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The Isolation of Mutagen-Sensitive nuv Mutants of Aspergillus nidulans and
Their Effects on Mitotic Recombination
Fekret Osman, Brian Tomsett and Peter Strike
Department of Genetics and Microbiology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England
Manuscript received September 25, 1992
Accepted for publication February 26, 1993
ABSTRACT
More than 200 mutants Aspergillus
of
nidulans were isolated as hypersensitiveto the monofunctional
alkylating agent MNNG and/or UV-irradiation (designated nuu mutants). Of these, 23 were selected
for further characterization. All were markedly hypersensitive to both MNNG and the quasi-UVmimetic mutagen 4-NQO. The hypersensitive phenotype of each mutant was shown to result from
mutation of a single gene. T h e nuu mutants exhibited a diverse range of growth responses on solid
media containing various concentrations of MNNG or 4-NQO. This suggested that they represented
many nonallelic mutations. Analysis to determine the dominance/recessiveness of the nuu mutations
with respect to hypersensitivity revealed that most were fully recessive, although several appeared to
be semidominant. A novel system to assay homologous mitotic recombination using simple plating
of the nuu mutations on
tests was developed. T h e system was exploited to determine the effects
mitotic recombination. Of the 23 mutations tested, 10 caused a hypo-recombination phenotype and
three a hyper-recombination phenotype,while 10 appeared to have no effecton recombination. T h e
hypo-rec effect of one of the mutations, nuv-117, appeared to be semidominant. Transcomplementation analysis between seven of the nuu mutations defined atleast six nonallelic loci.
D
ESPITE its fundamental importance for a wide
variety of biological processes, little is known of
the underlying molecular mechanisms involved in homologous genetic recombination in eukaryotic cells.
Much of our knowledge has relied on studiesin lower
eukaryotes, particularly a few species of fungithat
have genetic systems amenable for detecting, analyzing and assaying recombination events (WHITEHOUSE
1982;ORR-WEAVERand SZOSTAK1985;HASTINCS
1988).
Studies on the genetic control
of homologous recombination depend on the isolation and characterization of mutants defective in therecombination
pathway(s). DNA metabolic pathways in cells, includingreplication,transcription,recombination,repair
and mutagenesis, are not distinct biological processes
but are overlapping and, in part,underthe
same
genetic control. The isolation and characterization of
mutants hypersensitive to DNA damaging agents in
many organisms continuesto provide a diverse source
of mutations defective in these processes, as well as
revealing the complex interrelationships between
them. Hypersensitive mutants have been a particularly
useful source of mutants defective in homologous
recombination in bothprokaryotes (e.g., HOWARDFLANDERS and
THERIOT
1966; CLARK 1973) and eukaryotes (e.g., HOLLIDAY1967;BAKERandSMITH
1979; PRAKASH
et al. 1980). By far the best studied
lower eukaryote with respect to the mutational analysis of homologous recombination is the budding yeast
Genetics 134: 445-454 (June. 1993)
Saccharomyces cerevisiae. A relatively large number of
S. cerevisiae mutantsaffecting DNA repairand/or
recombination have been isolated and characterized
and many of the corresponding genes have been
cloned (HAYNES and
KUNZI98 1 ; GAME 1983;
COOPER
and KELLY1987; FRIEDBERC
1988, 1991). Work with
filamentous fungi has progressed much more slowly
such that only a relatively small number of such mutants have been isolated and characterized. In Aspergillus nidulans only nine UV-sensitive (uvs) mutants
have previously been extensively characterizedfor
effects onrecombination (reviewed by KAFER and
MAYOR1986).
There may be fundamentaldifferences between
DNA repair and recombination in S. cerevisiae and in
A. nidulans. Like mammalian cells but unlike S. cerevisiae, germinating A. nidulans cells spend asubstantial
part of their vegetative cell cycle in G2 (BAINBRIDGE
197 1; BERGENand MORRIS 1983). During G2 there
are two copies of each chromosome existing as sister
chromatids, which means there are greater opportunities for homologous recombination in haploid cells
of A . nidulans than S. cerevisiae. Recombinational repair pathways in A. nidulans may therefore be more
significant than in S. cerevisiae. This is the case with
Schirosaccharomyces pombe, which also spends a large
part of its vegetative cell cycle in G2 (PHIPPS,NASIM
and MILLER 1985). The pleiotropic phenotypes and
epistatic grouping of A. nidulans mutants defective in
DNA repair/recombination (KAFER and MAYOR,
446
F. Osman, B. Tomsett and P. Strike
1986) more resemble those of repair/recombination
mutants of other eukaryotes than do those of S. cerevisiae, implying that the genetic control of recombination in A. nidulans may be more typical. Indeed,
be
recentlycloned S. pombe rad genesthoughtto
involved in recombination (LEHMANet al. 199 1; SUBRAMANI 1991) show no extensive homology to any
cloned S. cerevisiae recombination genes.
(OSMAN et al. 1991) we deInapreviouspaper
A.
scribedthe isolation andcharacterizationofan
nidulans mutant, n u v l l , hypersensitive to N-methylN’-nitro-N-nitrosoguanidine
(MNNG) and 4-nitroquinoline-1-oxide (4-NQO), and with effects on meiotic
and mitotic recombination. We now describe the isolation of a further 23 mutants hypersensitive to these
damaging agents and, using simple plating tests, the
characterization of their effects on spontaneous mitotic intragenic recombination.
MATERIALS A N D METHODS
A. nidulans strains and media: A. nidulans strains used
in this work carried markers in general use that have been
described previously (CLUTTERBUCK
1974). The routine
genetic techniques used were modified after PONTECORVO
et al. (1 953). Standardmedia have beendescribed previously
(COVE 1966).“Difco” Bacto-Agar (Difco Laboratories, Detroit, Michigan) was used in all media. Minimal base (MB)
consisted of a carbon source, salts and trace elements. Minimal medium (MM) consisted of MB plus a nitrogen source
and supplements for auxotrophic markers. Mutagen-containing media were made up at pH 6.0.
Isolation of MNNG- and/or UV-sensitive mutants: Mutants wereisolated from three different parental strains,
each of which was phenotypically wild-type with respect to
mutagen sensitivity. Conidial (asexual uninucleate haploid
spore) suspensions of the threeparental strains, B608 ( P u A I ,
w A 3 , niiAniaD608), CS387 (yA2, luAI, niaD26) and L20
(pabaAI, wAj),were each treated with MNNG (0.5 pg/ml
final concentration) to giveless than 10% survival. Master
plates were prepared by overlaying mutagenized conidia at
a density of approximately 100 surviving conidia per plate
onto M M (to eliminate auxotrophic mutants) containing
0.08% sodium deoxycholate (SD) to induce compact colonies (MACKINTOSH and PRITCHARD1963). These plates
were incubated at 37“ for 3 days, by which time the mutagenized colonies had grown and sporulated. MNNG-sensitive and/or UV-sensitive colonies were identified by replica
plating using damp velvet pads. MNNG-sensitive colonies
were identified by comparing growth on two replica plates
of MM, one ofwhich contained 3.5 pg/ml MNNG. UVsensitivecolonieswere
scored as showing poor mycelial
growth and/or poor sporulation on replica plates preincubated for 7 hr and irradiated with 400 J/m’ U V (wavelength
254 nm) in comparison with growth of unirradiated replica
colonies.
Potential MNNG-and/or UV-sensitive mutants (designated nuv) were subcultured from the master plates to fresh
M M master plates, nine per plate together with the wildtype parental strain, in arrays suitable for 10-point wire
replication. These potential hypersensitive mutants were
rescreened by wire replication of conidia into MM plates
containing 0.75 and 1.0 pg/ml MNNG and 0.5 and 0.75
pg/ml4-NQO. The quasi-UV-mimetic mutagen 4-NQO was
used instead of UV for screening for sensitivity by wire
replication sinceresultsusing
U V were ambiguous and
inconsistent. Like MNNG, 4-NQO has the advantage that
it canbe added directly to media. I t gavemuch clearer
results than U V , although there is not 100% correlation
between U V and 4-NQOsensitivity. Hypersensitivemutants
were taken through two rounds of screening by wire replication, retaining only those that wereconsistently more
sensitive than conidia of the parental strain.
The MNNG, U V and 4-NQO dosesused to identify
hypersensitive mutants in these replica plating experiments
wereinitiallyshown
to beeffective at distinguishing the
previously isolated UV- and mutagen-sensitive A. nidulans
strains uusB, uvsC and uvsD (KAFERand MAYOR1986) from
wild-type.
Quantifying MNNG and 4-NQO sensitivity: A heavy
suspension of conidia was spread onto a M M plate using a
sterile glass spreader and incubated for 24 hr at 37” to
produce a nonsporulating mycelial “mat.” Mycelial “plugs”
were c u t out from the mats using the wide end of a sterile
Pasteur pipette. The plugswere transferred in duplicate
onto M M plates containing various concentrations of
M N N G or 4-NQO and incubated for 48 hr at 3 7 ” . The
diameters of the resulting colonies were measured under a
stereoscopic microscope to the nearest 0.1 nlm using a
micrometer. Radial growth was expressed as percentage of
average diameter of colonies on zero-dose plates.This technique avoids the problem of delayed germination found
with some mutagen treatments and ensures that the true
exponential phase growth rate is measured.
Performing the mitotic recombination assay: The assay
measuresmitotic recombination between nonoverlapping
deletions within the niaD gene. Full details of the assay will
be given i n REsuLrs. The mitotic recombination phenotype
was determined qualitatively by simply centrally inoculating
;I M M plate containing nitrate as sole nitrogen source with
0.5 pl of a suspension of conidia from the diploid under test.
The [dates were incubated at 37’ for 7 days before scoring.
RESULTS
Isolation of nuv mutants: By the end of the mutant
isolation program, 18 putativemutagen-hypersensitive mutants had been identified from screening about
4,800 mutagenized L20 colonies,
over
80 from
screeningabout 8,000 mutagenized B608 colonies,
and over 100 from screening about 10,000 mutagenized CS387 colonies. T h e mutant strains were designated L20nuv, B608nuv and CS387nuv, respectively,
followed by a unique distinguishing number.
After two rounds of screening, 23 of the mutants
were retained for further
investigation on the basis
that they were the most hypersensitive to
MNNG: it
had been found previously in both S. cerevisiae (BRENDEL, KHANand HAYNES
1970) andA. nidulans (KAFER
and MAYOR 1986) that hypersensitivity to monofunctional alkylating agents was especially pronounced in
mutants that affect recombination. The
23 mutants
selectedwereas
follows: ninemutantsisolated
in
B608-nuv-1, nuv-2, nuv-4, nun-6, nuv-7, nuv-8, nuv10, nuv-12, nuv-20; 12 from CS387-nuv-102, nuv103, nuv-107, nuv-109, nuv-110, nuv-11 I , nuv-112,
nuv-114, nuv-115, nuv-117, nuv-120, nuv-121;two in
A . nidulans Recombination Mutants
the L20 background”nuv-?29 and nuv-??4. All of
thesemutantswere
hypersensitive toboth M N N G
and 4 - N Q O .
Mutant nuu allele segregation: Each of the nuv
mutant phenotypes was shown to result from mutation
of a single gene by sexually crossing each mutant strain
with a strain phenotypically wild-type with respect to
mutagen-sensitivity. Segregation of the nuv-I0 phenotype was analyzed by the parasexual cycle, which
involved diploid construction and haploidization,
since sexual crosses heterozygous fornuv-10 produced
no viable hybrid sexual spores. Randomly chosen
progeny from each cross were screened for hypersensitivity to M N N G and 4 - N Q O by wire inoculation of
conidia into mutagen-containing media. In each case
there was a 1: 1 mutant/wild-type phenotypic segregation with respect to hypersensitivity. Analysis of the
sensitivities of the progeny did not
reveal any new
phenotypic class,all progenybeing clearly of one
parental type or the other, and with hypersensitivity
to M N N G and 4-NQO always segregated together.
Comparison of hypersensitivities of nuv strains to
MNNG and 4-NQO: The hypersensitive phenotype
of each nuv allele was tested in both B608 and CS387
genetic backgrounds, relative to the parental strains,
by measuringthe hyphal extension of germinating
mycelial plugs on solid minimal media containing various concentrations of M N N G or 4 - N Q O . T o make
direct comparisons between the different nuv strains,
thedeterminations were carriedout as two large
experiments, one for sensitivity to M N N G and the
other for sensitivity to 4 - N Q O , in which all the plates
at a particular mutagen concentration were made up
using the same mutagen-containing media. This was
necessary due to the inherent instability of the mutagens, particularly M N N G , and the variability when
making u p mutagen-containing media. A typical
growth response caused by one of the nuv mutations,
nuv-2, is represented graphically in Figure 1. Data on
thecomparative hypersensitivity to M N N G and 4N Q O of the different nuv mutants is presented in
Figure 2.
The nuv mutantsexhibiteda
variety of growth
responses on M N N G and 4 - N Q O relative to the wildtype parental strains, indicating that a diverse range
of mutantshad been isolated. Some mutants were
very hypersensitive to both M N N G and 4-NQO (e.g.,
nuv-110, nuv-??4). Others displayed relatively
greater sensitivity to M N N G than 4-NQO at the concentrations of mutagen used (e.g., nuv-l12), and vice
versa (e.g., nuv-6). Another class of mutants displayed
relatively low hypersensitivity to both M N N G and 4N Q O (e.g., nuv-107).
Dominance-recessiveness of the nuu alleles with
respect to hypersensitivity: This was determined by
comparing the sensitivity to M N N G and 4-NQO of
447
homozygous wild-type diploids with that of diploids
heterozygous and homozygous for each nuv mutation.
As before, to make direct comparisons between the
different nuv diploids, the determinations were carried out as two large experiments, one for sensitivity
to M N N G and the other for
sensitivity to 4 - N Q O .
Diploids were used instead of heterokaryons since
some of the mutants may have been defective in
nuclear-specific functions. Sensitivity was measured
using mycelial plugs inoculated onto M B media containing ammonium (selective for diploids) and various
concentrations of M N N G and 4 - N Q O .
The growth responses of several of the diploid
strains are represented graphically in Figure 3. In all
cases, except for nun-20, the homozygous nuv diploid
was markedly hypersensitive to M N N G and 4 - N Q O
relative to the corresponding heterozygous nuv and
homozygous wild-type diploids. An interesting result
was that the diploid homozygous for nuv-20 did not
appear to be hypersensitive to either M N N G or 4NQO. The homozygous nun-20 diploid was haploidized on benlate-containing media (HASTIE1970) and
the haploid segregants tested for hypersensitivity to
M N N G and 4 - N Q O . All segregants were hypersensitive confirming that the nuv-20 hypersensitive phenotype was haploid-specific.
In most cases the growth response of diploids heterozygous for the nuv mutations was similar to that of
homozygous wild-type diploids, suggesting complete
recessiveness of the nuv allele to its wild-type allele.
All of the mutations appeared to be recessive except
three: nuv-2, appeared to be semi-transdominant for
hypersensitivity to both M N N G and 4 - N Q O ; nuv-8
and nun-1 1I appeared to be semi-transdominant only
for hypersensitivity to 4 - N Q O . In these cases the
heterozygous diploid was intermediate in hypersensitivity relative to that of the corresponding homozygous nuv and homozygous wild-type diploids. None
of the mutants appeared tobe completely dominant.
Establishment of a novel assay for spontaneous
mitotic intragenic recombination: Mitotic intragenic
recombination between heteroalleles is most conveniently measured in the case of mutations conferring
auxotrophy. This allows rare prototrophic recombinants to be selected on medium lacking the appropriate nutrient. The mitotic recombination assay developed is based on the two A. nidulans strains, B608
(wA4, puA2, niiAniaD608) and CS387 (yA2, l u A I ,
niaD26), each carrying a non-overlapping heteroall e k deletion within the niaD gene on chromosome
VIII. The ends of the deletions had been previously
determined by fine-structure mapping (TOMSETT
and
COVE 1979). They are shown schematically in Figure
4, together with thestructuralorganization of the
nitrate assimilation genes. The two strains were deficient in nitratereductase,the
niaD geneproduct.
F. O m a n . B. Tomsett and P. Strike
448
+m
-+-mn*1
10
20
-
0.0
25
02
0.4
0.8
Od
1.0
FIGUREI .-Hypersensitivity
to
M N N G and 4-NQO caused by the
nuu-2 mutation. The growth response of germinating myceliaof
nuu-2 mutant strains i n comparison
with parental strains inoculated onto
solid media containing various concentrations of M N N G or 4-NQO.
The percentage hyphal growth represents the relative hyphal extension
of strains growing in the presence of
MNNG or 4-NQO in comparison
with controls growing i n the absence
of MNNG or 4-NQO. I t should be
noted that the linear growth response
of multinucleate hyphae to MNNG
and 4-NQO was determined in these
experiments. not conidial survival.
12
4NQo-bmJ
+1 pglrnl MNNG
n
f
f
I
4 . 5 pglml4NQO
70
a
2 3 0
0
I D 2 0
a
10
0
FIGURE
2.-Comparison of hypersensitivity to M N N G and 4-NQO of different nuu mutants and relationship to recombination ability.
The data represent the growth responses of germinating mycelia of B608 and B608nuu strains inoculated onto solid media containing either
0.5 pg/ml MNNG or 0.5 pg/ml 4-NQO. The percentage hyphal growth represents the relative hyphal extension of strains growing in the
presence of MNNG or 4-NQO in comparison with controls growing in the absence of M N N G or 4-NQO. For completeness sake. the figure
includes the data for the nuu-11 mutant, which has been described elsewhere (OSMAN
et al. 1991). In the bottom panel, the recombination
ability of each mutant is indicated. with the mutants being grouped phenotypically into Rec' (nuu-1 to nuu-329). hypo-rec (nuu-2 to nuu-117)
and hyper-rec (nuu-1I to nuu-334) categories. Recombination ability was assessed by a modification of the simple plate assay. which allows a
quantitative assessment of recombination competence to be made (F. OSMAN,
unpublished data).
They could not therefore utilize nitrate as sole nitrogen source. However,commercial agar contains traces
of reduced nitrogen, which allows nitrate nonutilizing
strains to grow o n nitrate-containing solid media with
a characteristic residual growthphenotype, namely
sparse, nonsporulating mycelial growth. This phenotype was exploited to qualitatively assay mitotic recombination within the niaD gene in the diploid B608/
CS387.
Centrally inoculated B608/CS387 diploid colonies
initially grew with the characteristic residual growth
phenotypeonnitrate-containing
media. After 4-5
days incubation,sporulating subcolonies were observed to arise within the sparse mycelial growth,
Figure 5 , plates A. As controls, heteroallelic diploids
were constructed between each test strain and strains
carrying an overlappingdeletion (see Figure 4); a
diploid was constructed between B608 and B144
(pabaA I ,JiuAl, niiAniaD526),and between CS387 and
B3 17 (biA I , niiAniaD564). In plating tests with these
449
A. nidulans Recombination Mutants
100
90
M
~
10 -
1
lo
0.00
FIGURE3.-Dominance/recessiveness of nuu mutations with respect to hypersensitivity to MNNG
and 4-NQO. This was determined by
measuringthegrowthresponse
of
germinatingmycelia of diploidsheterozygousandhomozygousforthe
nuu mutations in comparison with the
homozygouswild-type parental diploid.
0
0.50
1.00
1.50
MNNQ Cmcn.(pglmg
2.00
2.50
0.00 0.500.25
0.75
1.00
1.25
".(Wlml)
1w
90
M
sa
0.00
0.50
1.00
1.50
MNNQ
(puml)
2.00
2.50
Concn.
diploids no subcolonies arose, Figure 5 , plates B and
C.
The subcolonies were genetically analyzed and
shown to be still diploid andtrue nitrate-utilizing
recombinants. They were subcultured ontonitrate
plates and grew with a normal nitrate-utilizing phe-
notype. All those tested were shown to be diploid by
haploidization
benlate
on
plates (HASTIE1970). Each
produced
nitrate-utilizing
and
nitrate-nonutilizing
haploid segregants. Nitrate-utilizing segregants were
sexually crossed with either nitrate-utilizing or nonutilizing strains, and the progeny analyzed. The phe-
F. Osman, B. Tomsett and P. Strike
450
cmA-niiA-niaD GENE REGION ON CHROMOSOME Vm
CrnA
niiA
I
niaD
1
4
ni"ni-
4
nlU-nfaD608
4
nl~-nin~~281
1
notypic ratios of the progeny obtained were consistent
with the conclusion that the haploid segregants were
true niaD+recombinants: crosses with nitrate-utilizing
strains produced all nitrate-utilizing progeny;the
numbers of nitrate utilizers and nonutilizersfrom
crosses with nitrate-nonutilizing strains were not significantly different ( P = 70-5076) from a 1:l ratio.
Preliminary genetic analysis of the haploid segregants
from the nitrate-utilizing recombinants (L. IWANEJKO,
unpublished data) revealed that they could arise by
both reciprocal crossing over and nonreciprocal gene
conversion.
Effect of nuv mutations on mitotic recombination:
The assay described above was exploited to qualitatively determine the effect of heterozygous or homozygous nuv mutations on spontaneous mitotic recombination in B608/CS387 diploids. Each diploid was
tested on three separate plates. The results were consistent andreproducible. This assaywasespecially
effective at identifying nuv mutations causing a hyporecphenotype. Examples of plates from which the
results were obtained are shown in Figure 5 , plates
D-G. Three classes of nuv mutations were identified
on the basis of the mitotic recombination phenotype
of the homozygous nuv diploid: rec+ types (nuv-I,
nuv-10, nuv-12, nuv-20, nuv-103, nuv-107, nuv-109,
hypo-rec types (nuv-2,
nuv-115,nuv-120,nuv-121),
nuv-4, nuv-6, nuv-7, nuv-8,
nuv-110, nuv-112, nuv-114,
nuv-117, nuv-329)and hyper-rec types (nuv-102, nuvI I I, nuv-334). Rec+ types displayed a similar recombination phenotype to wild-type diploids. Hyper-rec
types produced a several-fold increase in the number
of niaD+ recombinants, the effect of nuv-102 being
the most marked followed by nuv-I11 and nuv-334.
The hypo-rec types produced a several-fold decrease
in the number of recombinants compared with wildtype. They could be classified into three groups: diploids homozygous for nuv-2, nuv-7, nuv-1I O , nuv-112
and nuv-329 produceda greater than 10-fold decrease; nuv-4 and nuv-I14 had an intermediate effect,
a less than IO-fold but greater than fourfold reduction; nuv-6,nuv-8 and nuv-117 caused a less than
fourfold decrease.The nuv-1 I7 mutation appeared to
have a semidominant effect; the heterozygous nuvI I7 diploid also displayed a hypo-rec phenotype but
not to the extent of the homozygous nuv-I17 diploid,
Figure 5 , plates F and G. The other nuv mutations
affecting recombination appeared to be fully reces-
1-
FIGURE4,"Schematic map of the c m A niiA-niaD gene cluster on chromosome V l l l
and the niaD and niiAniaD deletions. The
line for the niaD deletion depicts the full
extent of the region.
deleted
The niiAniaD
deletions extend from the niaD gene into
the niiA gene and only their right-hand limit
is shown.
sive, in each case the heterozygous nuv diploid displaying a similar recombination phenotype tothe
homozygous wild-type diploid.
Complementation analysis with some of the nuv
mutations: Complementation analysis is necessary to
determine the number of genes defined by the nuv
mutations. An initial study was carried out with the
previously described mutation n u v l l (OSMAN
et al.
1991) and six of the nuv mutations with effects on
mitotic recombination from this study: nuv-2, nuv-4,
nuv-102, nuv-1IO, nuv-I14 and nuv-117. Diploids heterozygous in trans for two nuv mutations were constructed for all possible pairwise combinations. Allelismwas determined by testing these heterozygous
diploids for hypersensitivity to MNNG and 4-NQO,
and for mitotic recombination phenotype using the
simple plating assay. The responses were compared
with that of the singly heterozygous nuv diploids (as
positive controls) and the homozygous nuv diploids
(as negative controls). The results are shown in Table
1. The results for trans complementation with respect
to hypersensitivity and to recombination phenotype
were consistent. They showed thatthe seven nuv
mutations analyzed defined at least six nonallelic loci.
The nuv-102 and nuvl17 mutations were noncomplementing for hypersensitivity, andthe nuv-I02/nuvI I7 diploid had the same hypo-rec phenotype as the
homozygous nuv-1 I7 diploid. T h e y thus appeared to
be allelic despitetheirdifferent
effects on mitotic
recombination. The diploids heterozygous for nuvI 1 7 and each of the other nuv mutations displayed a
hypo-rec phenotype
comparable
to
that
of the
nuvl17/wild-type diploid. This confirmed the semidominant hypo-rec phenotype of nuv-117. Diploids
heterozygous with nuv-2 were all hypersensitive to
MNNG and 4-NQO compared with wild-type, confirming its semi-transdominance for this phenotype.
Several of the nuv mutations were assigned to linkage groups (whole chromosomes) by mitotic analysis
(MCCULLY
and FORBES1965): nuv-2 to chromosome
V; nuv-4 to chromosome 11; nuv-102 and nuv-I14 to
chromosome VIII. Despite exhaustive attemptsthe
nuv-117 mutation could not be mapped to a specific
chromosome. This was also the case with some previand KAMENEVA
ously isolated uvs mutations (EVSEEVA
1977).None of the previously characterized mutations causing hypersensitivity to DNA damaging
agents,the uvs (KAFER and MAYOR 1986)and sag
A.
45 1
nidulans Mutants
Reconlbination
A
A
B
C
D
D
E
E
F
F
G
G
(SWIRSKI
et al. 1988) mutations, mapped to chromosome 11. T h e nuv-4 mutation therefore probably defines a new locus in A. nidulans. T h e nuu-2 mutation
FIGURE 5 . " A simple assay for mitotic recombination. Sporulatingsubcolonies arose spontaneously within
the characteristically sparse mycelium of centrally inoculated B608/
CS387 heteroallelic diploid colonies
growing on nitrate-containing medium (Plates A). These subcolonies
were shown to be nitrate-utilizing
niaD+ recombinants, and to have
arisen either by crossing over or gene
conversion. As controls, heteroallelic
diploids were constructed between
each test strain and a strain carrying
an overlapping niaD deletion. In plating tests with these diploids, B608/
B114 (Plate B) and B317/CS387
(Plate C), no nitrate-utilizing subcolonies arose. Recombinationdefective nuv mutants were identified by
comparing the frequency of nitrateutilizing subcolonies arising from
B608/CS387 diploids heterozygous
and homozygous for a nuv mutation.
Plates demonstrating the recessive
hyporec phenotype of nuv-2 and the
semi-transdominant hypo-rec phenot y p e of nuv-117 are alsoshown:
Plates D, B608nuv2/CS387;Plates E,
B608nuv2/CS387nuv2; Plates F,
B608/CS387nuvll7; Plates C,
B608nuv117/CS387nuvl17.
was analyzed for allelism with the uusl), uusE and uusJ
mutations also located on chromosome V. Complementation analysis was performed by scoring for hy-
452
F. Osman, B. Tomsett and P. Strike
TABLE 1
Trans complementation analysis of nuv mutants
nuv-117
nuv-114
nuv-110
inuv-102
nuv-11
nuu-4
nuv-2
nuv-4
nuu-11
nuv-102
nuv-110
nuu-114
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
i-
+
+
+
Complementation was determined by scoring: (i) sensitivity of
germinating mycelial plugs of heterozygous diploids to growth on
solid media containing 2.0 &ml MNNG or 0.75 pg/ml 4-NQO;
(ii) the mitotic intragenic recombination phenotype of heterozygous
diploids. + = complementation; - = noncomplementation.
persensitivity of diploids to MNNG and 4-NQO. The
results (data not shown) showed that nuv-2 was nonallelic to all of these uvs mutations. The nuv-2 mutation therefore also defines a new locus in A. nidulans.
DISCUSSION
The mutant isolation program was successful in
isolating a large number of mutants hypersensitive to
MNNG and/or UV-irradiation. Of the 23 nuv mutants chosen forfurther characterization, all were
hypersensitive to bothMNNG and 4-NQO and in
each case this phenotype appeared to be due to single
chromosomal gene mutation. The wide diversity of
growth responses to MNNG and 4-NQOexhibited by
the nuv mutants was desirable since this meant that
they were more likely to bemutations in different
genes. Initial complementation analysis seems to bear
this out.
From what is known from studies in other model
organisms (e.g., FRIEDBERG
1988), mutagen-hypersensitive mutants can result from mutations inactivating
structural genes for DNA repair activity, or those that
affect the regulation of expression of DNA repair
genes. For eukaryotic organisms, mutations in genes
involved in chromatin structure and regulationof the
cell cycle can also produce a hypersensitive phenotype
(HARRIS andBOYD1987; WEINERTand HARTWELL
1988).It is also importanttoconsiderthat
some
mutations may cause hypersensitivity due to indirect
consequences. Forexample,
sensitivity ofcells
to
MNNG has been previously shown to be affected by
intracellular thiol content (SEDGWICK
and ROBINS
1980) and pH (DELIC,HOPWOOD
and FRIEND 1970).
It can also be envisaged that a hypersensitive phenotype could be due to mutations affecting cell permeability, or causing enhanced damage tosome essential
cellular component other than DNA.
The tests for hypersensitivity of diploids heterozygous and homozygous for the nuv mutations relative
to wild-type were important for identifying recessive
and semi-transdominant nuv alleles. Mutant alleles
exhibiting dominance are of particular interest be-
cause they are candidatesfor specifying genes involved in regulation of repair functions. Alternatively
they could specify structural genes for repairenzymes,
which exhibit a gene dosage effect in diploids. The
semidominance of nuv-8 and nuv-Ill to 4-NQO but
not to MNNG is most readily explainable if it is
postulated that the function encoded by the wild-type
genes is a major regulatory or structural component
for repair oflesions caused by 4-NQO, but only a
minor component for the repair of MNNG-induced
lesions. The apparent haploid-specific sensitivity of
nuv-20 is interesting but not readily explainable in
molecular terms.
A test systemhas been developed in A. nidulans,
which is novel for this organism and which can assay
for mitotic intragenic recombination rapidly and effectively using simple plating tests. It directly measures recombinant subcolonies arising from independent events within the diploids, giving an accurate
measure of differences in the frequency of recombination events in different strains. Traditional assays
do not give such a direct measure of recombination
frequency. They rely on harvesting conidia from heteroallelic diploid colonies growing on nonselective
media and, hence, a recombinant cell can give rise to
a clonal population distorting the frequency (JANSEN
1970; FORTUIN197 1).
In this study we have investigated the effects on
mitotic recombination of mutations, which affect the
ability to repair general DNA damage. It seems likely
that those mutations thatdo show effects on the
intragenic recombination measured in our assay will
not only affect recombination in the niaD region but
will also affect recombination in other regions of the
genome. Among the mutants initially isolated as hypersensitive to DNA damaging agents, a number of
Rec phenotypes were observed. Of the 23 nuv mutations characterized in our assay, 10 appeared to have
no marked effects on mitotic recombination. These
nuv mutations were phenotypically similar to the previously characterized uvsA mutation (JANSEN 1967).
Ten nuv mutations appeared to be hypo-rec, similar
to the previously characterized uvsC and uvsE mutations (FORTUIN197 1;JANSEN 1970). Three appeared
to be hyper-rec, as were the uvsB, uvsD, uvsF, uvsH
and uvsJ mutations (LANIER,TUVESON
and LENNOX
1968;SHANFIELDand KAFER 1969; JANSEN 1970;
WRIGHTand PATEMAN 1970;FORTUIN197 1). nunI 1 7 had a semidominant hypo-rec phenotype, which
could imply a gene dosage effect for its product or a
mutation within a regulatory gene or sequences.
The possible defects of the nuv mutations affecting
mitotic recombination and the underlying functions
of the gene productscan to some extent be postulated
by extrapolation from what is known from mutational
studies in other organisms. Mutations causing a hypo-
A. nidulans Recombination Mutants
453
genes known to be involved in D N A repair and recomrec phenotype for spontaneous homologous chromobination in thisorganism. Thefurtherphenotypic
somal recombination are the most likely to be defecwith astive in structural or regulatory genes directly involved characterizationofthesemutantstogether
signment
to
epistatic
groups
will
be
important
in dein recombination. For example, the characteristic phetermining
their
functions.
The
availability
of
a
large
notypeofthe
S. cerevisiae RAD52 groupmutants,
number
of
mutants
should
facilitate
the
cloning
of the
which are thought to be defective in recombination
relevant
genes
by
complementation
and,
conseprocesses, is recombination deficiency (HAYNES
and
quently, purification and biochemical characterization
KUNZ 1981; GAME1983; COOPERand KELLY 1987;
of
the gene products. The correlationof mutant pheFRIEDBERC
1988, 1991). Mitotichyper-recmutants
notype
to biochemical activity will be the critical cricould also bedefective in primaryrecombination
terion
by
which the molecular mechanisms of homolfunctions, as is the rad50 mutant of S. cerevisiae (MAogous
recombination
will be elucidated. The number
LONE et UL. 1990). However, such a phenotype could
of
mutants
now
available
in A. nidulans make it atalso be the result of secondary effects caused by detractive
as
a
complementary
system to S. cerevisiae. I t
fects in genes involved in other aspects of D N A meis
likely
that
some
of
the
nuv
mutants
will define genes
tabolism. For example, these mutations could be deof
types
not
previously
characterized,
although the
fective in a repair mechanism, which causes the accuavailability
of
any
homologous
genes
from
two evomulation of recombinogenic lesions, o r lesions that
lutionarily
distinct
fungi
should
help
in
the
isolation
inducethe expressionofrecombinationenzymes.
of
homologues
from
higher
eukaryotes.
Some eukaryotic mutants characterized
as defective
in excision repair or error-prone replicativerepair
This work was supported by grantsfromthe
University of
havebeenshowntobehyper-recforspontaneous
Liverpool and the Wellcome Trust to P.S. and B.T. T h e helpful
mitoticchromosomalrecombination.Forexample,
is gratefully acknowledged.
advice of B. M. FAULKNER
the rad3 and rad6 mutants of S. cerevisiae, which are
deficient in excision repair and error-prone repair,
L I T E R A T U R E CITED
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