previously described growth conditions including those for inducing development were used for a. nidulans [ 17 , 18 ] .
expression of anea via niia promoter is induced by 0.6% sodium nitrate and repressed by 0.2% ammonium tartrate .
an anea disruption cassette ( dc ) was constructed according to methods described by yu et al . .
using genomic dna prepared from a. nidulans fgsc a4 , dna fragments for the dc were amplified by pcr by using appropriate primer sets ( anea - a1/-a2 , anea - b1/-b2 , and argb - for/-rev ) .
the complete dc was amplified by a nested - pcr primer set ( anea - c1/-c2 ) , purified , and then used to transform the tj1 strain .
nucleic acid preparation and northern blot analysis were performed as described previously . for southern blotting ,
genomic dna was treated with appropriate restriction enzymes and then separated on 1% agarose gels .
the gels were washed with distilled water ( dw ) and soaked twice in a depurination solution ( 250 mm hcl ) for 15 min . after the reaction , the gel was washed with dw again and then soaked in denaturation solution ( 1.0 m nacl and 0.4 m naoh ) for 1 hr . the genomic dna contained in the processed gel was transferred onto a hybond - n membrane ( amersham biosciences , pittsburg , pa , usa ) .
gene - specific probes were prepared from the pcr - generated fragments and were labeled using the ecl direct nucleic acid labeling system ( amersham biosciences ) .
previously described growth conditions including those for inducing development were used for a. nidulans [ 17 , 18 ] .
expression of anea via niia promoter is induced by 0.6% sodium nitrate and repressed by 0.2% ammonium tartrate .
an anea disruption cassette ( dc ) was constructed according to methods described by yu et al . .
using genomic dna prepared from a. nidulans fgsc a4 , dna fragments for the dc were amplified by pcr by using appropriate primer sets ( anea - a1/-a2 , anea - b1/-b2 , and argb - for/-rev ) .
the complete dc was amplified by a nested - pcr primer set ( anea - c1/-c2 ) , purified , and then used to transform the tj1 strain .
nucleic acid preparation and northern blot analysis were performed as described previously . for southern blotting ,
genomic dna was treated with appropriate restriction enzymes and then separated on 1% agarose gels .
the gels were washed with distilled water ( dw ) and soaked twice in a depurination solution ( 250 mm hcl ) for 15 min . after the reaction , the gel was washed with dw again and then soaked in denaturation solution ( 1.0 m nacl and 0.4 m naoh ) for 1 hr . the genomic dna contained in the processed gel was transferred onto a hybond - n membrane ( amersham biosciences , pittsburg , pa , usa ) .
gene - specific probes were prepared from the pcr - generated fragments and were labeled using the ecl direct nucleic acid labeling system ( amersham biosciences ) .
the dc for deletion of the anea gene was constructed and used to transform the recipient strain .
southern blot analysis of pst i - digested genomic dna from candidate strains with a pcr - amplified probe specific for the 5'-flanking region of anea confirmed the deletion of the chromosomal anea gene by revealing an expected band of 5.7 kb ( fig .
1 ) . to investigate the function of anea in development of a. nidulans , the pattern of hyphal growth including radial growth , septation , and asexual sporulation were examined in one of the deletion mutant strains .
however , we observed no differences between the wild - type and deletion mutant ( data not shown ) .
these results indicated that -cop is not essential for the viability of a. nidulans ; these results are similar to those obtained in yeasts .
recently , copi proteins were reported to play roles in responses to er stress and thermal stress in yeast [ 11 , 13 ] . here
, we tested the effect of -cop deletion on sensitivity to environmental stresses caused by treatment of the fungi with several drugs that are known to exert adverse effects on cellular processes related to the stress response .
no detectable changes were observed in the -cop - defective strain by treatment with drugs including inhibitors of cell - wall biosynthesis ( calcofluor white , congo red , and caspofungin ) , n - glycosylation ( tunicamycin ) , ergosterol biosynthesis ( terbinafine ) , and glycerol biosynthesis ( fludioxonil ) , an inducer of apoptosis ( farnesol ) , and a ca chelator ( egta ) .
thermal stress , which makes the -cop - defective yeast strain unviable , showed no effect on viability and vegetative growth in the -cop deletion strains ( data not shown ) . taken together ,
these results show that -cop in a. nidulans is neither essential for viability nor involved in stress responses unlike in yeast . in a. nidulans , a mutation in the sodc gene
copi depletion also results in the failure of cytokinesis and a reduction in the number of overlapping central spindle microtubules during meiotic divisions for spermatogenesis in drosophila . in the -cop - depleted ( anea ) a. nidulans strain , we observed no defects in asexual development ( data not shown ) , but significant defects in sexual development ( fig .
even after culturing in conditions favorable for cleistothecium development , with hypoxic treatment with 1% glucose in the dark , the anea strain showed more than 50% decrease of fruiting body formation ( table 3 ) .
in addition , in 1% glucose without hypoxic treatment , fruiting body formation was almost completely abolished by -cop depletion . in 2% lactose without hypoxic treatment , which predominantly induces the formation of fruiting bodies , -cop depletion showed no deleterious effect on fruiting body formation ( table 3 ) .
because the effect of c - sources and hypoxic treatment was tested in the presence of 0.1% sodium nitrate as a nitrogen source , we tested 0.2% yeast extract , which preferentially induces sexual development , and found that the defect in fruiting body formation on 1% glucose without hypoxic treatment was recovered in anea deletion strain ( data not shown ) .
although further experiments are required to determine the underlying mechanisms , our results suggested that -cop plays a role in sexual development in a. nidulans under certain environmental conditions , possibly by affecting cytokinesis and/or construction of er - based spindle envelopes , as observed in fruit fly spermatogenesis .
our previous study showed that the c - terminal domains of both -cop and -cop are essential for their interaction , and that the n - terminal wd40 motif of -cop and the tpr region of -cop are involved in controlling the interaction between these two cops in a. nidulans .
we also reported that a. nidulans -cop can substitute for s. cerevisiae -cop functions in vivo . in yeast , -cop is known to stabilize the thermo - sensitive -cop mutation .
thus , over - expression of -cop confers viability to the -cop mutant at elevated temperatures .
therefore , we investigated whether the phenotype of the sodc1 mutant was suppressed by over - expression of anea in a. nidulans . when a prg3-ama1 plasmid containing the anea gene encoding -cop was introduced into the sodc1 mutant , over - expression of anea
however , over - expression of -cop could not rescue the thermo - sensitive phenotype of the sodc1 mutant at a non - permissive temperature ( 42 ) ( fig .
these results indicate that -cop is able to interact with -cop , but does not stabilize -cop at elevated temperatures unlike in yeast . in summary , our results indicate that -cop in a. nidulans is not essential but related to formation of the sexual reproductive organ in response to particular environmental factor(s ) .
it is worth highlighting that recent reports indicate that the function of the copi complex ( and its subunits ) is not confined to intracellular vesicular trafficking .
the copi complex is also involved in other cellular events such as chromosome disjunction in a. nidulans , male development in chicken embryos , er stress responses in yeast , association with calcineurin during heat stress , meiotic divisions for spermatogenesis in drosophila , induction of productive autophagy and cellular survival , intercompartmental trafficking of specific rnas in neuronal cells , and neurite outgrowth .
although further studies are necessary to confirm the actual role of -cop ( and/or copi ) in sexual development of fungi , our results open up interesting avenues for further studies on the function of the copi complex .
the dc for deletion of the anea gene was constructed and used to transform the recipient strain .
southern blot analysis of pst i - digested genomic dna from candidate strains with a pcr - amplified probe specific for the 5'-flanking region of anea confirmed the deletion of the chromosomal anea gene by revealing an expected band of 5.7 kb ( fig .
1 ) . to investigate the function of anea in development of a. nidulans , the pattern of hyphal growth including radial growth , septation , and asexual sporulation were examined in one of the deletion mutant strains .
however , we observed no differences between the wild - type and deletion mutant ( data not shown ) .
these results indicated that -cop is not essential for the viability of a. nidulans ; these results are similar to those obtained in yeasts .
recently , copi proteins were reported to play roles in responses to er stress and thermal stress in yeast [ 11 , 13 ] . here
, we tested the effect of -cop deletion on sensitivity to environmental stresses caused by treatment of the fungi with several drugs that are known to exert adverse effects on cellular processes related to the stress response .
no detectable changes were observed in the -cop - defective strain by treatment with drugs including inhibitors of cell - wall biosynthesis ( calcofluor white , congo red , and caspofungin ) , n - glycosylation ( tunicamycin ) , ergosterol biosynthesis ( terbinafine ) , and glycerol biosynthesis ( fludioxonil ) , an inducer of apoptosis ( farnesol ) , and a ca chelator ( egta ) .
thermal stress , which makes the -cop - defective yeast strain unviable , showed no effect on viability and vegetative growth in the -cop deletion strains ( data not shown ) . taken together ,
these results show that -cop in a. nidulans is neither essential for viability nor involved in stress responses unlike in yeast .
in a. nidulans , a mutation in the sodc gene encoding -cop is responsible for non - disjunction of the chromosome during cell division .
copi depletion also results in the failure of cytokinesis and a reduction in the number of overlapping central spindle microtubules during meiotic divisions for spermatogenesis in drosophila . in the -cop - depleted ( anea ) a. nidulans strain , we observed no defects in asexual development ( data not shown ) , but significant defects in sexual development ( fig .
even after culturing in conditions favorable for cleistothecium development , with hypoxic treatment with 1% glucose in the dark , the anea strain showed more than 50% decrease of fruiting body formation ( table 3 ) .
in addition , in 1% glucose without hypoxic treatment , fruiting body formation was almost completely abolished by -cop depletion . in 2% lactose without hypoxic treatment , which predominantly induces the formation of fruiting bodies , -cop depletion showed no deleterious effect on fruiting body formation ( table 3 ) .
because the effect of c - sources and hypoxic treatment was tested in the presence of 0.1% sodium nitrate as a nitrogen source , we tested 0.2% yeast extract , which preferentially induces sexual development , and found that the defect in fruiting body formation on 1% glucose without hypoxic treatment was recovered in anea deletion strain ( data not shown ) .
although further experiments are required to determine the underlying mechanisms , our results suggested that -cop plays a role in sexual development in a. nidulans under certain environmental conditions , possibly by affecting cytokinesis and/or construction of er - based spindle envelopes , as observed in fruit fly spermatogenesis .
our previous study showed that the c - terminal domains of both -cop and -cop are essential for their interaction , and that the n - terminal wd40 motif of -cop and the tpr region of -cop are involved in controlling the interaction between these two cops in a. nidulans .
we also reported that a. nidulans -cop can substitute for s. cerevisiae -cop functions in vivo . in yeast , -cop is known to stabilize the thermo - sensitive -cop mutation .
thus , over - expression of -cop confers viability to the -cop mutant at elevated temperatures .
therefore , we investigated whether the phenotype of the sodc1 mutant was suppressed by over - expression of anea in a. nidulans . when a prg3-ama1 plasmid containing the anea gene encoding -cop was introduced into the sodc1 mutant , over - expression of anea
however , over - expression of -cop could not rescue the thermo - sensitive phenotype of the sodc1 mutant at a non - permissive temperature ( 42 ) ( fig .
these results indicate that -cop is able to interact with -cop , but does not stabilize -cop at elevated temperatures unlike in yeast . in summary , our results indicate that -cop in a. nidulans is not essential but related to formation of the sexual reproductive organ in response to particular environmental factor(s ) .
it is worth highlighting that recent reports indicate that the function of the copi complex ( and its subunits ) is not confined to intracellular vesicular trafficking .
the copi complex is also involved in other cellular events such as chromosome disjunction in a. nidulans , male development in chicken embryos , er stress responses in yeast , association with calcineurin during heat stress , meiotic divisions for spermatogenesis in drosophila , induction of productive autophagy and cellular survival , intercompartmental trafficking of specific rnas in neuronal cells , and neurite outgrowth .
although further studies are necessary to confirm the actual role of -cop ( and/or copi ) in sexual development of fungi , our results open up interesting avenues for further studies on the function of the copi complex .
 we have previously isolated -cop , the -cop interactor in copi of aspergillus nidulans , by yeast two - hybrid screening . to understand the function of -cop , the anea+ gene for -cop / anea was deleted by homologous recombination using a gene - specific disruption cassette . 
 deletion of the -cop gene showed no detectable changes in vegetative growth or asexual development , but resulted in decrease in the production of the fruiting body , cleistothecium , under conditions favorable for sexual development . unlike in the budding yeast saccharomyces cerevisiae , in a. nidulans , over - expression of -cop did not rescue the thermo - sensitive growth defect of the -cop mutant at 42. together , 
 these data show that -cop is not essential for viability , but it plays a role in fruiting body formation in a. nidulans . 
MATERIALS AND METHODS
Strains and growth conditions
Construction of a disruption cassette
Nucleic acid preparation, Northern blotting, and Southern blotting
RESULTS AND DISCUSSION
Effect of -COP depletion on growth and stress response
Effect of -COP depletion on development
Suppression of 