EXPERIMENTAL MYCOLOGY 11, 70-73 (1987)

BRIEF NOTE

A Microculture Hybridization Technique for the Detection of Specific DNA Sequences in Filamentous Fungi

PATRICK CROWLEYAND~TEPHEN G. OLIVER~

Department 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 detec- tion 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; micro- culture; transformation; technique.

The development of DNA-mediated transformation techniques for genetically well-characterized tilamentous fungi such as Aspergillus nidulans (Turner and Bal- lance, 1985) and Neurospora crassa (Case et al., 1979) has opened the way for the rapid development of fungal molecular ge- netics. Progress in this field, as in yeast molecular genetics, is dependent on the successful adaptation of many of the re- combinant DNA techniques developed for Escherichia coli. Among these techniques, colony hybridization (Grunstein and Hog- ness, 1975) is a tool that enables the identi- fication of transformants carrying partic- ular 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 should be addressed.

species. The fungal colonies do not grow well on nitrocellulose filter disks, and my- Celia1 debris is not easily washed off fol- lowing the protoplasting step, probably be- cause 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. ni- dulans transformants were picked from se- lective plates at random and transferred to the 96 wells of a microtiter tray, each con- taining 150 ~1 of 2x YEPD-PABA me- dium (4% yeast extract; 2% bacto-peptone; 2% glucose; 2 mM p-aminobenzoic acid).

70 0147-5975187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

A MICROCULTURE BLOTTING TECHNIQUE FOR FUNGI 71

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 ex- changed 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 in- cubation, 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 mi- crotiter 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 cap- illary action. The protoplasts from each mi- croculture 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

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 by using 1 M Tris-HCl, pH 7.6, an 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 hybridiaa- tion 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 inte- grated plasmid sequences into the Asper- gilllrs genome. We have exploited this tech- nique to detect unselected co-tra~$f~r~~~t “colonies” of A. ~idu~a~s with high effi- ciency. Figure 2 shows an example where A. nidulans FGSC237 has been co-trans- formed with pHY201 (Melton et al., 1984)

iNVERTED DRILLED MICROTITRE 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) recombi- nant carrying the A. niger glucoamylase gene (Boel et al., 1984). Trp + transfor- mants were selected and transferred to mi- crotiter 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. Trans- formed colonies were detected against a low background. This background is in- creased if the baking step is prolonged and can be reduced by a more stringent washing step following hybridization. However, a low level of background hy- bridization is quite desirable since it facili- tates the accurate location of positive “col- onies.” 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 con- tains 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 ex- actly analogous to colony hybridization with E. coli. In addition to the identifica- tion of transformants and co-transfor- mants, it may be used to exclude revertant colonies from a collection of putative transformants or to detect gene replace- ment events by screening for the excision of vector sequences from integrants. Al- though we have yet to apply it in this con- text, it could presumably be used to detect the synthesis of proteins encoded by cloned genes by using the Broome and Gil- bert (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 ear- marked studentship from the Biotechnology Direc- torate 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 TECHNIQUE FOR FUNGI 73

sequences in the glucoamylase gene from Asper- gilius 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 Neu- rosporn 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, M. J., CORRICK, C. M., AND KING, J. A. 1983. 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.

1982. Molecular Cloning: A Laboratory 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 fila- mentous 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~a- tiori 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, W. E. 1984. Transformation of Aspergilhs nidalarzs by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81: 1470-1474.