Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

Abstract

We have used a plasmid containing the argB gene to transform an Aspergillus nidulans argB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and approximately 3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.

Full text

ORIGINAL PAPER
O. Sa
Â
nchez á R. E. Navarro á J. Aguirre
Increased transformation frequency and tagging of developmental genes
in
Aspergillus nidulans
by restriction enzyme-mediated
integration (REMI)
Received: 22 August 1997 / Accepted: 20 November 1997
Abstract We have used a plasmid containing the argB
gene to transform an Aspergillus nidulans argB-deleted
strain in the presence of restriction enzymes and show a
20- to 60-fold increase in transformation frequency via
restriction enzyme-mediated integration (REMI). This
procedure was used to try to tag new genes involved in
the asexual development of this fungus. More than 2000
transformants isolated following electroporation of
conidia and 3700 transformants recovered following
protoplast fusion were screened for sporulation defects.
Unexpectedly, developmental mutants were obtained
only when the protoplast fusion approach was used.
Southern blot analysis of these mutants, and of randomly
selected transformants obtained by electropora tion, was
consistent with the occurrence of single plasmid inte-
gration events in 33 and 65% of the cases, respectively.
The argB marker was shown to be tightly linked to the
mutant phenotype in only 62% of the mutants analyzed
by sexual crosses. Partial DNA sequencing of a tagged
gene, whose mutation delays asexual sporulation and
results in a ¯uy phenotype, showed no homo logy to
previously reported sequences. Our results indicate that
REMI can be used in A. nidulans to increase the tra ns-
formation frequency and illustrate the advantages and
potential problems when using REMI to tag genes of
interest in this and other fungi.
Key words Aspergillus á Restriction enzyme-mediated
integration á Electroporation á Asexual development á
Gene tagging
Introduction
The integration of transforming DNA mediated by the
in vivo action of restriction enzymes (REMI) was ®rst
described in yeast (Schiestl and Petes 1991). More re-
cently, it has been used as a genetic tool to mutagenize
and tag genes involved in development in Dictyostelium
(Kuspa and Loomis 1992), in toxin production in Co-
chliobolus (Lu et al. 1994) and pathogenicity in Ustilago
(Bo
È
lker et al. 1995). The major advantage of REMI is
that it can provide a means to disrupt genes randomly by
plasmid insertion and the subsequent cloning of these
genes by plasmid rescue in Escherichia coli. Additionally,
in some but not all cases (Lu et al. 1994; Bo
È
lker et al.
1995), it can increase transformation frequencies.
The fungus Aspergillus nidulans has been an exten-
sively used experimental system for genetic studies of
fundamental biological problems such as gene regula-
tion and development (for an overview, see Martinelli
and Kinghorn 1994). Although the isolation of mutants
and their genetic characterization is easily achieved in
this fungus, cloning of genes by complementation is
limited by the low transformation frequencies routinely
obtained and by diculties found in recovering the
DNA used to complement a given mutation (Timberlake
1991). Here we show that REMI mutagenesis can be
used in A. nidulans signi®cantly to increase the trans-
formation frequency and, with some restrictions, to tag
and clone genes of interest.
The potential usefulness of REMI to mutagenize and
tag genes in A. nidulans was evaluated through the iso-
lation of mutants aected in the regulation of asexual
sporulation (conidiation). This developmental pathway
has been studied in great detail and the isolation of
mutants and the cloning of the corresponding genes has
been a powerful tool for the understanding of this pro-
cess (Clutterbuck 1969; Adams et al. 1988; Mirabito
et al. 1989).
Mol Gen Genet (1998) 258: 89±94
Ó
Springer-Verlag 1998
Communicated by E. Cerda
Â
-Olmedo
O. Sa
Â
nchez á R. E. Navarro á J. Aguirre (&)
Departamento de Gene
Â
tica Molecular,
Instituto de Fisiologõ
Á
a Celular,
Universidad Nacional Auto
Â
noma de Me
Â
xico,
Apartado Postal 70-242, 04510 Me
Â
xico, D. F., Me
Â
xico
Tel: +525-622-5651; Fax: +525-622-5630;
E-mail: jaguirre@ifcsun1.i®siol.unam.mx

Materials and methods
Strains, plasmids, media and transformation
To reduce the chances of plasmid integration by homologous re-
combination, we selected the argB -containing plasmid pDC1
(Aramayo et al. 1989) to transform the argB-deleted strain
RMSO11 (pabaA1, yA2; DargB::trpCDB; veA1, trpC801; Stringer
et al. 1991), which contains no detectable homology to pDC1.
This plasmid contains unique restriction sites for ClaI, XhoI,
NruI, SphI, SmaI, BamHI and KpnI at the polylinker. The alter-
native argB-deleted strain RMS010 (biA1; DargB::trpCDB;
metG1; veA1, trpC801; Stringer et al. 1991) and plasmids pMS12
and pILJ16 containing argB and other unique restriction sites are
available from the Fungal Genetics Stock Center (University of
Kansas Medical Center; http://www.kumc.edu/research/fgsc/
main.html).
The argB autonomously replicating plasmid pDHG25 (Gems
et al. 1991) was used as a transformation control.
Electrotransformation of swollen conidia (2 h) was carried out
as described before (Sa
Â
nchez and Aguirre 1996), except that the
time protocol was reduced by 1 h. Brie¯y, freshly harvested conidia
were used to inoculate Ka
È
fer's minimal nitrate medium (Ka
È
fer
1977) at a density of 10
7
conidia/ml. After a 2 h incubation at 37° C
with shaking, conidia were recovered by centrifugation, washed
with 400 ml of ice-cold sterile water and resuspended in 25 ml of a
20 mM ice-cold N-[2-hydroxyethyl]piperazine-N¢-[2-ethanesulfonic
acid] solution (adjusted to pH 8.0 with TRIS), containing 1% yeast
extract and 1% glucose.
Conidia were centrifuged again and resuspended in 2.5 ml
(about 1.6 ´ 10
9
conidia/ml ®nal) of ice-cold electroporation buer
(10 mM TRIS-HCl, 270 mM sucrose, 1 mM lithium acetate, pH
7.5) and kept on ice or frozen. 50 ll of this conidial suspension plus
DNA were adjusted to a ®nal volume of 60 ll with distilled water,
incubated on ice for 15 min and transferred to a 0.2 cm cuvette for
electroporation. A Bio-Rad (Hercules, USA) Gene Pulser and
pulse controller apparatus set to 1000 V, 25 lF and 400 W (pulse
length 5.1±5.8 ms) was used.
Transformation by protoplast fusion was carried out as re-
ported (Yelton et al. 1984). Sexual crosses between developmental
mutants and strain PW1 ( biA1; argB2; metG1; veA1; P. Weglenski;
Department of Genetics; University of Warsaw, Warsaw, Poland),
as well as diploid formation, were carried out using standard
methodology (Pontecorvo et al. 1953).
Molecular techniques
Plasmid pDC1 puri®ed on Qiagen (Hilden, Germany) columns was
digested with BamHI or KpnI (manufacturer's restriction buer, no
albumin included) and used immediately for transformation with
or without the addition of fresh extra enzyme. For electrotrans-
formation, 5 lg of pDC1 was digested with 30 U of BamHI for 2 h
in a 50 ll volume. Salts in the DNA preparations were removed by
using spin-columns packed with Sephadex G-50 equilibrated with
1 mM TRIS-HCl, pH 8.0, 0.1 mM EDTA buer or deionized
water. DNA was recovered in a volume of 50 ll, and 1 lg was used
to electroporate swollen conidia.
For protoplast fusion, 10 lg of plasmid pDC1 was digested for
3 h with 50 U KpnIorBamHI and 3 lg was incubated with the
protoplast suspension for 25 min before addition of polyethylene
glycol (average molecular weight 3350). Genomic DNA isolated
from Arg+ transformants (Timberlake 1980) was digested, trans-
ferred to Hybond-N nylon membranes (Amersham; Little Chal-
font, UK) and hybridized to pDC1 labeled with
32
P, using the
random priming system from Life Technologies (Gaithersburg,
USA). Plasmid recovery from some developmental mutants was
carried out by digesting genomic DNA, cleaning DNA using
Wizard clean up system minicolumns (Promega; Madison, USA),
ligation and electroporation of bacteria.
Results
The presence of restriction enzymes
during electroporation of conidia
increases transformation frequency
We have shown that electroporation of nondividing
swollen conidia from A. nidulans with an integrative
plasmid containing homologous sequences generated
transformants at similar frequencies to those obtained
by using protoplast fusion (10 transformants/lgof
plasmid; Sa
Â
nchez and Aguirre 1996). However, as shown
in Table 1, using the argB-containing integrative plas-
mid pDC1 to transform the argB-deleted strain RMS011
resulted in a transformation frequency of only 2 trans-
formants/lg of plasmid. In contrast, the similar argB-
containing plasmid pDHG25, which does not need to
integrate into the genom e to complement the RMS01 1
arginine auxotrophy, generated 656 transformants/lgof
DNA. We tested whether the presence of restriction
enzymes during electroporation of pDC1 could increase
the number of transformants obt ained, as has been
shown for other fungi (Lu et al. 1994). Results in Table 1
show that, when pDC1 was digested with BamHI and
used immediately for electroporation, the transforma-
tion frequency increased about 60-fold. This increase
was only 19-fold when the enzyme was partially inacti-
vated by heating. The inclusion of extra amounts of
fresh BamHI (1 U) during electroporation slightly in-
creased the transformation frequency (not shown) but
there were detrimental eects when adding higher con-
centrations of enzyme (10 U; Table 1). Similar eects in
transformation frequency were observed when the en-
zyme KpnI was used instead of BamHI (not shown).
DNA from randomly selected transformants ob-
tained with pDC1, in the presence or absence of BamHI,
was digested with the enzyme PstI (which does not cut in
pDC1) or with BamHI, which linearizes this plasmid,
and analyzed by Southern blotting using pDC1 as a
probe. The results presented in Fig. 1 show that, in
contrast to the recipient strain RMS011 (Fig. 1A,
lane 1), all transformants contained argB-hybridizing
sequences. Most of the integration events resulted in
Table 1 The presence of a restriction enzyme during electropora-
tion increases transformation frequency. 1 lg of freshly digested
pDC1 was used to electroporate swollen conidia from strain
RMS011 without further treatment, after being heated at 65° C for
15 min, or after adding 10 extra units of BamHI before electro-
poration. pDHG25 was included as a control.Numbers represent
mean values from the two independent experiments indicated in
parentheses
Plasmid/treatment Arg+ transformants/lg
pDC1 2 (4+0)
pDC1(BamHI) 128 (115+141)
pDC1(BamHI, 65° C) 38 (35+41)
pDC1(BamHI)+10 U 7 (8+6)
pDHG25 656 (650+662)
90

dierent-sized Pst I fragments, as expected if integration
was at dierent single sites in the individual transform-
ants (Fig. 1A). Transformants obtained with intact
pDC1 (lanes 2±4) show hybridization bands equal to or
smaller than pDC1 in the BamHI digest. Smaller bands
could result from rearrange ments and/or deletions dur-
ing integration (Fig. 1B).
Of the transformants obtained in the presence of
BamHI (lanes 5±18), numbers 7, 8, 9, 11, 15 and 17 show
a single 4.5 kb BamHI fragment that comigrates with
BamHI-digested pDC1 (Fig. 1B). This indicates the in-
sertion of pDC1 into genomic BamHI sites and their
preservation. This phenomenon has been described in
other microorganisms as REMI (Schiestl and Petes
1991; Kuspa and Loomis 1992; Lu et al. 1994; Bo
È
lker
et al. 1995). Single hybridization signals larger than
pDC1 (transformants 5, 6 and 14) would be consistent
with integrations at BamHI sites without site regenera-
tion or not at a BamHI site. Finally, the hybridization
patterns of transformants 10, 12, 13 and 18 are consis-
tent with tandem or duplicate pDC1 fragment integra-
tions at BamHI sites, without regeneration of all sites at
the integration place.
Tagging of A. nidulans developmental genes
using REMI and insertional mutagenesis
The possibility of using REMI, and insertional muta-
genesis in general, to tag genes invo lved in the regulation
of asexual development in A. nidulans was tested by
electroporating conidia from strain RMS011 with plas-
mid pDC1, in the presence of the restriction enzymes
BamHI or KpnI, and screening for morphological mu-
tants aected in conidiation. Unexpectedly, not a single
developmental mutant was detected after screening
2000 transformants generated by this method. For this
reason, we decided to transform protoplasts derived
from vegetative mycelium in the presence of restriction
enzymes and screen the resulting transformants for de-
velopmental defects.
Transformation frequencies obtained using this ap-
proach were similar to those observed using electropo-
ration (Table 2). Also, the presence of restriction
enzymes during protoplast fusion increased the fre-
quency of integrative transformation. In contrast to the
electroporation experiments, the appearance of trans-
formants with aberrant morph ologies was readily evi-
dent. Table 2 indicates the number of transformants
that had either limited growth, or normal growth but
atypical conidiation obtained in two independent ex-
periments using KpnIorBamHI. Of these, 23 mutants
showing a reduced number or a total lack of conidia are
shown in Fig. 2. The ®rst 20 were char acterized further.
Mutants KL001 and BL0 04 lacked conidiophore struc-
tures besides the stalks and presented the typical mor-
phology of brlA null developmental mutants
(Clutterbuck 1969). Mutants B1001 and BL002 pre-
sented the classic morphology of null abaA mutants
(Clutterbuck 1969), diering among them by the degree
of conidiophore pigmentation (Fig. 2). In fact, diploids
made between these and abaA2 mutants conserved the
abacoid morphology, further indicating that mutants
B1001 and BL002 are aected in abaA.
Mutants BL001, BL003, K5003, K5009, K5012 and
K5014 presented a typical ¯uy morphology (Dorn
1970), characterized by a notable retardation in con-
idiophore formation, although the conidiophore mor-
phology was not altered in any of them. Results from
diploid complementation tests between mutants BL001,
K5012, K5003 and other ¯uy mutants aected in the
¯uG and ¯bA±E (Wieser et al. 1994) genes, indicated that
all three contain recessive mutations, BL001 being af-
fected in a gene dierent from ¯uG/¯bA±E, muta nt
K5012 containing a mutation allelic to ¯bB, and mutant
K5003 being aected in a gene dierent from ¯bA±C.
These results indicate that at least ®ve dierent genes are
represented by the mutant s shown in Fig. 2.
To analyze the types of integration events that had
occurred, genomic DNA from dierent develop mental
mutants was hybridized to pDC1 sequences. Mutants
Fig. 1A, B Southern blot analysis of transformants obtained by
electroporation in the presence of BamHI. Swollen conidia (2 h) from
strain RMS011 were electroporated with intact pDC1 (lanes 2±4) or
pDC1 freshly digested with BamHI (lanes 5±18). Genomic DNAs
from randomly selected Arg+ transformants were digested with PstI,
which does not cut pDC1 (A), or with BamHI, which cuts once in
pDC1(B), and analyzed by Southern blotting, using pDC1 as a probe
91

obtained in the presence of KpnI were digested with PstI
(Fig. 3A), which does not cut pDC1, or KpnI (Fig. 3B),
which cuts once in the plasmid. The genomic DNA from
mutants obtained in the presence of Bam HI was digested
with Pst I (FIg. 4A), or BamHI (Fig. 4B), and analyzed
as indicated before. When digested with PstI, the mu-
tants obtained with KpnI showed a hybridization pattern
that indicated that pDC1 had been inserted at single
locations in the individual transformants (Fig. 3A). The
KpnI digest showed single plasmid integration events for
transformants KL002, KL003, K5003 and K5009
(Fig. 3B), the pattern for KL002 being consistent with
the integration of a single pDC1 molecule and the re-
generation of the KpnI sites. The other ®ve mutants
analyzed showed patterns that could be explained by
tandem inte grations without regeneration of some KpnI
sites at the original integration site. Similar results were
obtained when mutants, generated in the presence of
BamHI, were analyzed by Southern blotting. The results
of PstI digestion indicated insertion at dierent single
sites in the di erent transformants (Fig. 4A). The
BamHI digestion showed that only transformants B1001
and BL001 had undergone a single plasmid integration
event, whereas the other mutants analyzed presented
more complex integration patterns (Fig. 4B).
To test whether or not the mutations respon sible for
altered morphology were linked to the plasmid insertion
site, we tried to cross 19 of these mutants to the argB)
strain PW1 and plated the ascospores in medium con-
taining 0, 0.5 and 5 mM argi nine. Thirteen mutants were
able to cross, but in only eight of them did the mutant
phenotype segregate with ArgB+, indicating that the
developmental defects resulted from pDC1 insertion in
only 62% of these cases. PDC1-derived plasmids con-
taining inserts were recovered from three dierent de-
velopmental mutants. Partial sequencing of a plasmid
recovered from transformant BL001 showed no ho-
mology to previously reported sequences.
Discussion
Here we have used an argB-containing integrative plas-
mid with no detectable homology to an argB-deleted
A. nidulans strain to transform such a strain in the
presence or absence of restriction enzymes. In the ab-
sence of restriction enzymes, electroporation of conidia
or protoplast fusion protocols gave transformation fre-
quencies of 2±5 transformants/lg. With both protocols,
the presence of restriction enzymes notably increased
transformation frequencies (Tables 1 and 2). In contrast,
a similar autonomously replicating argB-containing
Table 2 The presence of restriction enzymes during protoplast
transformation increases transformation frequency and results in
mutation. 3 lg of plasmid pDC1 digested with KpnIorBamHI was
incubated with protoplasts for 25 min before of polyethyleneglycol
(PEE) baddition. Arg+ transformants were screened for growth or
developmental defects. Plasmid pDHG25 was used as reference
Plasmid/treatment Arg+ transformants per 3 l g of DNA
a
Detected mutants
Exp. 1 Exp. 2 Exp. 1 Exp. 2
pDC1 15 15 0 0
pDC1(KpnI) ND
b
306 ND 4
pDC1(KpnI)+1U
c
ND 420 ND 2
pDC1(KpnI)+5 U ND 720 ND 7
pDC1(BamHI) 234 372 0 2
pDC1(BamHI)+1 U 183 609 3 2
pDC1(BamHI)+5 U 132 168 3 5
pDC1(BamHI)+10 U 75 ND 3 ND
pDC1(BamHI)+30 U 60 ND 2 ND
pDHG25 384 486 0 0
Fig. 2 Developmental mutants isolated after transformation of
protoplasts in the presence of restriction enzymes. Colonies labeled
from top to the bottom and left to right are: KL001, K1002, KL004,
KL003, KL002, K5014, K5012, K5009, K5005, K5003, B5005,
B1003, B1001, BL004, BL003, BL002, BL001, B5008, B5007, B5006,
PL003, PL002, PL001. Recipient strain RMS011 is shown for
comparison (insert)
a
Transformation frequency is referred to 3 lg of DNA to show the
total number of transformants screened in relation to the number
of mutants obtained for each treatment
b
Not done
c
Fresh extra enzyme (1±30 U) was added before PEG treatment
92

plasmid transformed at much higher frequencies in the
absence of restriction enzymes. The fact that two similar
plasmids, diering by their ability to propagate auton-
omously, transform at such disparate frequencies, as
well as the increase in transforma tion frequency for the
integrative plasmid when in the presence of a restriction
enzyme, suggests that linearization of genomic and
plasmid DNAs, prior to integration, are limiting events
for transformation in A. nidulans.
Important dierences in the kind of transformants
obtained by electroporation of conidia versus protoplast
fusion, in the presence of restriction enzymes, were ob-
served. In the ®rst case, about two-thirds of transform-
ants contained single plasmid integration events,
whereas in the second case, this proportion corre-
sponded to about one-third and most transformants had
undergone more complex integration events. A more
striking dierence became evident after we tried to use
electroporation of conidia and REMI to isolate mutants
aected in asexual development. No such mutants were
detected after screening 2000 transformants obtained
by electroporation, whereas morphological mutant
strains were easily detected after screening 3700
transformants obtained by protoplast fusion. These re-
sults suggest that the same target genes are not equally
available for plasmid integration in conidia and proto-
plasts. This is conceivable if one considers that nuclei
present in 2 h swollen conidia have not undergone their
®rst mitotic division and perhaps contain more densely
packed chromatin, which could limit restriction enzyme
function in vivo. On the other hand, the relatively high
frequency of develop mental mutants obtained after
protoplast fusion (0.6%), and the fact that two likely
brlA and two abaA mutants were obtained in this
screening, could suggest the existence of hot spots for
REMI in protoplasts. Alternatively, the res triction en-
zymes and the morphological screen used could bias the
number and kind of mutants obtained.
In our experiments, we did not see a clear correlation
between enzyme type or concentration and the number
of mutants, or the types of integrations obtained (Ta-
ble 2, Figs. 3 and 4). Similar conclusions have been
made independently after testing dierent restriction
enzymes and enzyme concentrations, using protoplast
fusion and a dierent plasmid/strain system (S. Eckert
and G. Braus, personal communication).
We have been able to iso late 23 develop mental mu-
tants after screening 3700 transformants. This number is
Fig. 3A, B Southern blot analysis of developmental mutants obtained
in the presence of KpnI. Genomic DNAs from the indicated Arg+
morphological mutants obtained by transformation in the presence of
KpnI (Fig. 2) were digested with PstI(A), or KpnI(B), and analyzed
by Southern blotting, using pDC1 as a probe
Fig. 4A, B Southern blot anal-
ysis of developmental mutants
obtained in the presence of
BamHI. Genomic DNAs from
the indicated Arg+ morpho-
logical mutants (Fig. 2) ob-
tained by transformation in the
presence of BamHI were di-
gested with PstI(A), or BamHI
(B), and analyzed by Southern
blotting, using pDC1 as a probe
93

easy to achieve given the inherent increase in transfor-
mation frequency provided by the restriction enzymes.
From 19 mutants tested in sexual crosses, only 13 pro-
duced sexual spores and 8 resulted from actual plasmid
insertion. This indicates that in some cases a mutation
arises from in vivo enzyme action, without plasmid in-
tegration, perhaps followed by imperfect repair.
Mutants speci®cally blocked in conidiation have been
a powerful tool to understand development in A. nidul-
ans (Clutterbuck 1969; Adams et al. 1988; Mirabito et al.
1989) and have led to the characterization of the brlA
function as a fundamental regulatory point. Mutants
showing a notable delay in conidiation, or ¯ues, have
been identi®ed as important for under standing growth
and brlA regulation (Dorn 1970; Aguirre et al. 1993;
Wieser et al. 1994; Yu et al. 1996). The number of
mapped genes in which mutation confers a ¯uy phe-
notype (Clutterbuck 1994) is larger than the number of
genes currently under study (Wieser et al. 1994; Yu et al.
1996). We have recovered pDC1-derived plasmids with
inserts from three dierent ¯uy mutants and are in the
process of characterizing them. In fact, partial se-
quencing of a plasmid recovered from transformant
BL001, a case of RE MI in which the mutant phenotype
was linked to argB, shows no homology to known se-
quences in GenBank. Currently, we are trying to isolate
more ¯uy mutants using dierent plasmids/restriction
enzymes. In ad dition to strain RMS011, we are using the
alternative argB-deleted strain RMS010, which carries
dierent genetic markers. Mutants obtained with dif-
ferent recipient strains can be used for direct diploid
complementation tests.
We detected some potential problems in using REMI
as a general mutagenesis/gene-tagging procedure in
A. nidulans (i.e., mutations not linked to plasmid inser-
tions, tandem integrations and possible bias in plasmid
insertion). However, the experimental facilities of this
fungus make it possible to solve most of these problems
(i.e., sexual crosses to select only mutations caused by
plasmid insertion, production of self-cleistothecia to re-
solve tandem integrations, transformation by electro-
poration versus protoplast fusion, etc), making this
approach a feasible one for cloning genes of interest in
this organism. The potential problems described here
would have to be considered when trying to apply
REMI to other fungi, particularly to other Aspergilli.
Acknowledgements This work was partially supported by grants
0708-N9109 from CONACyT, and IN200192 from DGAPA-
UNAM, Mexico. We thank Gabriela Soid for the diploid com-
plementation tests. We also thank Thomas Adams and Wilhelm
Hansberg for critical reading of the manuscript.
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