondary productsof malsegre- mutants accounts in part for differences in genetic gation in monosomic nuclei. However, in Aspergillus background. Regardless of the causes, both theresults diploids, such events are exceedingly rare andnondis- obtainedhere, and those of FORTUIN,fit current junctional diploid segregants normally originate from models of recombination considerably betterthan trisomic types (KAFER 1977). those of PRITCHARD.This conclusion assumes that the All considered, the musL mutant may be the only main features of mechanisms proposed for meiotic strain worth testing for the recombination endo-exo- recombination also apply to mitotic recombination; nuclease. It shares several characteristics with the namely formation of “hybrid” DNA corresponding to rad52 mutant of yeast that practically lacks the cor- conversion stretches, and closely associated intergenic responding enzyme (CHOWand RESNICK1988). Meas- crossing over in a substantial fraction of cases (FOGEL urements of endo-exonuclease activities in UVSCand et al. 1979; NICOLASet al. 1989; BORTSand HABER uvsE strains of Aspergillus, have not revealed an ob- 1987). Our results for wild type fit expectations based vious difference to wild type (KOA,FRASER and KAFER on these models, provided associated crossing over is 1990). However, antibody tests, required to demon- assumed to be less frequent in mitosis than meiosis. mus Effect on Mitotic Recombination 727 The frequency obtained here (15%) is very similar to out, possiblymaking use of information from the the 23% observed for mitotic recombination in yeast cloning of the yA region (TIMBERLAKE1990). by HABERand HEARN(1985). However, it has been We wish to thank DOROTHYLUK forexcellent technical assistance found that values can vary considerably in different in the genetic analysis of the mutagen sensitive strains. The main systemsof this species (ORR-WEAVERand SZOSTAK results presented here were obtained by P.Z. in partial fulfillment 1985). of the requirements for an M.S. degree. This project was supported Whatever the correct interpretation of the apparent by operating grant A2564 of the Natural Science and Engineering mitotic crossovers between alleles inmus+ diploids of Research Council of Canada. Aspergillus, it does not affect our finding of striking LITERATURECITED differences between mus mutants and wild type. It was evident that not only overallfrequencies were altered, AGUILERA, A,,and H. L. KLEIN,1990 HPRI, a novel yeast gene but also the proportions of different types. For two that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomycescerevisiae mus mutations very specific effects could be deduced TOPZ gene. Mol. Cell. Biol. 10 1439-1451. from the genotypes of intragenic (ad+)recombinants. BOLLAG, R. J., A. S. WALDMANand R. M. LISKAY,1989 They showed that the hyperrec musN mutation in- Homologous recombination in mammalian cells. Annu. Rev. creased mainly reciprocal crossing over, not only in Genet. 23: 199-225. inter- but also in intragenic recombination while sim- BORTS,R. H., and J. E. HABER, 1987 Meiotic recombination in yeast: alteration by multiple heterozygosities. Science 237: ple convertants remained at wild-type level. The hy- 1459-1465. porec musL mutation, on the other hand, decreased CHEN,D. S., andH. BERNSTEIN,1988 Yeast gene RAD52 can simple convertants, especially ofthe centromere-distal substitute for phage T4 gene 46 or 47 in carrying out recom- allele (adE8).This was however “compensated”for by bination and DNA repair. Proc. Natl. Acad. Sci.USA 85: 6821-6825. relatively high levels of a single type of ad+ recombi- CHOW,T. Y.-K., and M. A. RESNICK,1988 An endo-exonuclease nants which couldrepresent unusual adE8 convertants activity of yeast that requires a functional RAD52 gene. Mol. with distal marker exchange, or possibly crossovers Gen. Genet. 211: 41-48. between the two alleles. In either case, segregation of CLUTTERBUCK,A. J., 1987 Aspergillus nidulans, nuclear genes, pp. the ad++ strand must have been exclusively with the 325-335 in Genetic Maps, Vol. 4, edited by S. J. O’BRIEN.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. parental-type opposite homolog. Similar disparitywas Cox, M. M., and I. R. LEHMAN,1987 Enzymes of general recom- described inyeast for mitotic recombinants from bination. Annu. Rev. Biochem. 56 229-262. rad52 diploids by HABERand HEARN(1 985). These ESPOSITO,M. S., 1978 Evidence that spontaneous mitotic recom- authors postulated that an imperfect, RAD5Zinde- bination occurs at the two-strand stage. Proc. Natl. Acad. Sci. pendent recombination processmight be activated USA 75 4436-4440. FISHER,R. A., 1954 Statistical Methodsfor Research Workers, Ed. 12. which regularly caused lethality of one of the two Oliver & Boyd, Edinburgh. participating crossover chromatids. FOGEL,S., R. MORTIMER,K. LUSNAKand F. TAVARES, 1979 For the corresponding ad+ recombinants from musL Meiotic gene conversion: a signal of the basic recombination diploids and similartypes from musQ, however, an event in yeast. Cold Spring Harbor Symp. Quant. Biol. 43: 1325-1341. alternate mechanism could explain the observed un- FORTUIN,J. J. H., 1971 Another two genes controlling mitotic usually frequent recombinants. Sincethey all are intragenic recombination and recovery from UV damage in homozygous for adE8+ and biA, which were in cou- Aspergillus nidulans. IV. Genetic analysis of mitotic intragenic pling in the original strand, they could have resulted recombinants from uvs+/uvs+, uvsD/uvsD and uvsEluvsE dip from coconversion in an unusually long conversion loids. Mutat. Res. 13: 137-148. FRASER,M. J., H. KOA and T. Y.-K. CHOW,1990 Neurospora stretch, extending for 6 cMo distal from adE to biA. endo-exonuclease is immunochemically related to the recC gene Surprisingly similar results by FORTUIN(1 97 1) for product ofEscherichiu coli. J. Bacteriol. 172: 507-510. uvsE also fit this hypothesis, provided it is assumed FRIEDBERG,E. C., 1988 Deoxyribonucleic acid repair in the yeast that coconversion canextend over 20 cM (from pabaA Saccharomyces cerevisiae. Microbiol. Rev. 52: 70-102. to biA). GATTI,M., 1979 Genetic control of chromosome breakage and rejoining in Drosophila melanogaster: spontaneous chromosome This interpretation is attractive in the light ofrecent aberrations in X-linked mutants defective in DNA metabolism. findings in yeast which identified longer conversion Proc. Natl. Acad. Sci. USA 76: 1377-1381. stretches in mitotic recombinants than in meiotic re- HABER,J. E., and M. 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