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A microculture hybridization technique for the detection of specific DNA sequences in filamentous fungi
EXPERIMENTAL MYCOLOGY 11, 70-73 (1987)
BRIEF NOTE A Microculture
Hybridization Technique for the Detection DNA Sequences in Filamentous Fungi PATRICK CROWLEYAND~TEPHEN
Department
of Specific
G. OLIVER~
of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 IQD, England Accepted for publication November 18, 1986
CROWLEY, P., AND OLIVER, S. G. 1987. A microculture hybridization technique for the detection of specific DNA sequences in filamentous fungi. Experimental Mycology 11, 70-73. A procedure that permits the successful application of the technique of colony hybridization to the filamentous fungi is described. The method involves the growth of microcultures in the wells of a microtiter tray using spore inocula. These microcultures are protoplasted in situ and transferred to a nylon falter through a device made by drilling out the wells of a second microtiter tray. The transferred protoplasts are confined to defined areas of the membrane, where they may be lysed and subjected to hybridization with a radioactive probe. The technique gives highly reproducible results and is of sufficient sensitivity to detect the integration of a single copy of a transforming plasmid into the genome of Aspevgillus nidulans. o 1987 Academic PESS, hc. INDEX DESCRIPTORS: Aspergillus; colony hybridization; protoplast; filamentous fungi; microculture; transformation; technique.
The development of DNA-mediated transformation techniques for genetically well-characterized tilamentous fungi such as Aspergillus nidulans (Turner and Ballance, 1985) and Neurospora crassa (Case et al., 1979) has opened the way for the rapid development of fungal molecular genetics. Progress in this field, as in yeast molecular genetics, is dependent on the successful adaptation of many of the recombinant DNA techniques developed for Escherichia coli. Among these techniques, colony hybridization (Grunstein and Hogness, 1975) is a tool that enables the identification of transformants carrying particular DNA sequences. An attempt to transfer this technique to the filamentous fungi was made using Neurospora crassa (Stohl and Lambowitz, 1983) but has not found general utility with other fungi. We have encountered a number of problems in using this technique with Aspergillus i To whom correspondence
species. The fungal colonies do not grow well on nitrocellulose filter disks, and myCelia1 debris is not easily washed off following the protoplasting step, probably because the hyphae grow through the filter into the nutrient substratum. Moreover, the lysed colonies tend to run into each other so that only a few colonies per plate may be analyzed in an unambiguous manner. In this communication we report a modified method that overcomes these problems by growing fungal microcultures in the wells of a microtiter tray. We have made use of this technique with both Aspergillus niger and A. nidulans and present here data using the A. nidulans strain FGSC237 (pabaA yA2 trpC801; Yelton et al., 1984). Spores from A. nidulans transformants were picked from selective plates at random and transferred to the 96 wells of a microtiter tray, each containing 150 ~1 of 2x YEPD-PABA medium (4% yeast extract; 2% bacto-peptone; 2% glucose; 2 mM p-aminobenzoic acid).
should be addressed. 70
0147-5975187 $3.00 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
A MICROCULTURE
BLOTTING
The tray was then incubated at 37°C for 24 h before the medium was removed by using a suction device and replaced with 200 ~1 of 1.2 M MgSO, in 0.1 M phosphate buffer at pH 5.6 (PBM). The buffer was then exchanged for 200 ~1 of a 5 mg/ml solution of Novozym 234 in PBM. The microcultures were protoplasted by incubation at 37°C for 3 h with gentle shaking. Following this incubation, the mycelia were not completely converted to protoplasts but were highly sensitive to reduced osmotic pressures. A multipipet was employed to transfer the protoplasted microcolonies into a transfer device. The latter consists of a 96well microtiter tray with a 6.4 mm hole drilled through its base at the center of each well. The transfer device was placed face down on a nylon membrane, wetted with distilled water, that was lying on top of several layers of filter paper. The whole apparatus was held together with rubber bands (Fig. 1). The transfer device provides channels along which the protoplasts can pass as they are drawn onto the nylon filter by capillary action. The protoplasts from each microculture are thus confined to a defined area of the filter and do not spread out and coalesce with material from adjoining wells. This result cannot be achieved by simply inverting the original microtiter tray onto the membrane, because the vacuum
TECHNIQUE
FOR FUNGI
71
created in the wells prevents efficient transfer of the protoplasts. The membrane was removed from the transfer device and dried briefly on blottang paper. The protoplasted microcolonies were then lysed in situ by transferring the nylon membrane to a stack of blotting paper soaked in 0.2 M NaOH, 0.6 M NaCl and leaving it for 4 min. The lysis step was repeated after briefly drying the membrane on fresh, dry blotting paper. Following lysis, a neutralization step was pe~o~med pH 7.6, an by using 1 M Tris-HCl, membrane was dried in air before bak 80°C for no more than 3 h. A prewash step, which involved an overnight incubation m 3 x SSC, 0.2% SDS at room temperature, was essential before carrying out hybridiaation in sealed polyethylene bags by ing nick-translated probes (Rigby et a!., 77) in a standard manner (Maniatis et ak., 1982). This technique gives highly reprod~c~b~$ results and is sensitive enough to detect transformation events which have integrated plasmid sequences into the Aspergilllrs genome. We have exploited this technique to detect unselected co-tra~$f~r~~~t “colonies” of A. ~idu~a~s with high efficiency. Figure 2 shows an example where A. nidulans FGSC237 has been co-transformed with pHY201 (Melton et al., 1984)
iNVERTED MICROTITRE
DRILLED TRAY
NYLON FILTER
BLOTTlNG
PAPER
FIG. 1. The apparatus used to transfer fungal protoplasts onto the nylon membrane, confining them to a defined area. The drilled microtiter tray is inverted onto the filter to provide a better seal. The arrows indicate the drill holes made in the base of the microtiter tray.
72
CROWLEY
AND OLIVER
FIG. 2. An autoradiograph of 96 microcolonies of A. nidulans which had been co-transformed with plasmids pHY201 and p3SR2 recombinant selecting for Trp+ transformants. The microcolonies were probed for the unselected glucoamylase gene using the technique described; positive co-transformants are easily detected against the background of microcolonies transformed with trpC alone.
which carried the A. nidulans trpC gene and a p3SR2 (Hynes et al., 1983) recombinant carrying the A. niger glucoamylase gene (Boel et al., 1984). Trp + transformants were selected and transferred to microtiter trays for culture, protoplasting, and transfer. The frequency of reversion of the tvpC mutation in the host strain is ca. 1 x 10e7 (Yelton et al., 1984). In the figure, the microculture blot has been probed with an EcoRl/EcoRV fragment containing the glucoamylase coding sequence. Transformed colonies were detected against a low background. This background is increased if the baking step is prolonged and can be reduced by a more stringent washing step following hybridization. However, a low level of background hybridization is quite desirable since it facilitates the accurate location of positive “colonies.” Differences in the intensities of the hybridization signals obtained from the positive colonies were found to reflect the numbers of copies of the plasmid which had been integrated into the fungal genome. In Fig. 2, transformant B2 contains ca. three copies of the glucoamylase
gene, whereas transformant H4 contains a single copy. The technique of microculture blotting described can be applied in a manner exactly analogous to colony hybridization with E. coli. In addition to the identification of transformants and co-transformants, it may be used to exclude revertant colonies from a collection of putative transformants or to detect gene replacement events by screening for the excision of vector sequences from integrants. Although we have yet to apply it in this context, it could presumably be used to detect the synthesis of proteins encoded by cloned genes by using the Broome and Gilbert (1978) procedure. ACKNOWLEDGMENTS We are grateful to M. Yelton for supplying the trpC vector and host A. nidulans strains and to E. Boel for the glucoamylase clone. P.C. is the recipient of an earmarked studentship from the Biotechnology Directorate of the SERC. REFERENCES BoEL,E.,HANsEN,M. T.,HJoRT,I.,HEGH,I.,AND FIIL, N. P. 1984. Two different types of intervening
A MICROCULTURE
BLOTTING
sequences in the glucoamylase gene from Aspergilius niger. EM30 J. 3: 1581-1585. BROOME, S., AND GILBERT, W. 1978. Immunological screening method to detect specific translation products. Proc. Natl. Acad. Sci. USA 15: 2746-2749. CASE, M. E., SCHWEIZER, M., KUSHNER, S. R., AND GILES, N. H. 1979. Efficient transformation of Neurosporn cozzssu by utilising hybrid plasmid DNA. Puoc. Natl. Acad. Sci. USA 16: 5259-5263. GRUNSTEIN, M. G., AND HOGNESS, D. S. 1975. Colony hybridisation: A method for the detection of cloned DNAs that contain a specific gene. Proc.
Natl. Acad. Sri. USA 72: 3961-3965. HYNES, 1983.
M. J., CORRICK, C. M., AND KING, J. A. Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations.
Mol. Cell. Biol. 3: 1430-1439. MAN~ATIS,
T.,
FRITSCH,
E. F., AND
SAMBROOK,
J.
TECHNIQUE
1982. Molecular
FOR
FUNGI
Cloning: A Laboratory
73
Manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. RIGBY, I? W., DIECKMANN, M., RHODES, C., a~\:1) BERG, P. 1977. Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. 1. Mol. Biol. 113: 237-251. STOHL, L. L., AND LAMBOWITZ, A. colony filter hybridisation procednre for the filamentous fungus Neurospora crassa. Anal. Bio&em. 134: 82-85. TURNER, G.. AND BALLANCE, D. J. 1985. Cloning and transformation in Aspergillus. In Gene ~a~l~pa~atiori in Fmgi (J. W. Bennett and E. L. Lasure, Eds.), pp. 259-278. Academic Press, New York1 London.
YELTON, MM.,
HAMER, J.E., AND TIMBERLAKE, of Aspergilhs nidalarzs Proc. Natl. Acad. Sci. USA 81: 1470-1474.
W. E. 1984. Transformation by using a trpC plasmid.
BRIEF NOTE A Microculture
Hybridization Technique for the Detection DNA Sequences in Filamentous Fungi PATRICK CROWLEYAND~TEPHEN
Department
of Specific
G. OLIVER~
of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 IQD, England Accepted for publication November 18, 1986
CROWLEY, P., AND OLIVER, S. G. 1987. A microculture hybridization technique for the detection of specific DNA sequences in filamentous fungi. Experimental Mycology 11, 70-73. A procedure that permits the successful application of the technique of colony hybridization to the filamentous fungi is described. The method involves the growth of microcultures in the wells of a microtiter tray using spore inocula. These microcultures are protoplasted in situ and transferred to a nylon falter through a device made by drilling out the wells of a second microtiter tray. The transferred protoplasts are confined to defined areas of the membrane, where they may be lysed and subjected to hybridization with a radioactive probe. The technique gives highly reproducible results and is of sufficient sensitivity to detect the integration of a single copy of a transforming plasmid into the genome of Aspevgillus nidulans. o 1987 Academic PESS, hc. INDEX DESCRIPTORS: Aspergillus; colony hybridization; protoplast; filamentous fungi; microculture; transformation; technique.
The development of DNA-mediated transformation techniques for genetically well-characterized tilamentous fungi such as Aspergillus nidulans (Turner and Ballance, 1985) and Neurospora crassa (Case et al., 1979) has opened the way for the rapid development of fungal molecular genetics. Progress in this field, as in yeast molecular genetics, is dependent on the successful adaptation of many of the recombinant DNA techniques developed for Escherichia coli. Among these techniques, colony hybridization (Grunstein and Hogness, 1975) is a tool that enables the identification of transformants carrying particular DNA sequences. An attempt to transfer this technique to the filamentous fungi was made using Neurospora crassa (Stohl and Lambowitz, 1983) but has not found general utility with other fungi. We have encountered a number of problems in using this technique with Aspergillus i To whom correspondence
species. The fungal colonies do not grow well on nitrocellulose filter disks, and myCelia1 debris is not easily washed off following the protoplasting step, probably because the hyphae grow through the filter into the nutrient substratum. Moreover, the lysed colonies tend to run into each other so that only a few colonies per plate may be analyzed in an unambiguous manner. In this communication we report a modified method that overcomes these problems by growing fungal microcultures in the wells of a microtiter tray. We have made use of this technique with both Aspergillus niger and A. nidulans and present here data using the A. nidulans strain FGSC237 (pabaA yA2 trpC801; Yelton et al., 1984). Spores from A. nidulans transformants were picked from selective plates at random and transferred to the 96 wells of a microtiter tray, each containing 150 ~1 of 2x YEPD-PABA medium (4% yeast extract; 2% bacto-peptone; 2% glucose; 2 mM p-aminobenzoic acid).
should be addressed. 70
0147-5975187 $3.00 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
A MICROCULTURE
BLOTTING
The tray was then incubated at 37°C for 24 h before the medium was removed by using a suction device and replaced with 200 ~1 of 1.2 M MgSO, in 0.1 M phosphate buffer at pH 5.6 (PBM). The buffer was then exchanged for 200 ~1 of a 5 mg/ml solution of Novozym 234 in PBM. The microcultures were protoplasted by incubation at 37°C for 3 h with gentle shaking. Following this incubation, the mycelia were not completely converted to protoplasts but were highly sensitive to reduced osmotic pressures. A multipipet was employed to transfer the protoplasted microcolonies into a transfer device. The latter consists of a 96well microtiter tray with a 6.4 mm hole drilled through its base at the center of each well. The transfer device was placed face down on a nylon membrane, wetted with distilled water, that was lying on top of several layers of filter paper. The whole apparatus was held together with rubber bands (Fig. 1). The transfer device provides channels along which the protoplasts can pass as they are drawn onto the nylon filter by capillary action. The protoplasts from each microculture are thus confined to a defined area of the filter and do not spread out and coalesce with material from adjoining wells. This result cannot be achieved by simply inverting the original microtiter tray onto the membrane, because the vacuum
TECHNIQUE
FOR FUNGI
71
created in the wells prevents efficient transfer of the protoplasts. The membrane was removed from the transfer device and dried briefly on blottang paper. The protoplasted microcolonies were then lysed in situ by transferring the nylon membrane to a stack of blotting paper soaked in 0.2 M NaOH, 0.6 M NaCl and leaving it for 4 min. The lysis step was repeated after briefly drying the membrane on fresh, dry blotting paper. Following lysis, a neutralization step was pe~o~med pH 7.6, an by using 1 M Tris-HCl, membrane was dried in air before bak 80°C for no more than 3 h. A prewash step, which involved an overnight incubation m 3 x SSC, 0.2% SDS at room temperature, was essential before carrying out hybridiaation in sealed polyethylene bags by ing nick-translated probes (Rigby et a!., 77) in a standard manner (Maniatis et ak., 1982). This technique gives highly reprod~c~b~$ results and is sensitive enough to detect transformation events which have integrated plasmid sequences into the Aspergilllrs genome. We have exploited this technique to detect unselected co-tra~$f~r~~~t “colonies” of A. ~idu~a~s with high efficiency. Figure 2 shows an example where A. nidulans FGSC237 has been co-transformed with pHY201 (Melton et al., 1984)
iNVERTED MICROTITRE
DRILLED TRAY
NYLON FILTER
BLOTTlNG
PAPER
FIG. 1. The apparatus used to transfer fungal protoplasts onto the nylon membrane, confining them to a defined area. The drilled microtiter tray is inverted onto the filter to provide a better seal. The arrows indicate the drill holes made in the base of the microtiter tray.
72
CROWLEY
AND OLIVER
FIG. 2. An autoradiograph of 96 microcolonies of A. nidulans which had been co-transformed with plasmids pHY201 and p3SR2 recombinant selecting for Trp+ transformants. The microcolonies were probed for the unselected glucoamylase gene using the technique described; positive co-transformants are easily detected against the background of microcolonies transformed with trpC alone.
which carried the A. nidulans trpC gene and a p3SR2 (Hynes et al., 1983) recombinant carrying the A. niger glucoamylase gene (Boel et al., 1984). Trp + transformants were selected and transferred to microtiter trays for culture, protoplasting, and transfer. The frequency of reversion of the tvpC mutation in the host strain is ca. 1 x 10e7 (Yelton et al., 1984). In the figure, the microculture blot has been probed with an EcoRl/EcoRV fragment containing the glucoamylase coding sequence. Transformed colonies were detected against a low background. This background is increased if the baking step is prolonged and can be reduced by a more stringent washing step following hybridization. However, a low level of background hybridization is quite desirable since it facilitates the accurate location of positive “colonies.” Differences in the intensities of the hybridization signals obtained from the positive colonies were found to reflect the numbers of copies of the plasmid which had been integrated into the fungal genome. In Fig. 2, transformant B2 contains ca. three copies of the glucoamylase
gene, whereas transformant H4 contains a single copy. The technique of microculture blotting described can be applied in a manner exactly analogous to colony hybridization with E. coli. In addition to the identification of transformants and co-transformants, it may be used to exclude revertant colonies from a collection of putative transformants or to detect gene replacement events by screening for the excision of vector sequences from integrants. Although we have yet to apply it in this context, it could presumably be used to detect the synthesis of proteins encoded by cloned genes by using the Broome and Gilbert (1978) procedure. ACKNOWLEDGMENTS We are grateful to M. Yelton for supplying the trpC vector and host A. nidulans strains and to E. Boel for the glucoamylase clone. P.C. is the recipient of an earmarked studentship from the Biotechnology Directorate of the SERC. REFERENCES BoEL,E.,HANsEN,M. T.,HJoRT,I.,HEGH,I.,AND FIIL, N. P. 1984. Two different types of intervening
A MICROCULTURE
BLOTTING
sequences in the glucoamylase gene from Aspergilius niger. EM30 J. 3: 1581-1585. BROOME, S., AND GILBERT, W. 1978. Immunological screening method to detect specific translation products. Proc. Natl. Acad. Sci. USA 15: 2746-2749. CASE, M. E., SCHWEIZER, M., KUSHNER, S. R., AND GILES, N. H. 1979. Efficient transformation of Neurosporn cozzssu by utilising hybrid plasmid DNA. Puoc. Natl. Acad. Sci. USA 16: 5259-5263. GRUNSTEIN, M. G., AND HOGNESS, D. S. 1975. Colony hybridisation: A method for the detection of cloned DNAs that contain a specific gene. Proc.
Natl. Acad. Sri. USA 72: 3961-3965. HYNES, 1983.
M. J., CORRICK, C. M., AND KING, J. A. Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations.
Mol. Cell. Biol. 3: 1430-1439. MAN~ATIS,
T.,
FRITSCH,
E. F., AND
SAMBROOK,
J.
TECHNIQUE
1982. Molecular
FOR
FUNGI
Cloning: A Laboratory
73
Manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. RIGBY, I? W., DIECKMANN, M., RHODES, C., a~\:1) BERG, P. 1977. Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. 1. Mol. Biol. 113: 237-251. STOHL, L. L., AND LAMBOWITZ, A. colony filter hybridisation procednre for the filamentous fungus Neurospora crassa. Anal. Bio&em. 134: 82-85. TURNER, G.. AND BALLANCE, D. J. 1985. Cloning and transformation in Aspergillus. In Gene ~a~l~pa~atiori in Fmgi (J. W. Bennett and E. L. Lasure, Eds.), pp. 259-278. Academic Press, New York1 London.
YELTON, MM.,
HAMER, J.E., AND TIMBERLAKE, of Aspergilhs nidalarzs Proc. Natl. Acad. Sci. USA 81: 1470-1474.
W. E. 1984. Transformation by using a trpC plasmid.