Proc. Natl. Acad. Sci. USAVol. 81, pp. 1470-1474, March 1984Genetics

Transformation of Aspergillus nidulans by using a trpC plasmid(hybrid plasmid/gene transfer/chromosome integration)

M. MELANIE YELTON, JOHN E. HAMER, AND WILLIAM E. TIMBERLAKE*Department of Plant Pathology, University of California, Davis, CA 95616

Communicated by Charles Yanofsky, November 10, 1983

ABSTRACT We constructed a chimeric plasmid carryinga complete copy of the trifunctional trpC gene from the Asco-mycete fungus Aspergillus nidulans. This plasmid, designatedpHY201, replicates in Escherichia coli, where it confers resis-tance to ampicillin and chloramphenicol and complementsthpC mutants lacking phosphoribosylanthranilate isomeraseactivity. We used pHY201 to transform an A. nidulans trpC-strain to trpC' at frequencies of >20 stable transformants per,ug of DNA. Southern blot analysis of DNA from transfor-mants showed that pHY201 DNA had integrated into the A.nidulans chromosomes in a majority of cases. Most of the inte-gration events appeared to occur at the site of the tipC- alleleof the recipient strain. In several instances, we succeeded inrecovering pHY201, or derivatives thereof, from A. nidulanstransformants by restriction endonuclease digestion of chro-mosomal DNA, ligation, and transformation of E. coli.

The Ascomycete fungus Aspergillus nidulans has been usedextensively for the study of eukaryotic gene structure, orga-nization, and regulation (1-4) because features of its life cy-cle make it particularly amenable to biochemical and geneticanalysis. A. nidulans has been especially valuable for inves-tigating the genetic and molecular processes controlling fun-gal cell differentiation (5-9). In contrast to the situation withits close relatives Saccharomyces cerevisiae (yeast) andNeurospora crassa, however, investigations of A. nidulanshave not been facilitated by the availability of a tractableDNA-mediated transformation system.We recently isolated the trifunctional trpC gene from A.

nidulans (10) for use as a selective marker in transformationexperiments with this organism. Here we report construc-tion of a chimeric plasmid, designated pHY201, consisting ofa complete wild-type copy of the Aspergillus trpC gene in-serted into the unique Sal I site of pBR329 (11). We find thatpHY201 DNA, either in circular or linear form, transforms atrpC- A. nidulans strain to trpC+ at frequencies of >20 sta-ble transformants per pg of DNA. The transforming DNAbecomes integrated into the host genome, frequently at thesite of the resident gene. In several instances, we were ableto recover pHY201 in native or altered form from Aspergil-lus transformants by restriction endonuclease digestion ofchromosomal DNA, ligation, and transformation of Esche-richia coli to ampicillin resistance. Thus, pHY201 has prop-erties that suggest that it may serve as a valuable prototypefor the development of more sophisticated Aspergillus clon-ing vectors.

MATERIALS AND METHODSMaterials. MgSO4, /3glucuronidase (Type H2S), bovine

serum albumin (essentially fatty acid free), D-sorbitol, andTrizma were purchased from Sigma; CaC12, from Mallinck-rodt; polyethylene glycol 4000 and NaDodSO4 (product code44215), from BDH; Mira-Cloth, from Calbiochem; Gene-

Screen membrane, from New England Nuclear; [a-32P]de-oxynucleotide triphosphates (400 Ci/mmol), from Amer-sham; and restriction enzymes, T4 DNA ligase, and E. coliDNA polymerase I, from Bethesda Research Laboratories.Novozyme 234 was a gift from Novo Industries.E. coli Strains. E. coli K-12 strain MC1066 (Alac-

(IPOZYA)X74, galU, galK, strAR, hsdR-, trpC9830, leuB6,pyrF74::TnS (KmR); ref. 12) was used for selection of theAspergillus trpC gene by complementation. E. coli HB101(13) was used for routine propagation of plasmids, andDP50:SupF (14) was used for propagation of X phages.

Aspergillus Strains. A. nidulans strain FGSC237 (pabaAlyA2; trpC801) was used as the recipient in transformationexperiments. The source of the wild-type copy of the A. ni-dulans trpC gene was strain FGSC4 (Glasgow wild type).

Plasmids and Phages. Aspergillus trpC clones were initiallyselected by lytic complementation (15) of E. coli MC1066 byusing a recombinant phage library formed between A. nidu-lans nuclear DNA and X Charon 4 (9, 10). We determinedthat a 4.1-kilobase Xho I restriction fragment present in aphage designated XAn trpC12 contained the entire trpC geneplus about 0.4 kilobase of 3' and 5' flanking sequences (un-published results). This fragment was recloned into theunique Sal I site of pBR329 (11) to form pHY201 (Fig. 1).

Isolation and Labeling of DNA. Phage and plasmid DNAswere isolated by using standard procedures (16). DNA waslabeled by nick-translation (17, 18) to a specific radioactivityof 1-2 x 108 cpm/,g. Aspergillus DNA was prepared by us-ing a modification of the rapid isolation procedure developedfor yeast by Davis et al. (19). Flasks containing 50 ml of min-imal nitrate medium (20) were inoculated with 5 x 107 conid-ia and grown at 37°C with agitation for 48-72 hr. The myceli-um was harvested by filtration through Mira-Cloth, washedwith cold deionized H20, and frozen and powdered in liquidN2. The cells were rapidly suspended in 5 ml of 50 mM sodi-um EDTA, pH 8.5/0.2% NaDodSO4 containing 5 ,ul of dieth-yl oxydiformate and shaken for 1 min at room temperature.The lysate was heated to 68°C for 15 min, cooled to roomtemperature, and centrifuged for 15 min at 12,000 x g; 4 mlof the supernatant was transferred to a new centrifuge tube.The tube was placed on ice, 0.25 ml of 8.0 M potassium ace-tate (pH 4.2) was added, and the solutions were mixed thor-oughly. After incubation on ice for 1 hr, the tube was centri-fuged at 25,000 x g at 4°C for 15 min, 3 ml of the supernatantwas transferred to a fresh tube, and nucleic acids were pre-cipitated at room temperature by the addition of 3 ml of 2-propanol. The precipitate was collected by centrifugationand dissolved in 0.6 ml of TER buffer [10 mM TrisHCl, pH7.6/1 mM Na2EDTA/10 ,ug of RNase A per ml (previouslyheated to 90°C for 10 min)] at room temperature. The solu-tion was immediately transferred to a 1.5-ml microcentrifugetube, and the DNA was precipitated at room temperature bythe addition of 0.6 ml of 2-propanol and collected by centrif-ugation for 1 min. The pellet was finally dissolved in 50 ,ud ofTER buffer. We used 5 ,ul of the DNA solution (-1 pgg of

Abbreviation: ccc DNA, covalently closed circular DNA.*To whom reprint requests should be addressed.

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Sol I (XhoI)T 8 .2 Kilobases Sol I (XhoI)

Born Hi \TCR -cR

Hind \ CmR

EcoRI AR

PVU I

FIG. 1. Restriction map of plasmid pHY201. The plasmid was

constructed by ligating a 4.1-kilobase Xho I fragment containing a

complete wild-type copy of the A. nidulans trpC gene to Sal I-digest-ed pBR329 DNA, thereby interrupting the tetracycline-resistance(TcR) gene. The plasmid confers resistance to ampicillin (ApR) andchloramphenicol (CmR) to E. coli.

DNA) for restriction endonuclease digestions and gel analy-sis.

Gel Blotting. Partially depurinated DNA was transferredto Gene-Screen membrane using the procedures recom-

mended by the manufacturer.Preparation of Protoplasts. Aspergillus protoplasts were

prepared by using a modification of the procedures of Pe-berdy and Isaac (21) and Tilburn et al. (22). Siliconized 1-liter flasks containing 400 ml of minimal medium plus 4 mMfilter-sterilized L-tryptophan were inoculated with 8 x 108conidia and shaken at room temperature for 18 hr. The my-

celium was harvested by filtration through Mira-Cloth,washed with 0.6 M MgSO4, and squeezed and blotted withpaper towels to remove excess liquid. The cells were sus-

pended in filter-sterilized osmotic medium (1.2 M MgSO4/10mM sodium phosphate, pH 5.8; 5 ml/g of mycelium) by vig-orous mixing, transferred to a 250-ml flask, and placed on

ice. Filter-sterilized solutions ofp-glucuronidase (0.2 ml/g ofmycelium) and Novozyme 234 (20 mg/ml in osmotic medi-um; 1 ml/g of mycelium) were added, and the cells were in-cubated on ice for 5 min. A filter-sterilized solution of bovineserum albumin (12 mg/ml in osmotic medium; 0.5 ml/g ofmycelium) was added, and the cell suspension was shaken at80 rpm at 300C for 90 min. The suspension was transferred toa centrifuge tube, overlayed with 10 ml of ST buffer (0.6 Msorbitol/100 mM Tris-HCl, pH 7.0), and centrifuged in aswinging bucket rotor at 4,000 x g at 40C for 15 min. Proto-plasts at the buffer interface were removed by using a bentPasteur pipet and placed on ice. The remaining ST bufferwas removed, the mycelial pellet was resuspended, fresh STbuffer was added as before, and protoplasts were againbanded by centrifugation. The protoplasts were pooled, di-luted with 2 volumes of STC buffer (1.2 M sorbitol/10 mMTris HCl, pH 7.5/10 mM CaCl2), and pelleted by centrifuga-tion at 8,000 x g at 40C for 5 min. They then were washedtwice by centrifugation with 10 ml of STC buffer and resus-

pended in 1/1000th of the original culture volume of STCbuffer. Each 400-ml culture yielded 5-10 x 107 protoplasts,20-30% of which were capable of regeneration when plateddirectly onto regeneration medium.

Transformation. The transformation procedure used was a

modification of that of Hinnen et al. (23). DNA, dissolved in25,1 of STC buffer, was mixed with 100 Al of protoplasts ina disposable plastic centrifuge tube and incubated at room

temperature for 25 min. Then 0.2 ml of 60% polyethyleneglycol 4000/10 mM Tris HCl, pH 7.5/10 mM CaCl2 was add-ed, and the tube was agitated gently by hand. This was fol-lowed by a second addition of 0.2 ml and a third addition of0.85 ml of the polyethylene glycol solution, with gentle mix-ing after each addition. The protoplasts were incubated for20 min at room temperature and pelleted at 8,000 x g at 40Cfor 5 min. The supernatant was decanted, and droplets ofsolution adhering to the tube were removed with a cottonswab. The protoplasts were suspended in 2.5 ml of 0.5%yeast extract/2.0% D-glucose/1.2 M sorbitol and incubatedat 370C on a rotary shaker at 150 rpm for 2 hr. They werethen pelleted at 8,000 x g at 40C for 5 min, the supernatantwas decanted, and droplets of medium adhering to the tubewere removed with a cotton swab. The protoplasts were fi-nally suspended in 0.15 ml of STC buffer, diluted appropri-ately in the same buffer, spread onto medium containing 1.2M sorbitol and 1.5% agar, and incubated at 370C. The viabili-ty of the protoplasts following these treatments was 1-7%.Plating the protoplasts in agar overlays had no effect on re-generation frequency.

RESULTSCharacteristics of pHY201. pHY201 is 8.2 kilobases in

length and contains intact genes for chloramphenicol andampicillin resistance, derived from pBR329 (11), and a com-plete wild-type copy of the A. nidulans trpC gene, coding fora trifunctional polypeptide having glutamine amidotransfer-ase (GAT), indoleglycerolphosphate synthase (IGPS), andphosphoribosylanthranilate isomerase (PRAI) activities (24)(see Fig. 1). We found that pHY201 is capable of transform-ing E. coli MC1066 (PRAI-) to tryptophan prototrophy andchloramphenicol and ampicillin resistance at high frequency.

Transformation of A. nidulans. We used A. nidulans FGSC-237 as the recipient for transformation experiments. Thisstrain has a spontaneous reversion rate to trpC+ of 1 x 10-7.Approximately 2 x 106 regenerable protoplasts were mixedwith various amounts of pHY201 DNA and treated with Ca2+and polyethylene glycol as described. In these experiments,the total amount of DNA added per transformation was heldconstant (10 ,g) by the addition of carrier pBR329 DNA.Treated protoplasts were spread onto regeneration mediumlacking L-tryptophan and incubated at 37°C. In those caseswhere pHY201 DNA was added, small colonies could beseen with the aid of a dissecting microscope after 12 hr ofincubation, and conidiating colonies were evident after 36hr. No colonies were obtained from protoplasts that hadbeen treated with pBR329 DNA alone. We noted that themajority of the colonies visible after 12 hr of incubation didnot give rise to stable conidiating colonies. The reasons forthis abortive growth pattern are presently unknown.

Fig. 2 shows the relationship between the amount of cova-lently closed circular (ccc) pHY201 DNA added per transfor-mation and the number of stable trpC+ colonies obtained.The number of putative transformants increased with in-creasing amounts of pHY201 DNA added. Transformationfrequencies varied from .300 transformants/,ug when O0.05,ug of DNA was added to "20 transformants/,g when 10 ,ugof DNA was added. The frequencies of transformation withrespect to viable protoplasts ranged from 1.6 x 10-6 trans-formants per protoplast when 0.01,ug of DNA was added to1.7 x 10-4 transformants per protoplast when 10,g of DNAwas added.

In a separate experiment, we used pHY201 DNA that hadbeen linearized by digestion with Sst I, which cuts the plas-mid once within the trpC protein coding region (Fig. 1). Thetransformation frequencies obtained were similar to thoseobserved with ccc DNA. Ten putative transformants ob-tained with ccc DNA and five putative transformants ob-

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1472Genetics:Yeltonetal.~~~Proc.NatL. AcadJ ScL USA 81 (1984)

U,

0E0

0

0

(IV

1501

00o

501~

2 4 6 8pHY2OI DNA Added, ,.xg

10

FIG. 2. Transformation of an A. nidulans trpC- strain to trpC'.Approximately 2 x 106 regenerable protoplasts from A. nidulansstrain FGSC237 were treated with various amounts of pHY2O1DNA, diluted appropriately, and spread onto regeneration mediumlacking L-tryptophan as described. The total amount of DNA addedper transformation (10 u±g) was held constant by addition of carrierpBR329 DNA. Conidiating colonies were counted after 2 days ofincubation at 370C.

tamned with linear DNA were selected for further analysisand purified by two cycles of single-spore isolation underselective conditions.

Hybridization Analysis. The 15 selected transformantswere grwn in medium lacking L-tryptophan, and total DNAwas isolated and subjected to hybridization analysis. We hy-bridized gel blots of undigested, Xho I-digested, and Sst I-digested DNA with either radiolabeled pBR329 or XAntrpCl2 DNA. Representative results from these experimentsare shown in Fig. 3. As expected, DNA from the recipientstrain contained no sequences complementary to pBR329.The XAn trpC12 probe hybridized with the 4.1-kilobase XhoI fragment containing the trpC gene and with two adjacentXho I fragments, 6.3 and 7.9 kilobases in length (see Fig. 4).Three transformants, represented by HY201-3 in Fig. 3,yielded hybridization patterns identical to the recipientstrain. These presumably arose as the result of either doublecrossover events at the site of the resident gene or singlecrossover events followed by precise excision of the trpC-allele and plasmid -DNA sequences (type III integrationevents by the classification system of Hinnen et al.; see ref.23 and footnotes to Table 1), although it is also possible thatthese strains arose by reversion. However, we did not ob-serve revertants in control transformations where pBR329DNA was substituted for pHY2O1 DNA.

The remainder of the transformants we examined con-tained DNA sequences complementary to pBR329. With un-digested DNA, hybridization with pBR329 occurred at theposition of chromosomal DNA in all cases (Fig. 3), indicat-ing that the pBR329 sequences had been integrated into thehost genome. We did not observe hybridization to lower mo-lecular weight DNA fragments, even when autoradiogramswere exposed for extended periods of time, suggesting thatunintegrated copies of pHY2O1 were not present in the trans-formants. Further analysis of these transformants showedthat they arose by integration either at the site of the residenttrpC gene or elsewhere in the genome.

Fig. 4 shows that integration of pHY2O1 DNA into thehost genome by a single homologous recombination event(type I integration event by the classification system of Hin-nen et al.; ref. 23) would result in the conversion of the 4.1-kilobase Xho I fragment containing the trpC gene to an Xho Ifragment 12.3 kilobases in length. Three of the transformantswe examined, represented by HY201-9 in Fig. 3, were of thistype. Xho I-digested DNA from HY201-9 had the expected6.3- and 7.9-kilobase trpC flanking sequences but lacked the4.1-kilobase sequence, which was replaced by a fragment ofabout the predicted length; Only the largest Xho I fragmenthybridized with pBR329 DNA. Sst I digestion of HY201-9DNA yielded a single 8.2-kilobase fragment that hybridizedwith pBR329 DNA (Fig. 3) and an 8.2-kilobase fragment andseveral other fragments that hybridized with XAn trpCl2DNA (data not shown). Because pHY2O1 contains a singleSst I site in the trpC coding region, these results are consis-tent with integration of one copy of the plasmid into the hostby a single crossover event at the site of the resident trpCgene (Fig. 4).

Several other transformants, represented by HY201-10and HY201-6 in Fig. 3, are similar to transformant HY201-9in that the 4.1-kilobase Xho I fragment was replaced by amuch larger Xho I fragment that hybridized with pBR329 andwith XAn trpCl2 DNA. However, with these transformants,the novel Xho I fragments were larger than predicted by inte-gration of a single copy of pHY2O1. That multiple copies ofthe plasmid were present in the genome was confirmed bySst I digestion, which yielded 8.2-kilobase fragments that hy-bridized with pBR329 DNA, as well as larger fragments.These results are consistent with the presence of tandem in-tegrated plasmid copies, some of which have been modifiedby rearrangements or insertions. They are also consistentwith integration of one or more copies of the plasmid at thesite of the resident trpC gene and integration of one or morecopies elsewhere in the genome.The final class of transformants we obtained is represent-

ed by HY201-5 in Fig. 3. Xho I digestion of HY201-5 DNAproduced the 4.1-, 6.3-, and 7.9-kilobase fragments present

1 2 34 1 2 34 1 2 34 1 2 34 1 2 34 1 2 34FIG. 3. Hybridization analysis of

* ~~~~~~~~~selectedtrpC' transformants. DNAwas isolated from transformants as

23.1 - Now- W described and fractionated by electro-9.4- poei n1 grs esete e

go ~ _ hrssi % grs esete e6.6-two ~~~~~~~~~~~~~~~~~~~~~forerestriction endonuclease digestion4.4 (lanes 1) or after digestion with Xho I- ~~~~~~~~~~~~~~~~~~~~~~(lanes2 and 3) or Sst I (lanes 4). The

gels were blotted, and the blots were

2 3- hybridized with either radiolabeled2.0- PBR329 (lanes 1, 3, and 4) or XAn

trpCl2 DNA (lane 2), as indicated,washed at 680C in a buffer containing0.04 M Na', and autoradiographed.

0.6- The sizes of molecular weight stan-dards (HindIII fragments of bac-teriophage XCI857) are given in kilo-

FGSC 237 HY201-3 HY201-9 HY201-10 HY201 6 HY201-5 bases.

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XAn trpC12 Probe

Sal I(Xho

-p8R329 DNA

Sst I SOt IXho I Xho I 0SaIIMho) SalM(Xo)l Xho I Xho I

1 8.2 kiloboses -* 12.3 kiloboses - i

pBR 329Probe

FIG. 4. Integration of pHY201 DNA by homologous recombina-tion. The predicted structure of the trpC region after a single ho-mologous recombination event with pHY201 DNA is shown. TheDNA probes used for the hybridization analysis shown in Fig. 3 areindicated.

in the recipient strain plus a larger fragment that hybridizedwith both pBR329 and XAn trpC12 DNA. Sst I digestion ofHY201-5 DNA yielded three fragments, approximately 7.3,8.2, and 9.0 kilobases in length, that hybridized with pBR329(Fig. 3) and with XAn trpC12 (data not shown). Thus, thistransformant contained multiple copies of the plasmid, someof which may have been modified by rearrangements or in-sertions. These data indicate that integration of pHY201DNA into the host genome occurred at a site other than theresident trpC gene (type II integration event by the classifi-cation system of Hinnen et al.; ref. 23). They also could beexplained by homologous recombination in a recipient straincontaining a duplicated trpC region. Arguing against the sec-ond possibility, however, is the observation that the 4.1-,6.3-, and 7.9-kilobase Xho I fragments shown in Fig. 3 werepresent in approximately a 1:1:1 molar ratio, as determinedby densitometry of the autoradiogram. The tentative classifi-cations of the 15 transformants we subjected to hybridizationanalysis are summarized in Table 1.

Stability of trpC' Transformants. The transformants usedfor the hybridization analysis shown in Fig. 3 were tested formitotic stability by growing colonies from single conidia onmedium containing L-tryptophan and then replica-plating atleast 50 isolates onto selective and nonselective media. NotrpC- segregants were observed with any of the transfor-mants. The same transformants were tested for meiotic sta-bility by allowing them to self (A. nidulans is homothallic)under nonselective conditions. At least 50 colonies derivedfrom single ascospores were then replica-plated onto selec-tive and nonselective media. No trpC- segregants were ob-

Table 1. Classification of transformants

Transformants, no.

Integration event* ccc DNAt Linear DNAtType I 5 4Type II 3 0Type III 2 1

*Classification system of Hinnen et al. (23). The integration eventsare as follows: type I, integration of plasmid at the site of the resi-dent trpC gene; type II, integration of plasmid at a site other thanthe resident trpC gene; type III, integration of Aspergillus DNAsequences only at the site of the resident trpC gene.

tRefers to the form of DNA used to obtain the transformants.

tained with transformant HY201-3. With the other transfor-mants, trpC- segregants arose at frequencies ranging from2% (HY201-6) to 12% (HY201-10). We have not subjectedthe trpC- segregants to hybridization analysis.Recovery of Plasmids from Aspergillus Transformants. The

results shown in Fig. 3 predicted that it would be possible torecover pHY201 from some of the Aspergillus transformantsby back transformation of E. coli. To test this possibility,DNA from transformants HY201-9 and HY201-10 was di-gested with Sst I, ligated, and used to transform E. coliMC1066 to ampicillin resistance. In both cases, we obtainedampicillin-resistant colonies. All of the clones we testedwere also trpC'. Plasmid DNA was prepared from a numberof these colonies and analyzed by restriction endonucleasedigestion and agarose gel electrophoresis. Fig. 5 shows theresults from some of these experiments. Several of the plas-mids we recovered appeared to be identical to pHY201(pHY201/9-1, 9-2, and 10-3), whereas several others(pHY201/10-1, 10-2, and 10-4) did not. The latter plasmidsdiffered from pHY201 in that the 2.4-kilobase EcoRI frag-ment, derived from the trpC coding region (Fig. 1), was nolonger present, being replaced by a larger EcoRI fragmentthat lacked Sal I sites. The mechanisms giving rise to thesemodifications have not been investigated. It should be notedthat these alterations were not expected to influence com-plementation of the trpC9830 mutation of E. coli MC1066because we previously have shown that the 1.8-kilobaseEcoRI-Xho I fragment of pHY201 (Fig. 1) is sufficient forcomplementation of this strain (10).

DISCUSSIONWe isolated the trpC gene from a wild-type strain of A. nidu-lans and inserted it into the bacterial plasmid pBR329 toform plasmid pHY201. Our results demonstrate that pHY201

+ 4. + 4. 4. 4. .L Lii L ai W1 W W LiiJ L ii W La

23.1 -

94--

6.6 -

4.4 -

2.3 -2.0-

L I.,, L.... j I

pHY 201 9-I 9-2 10-1 10-2 10-3 10-4

FIG. 5. Analysis of plasmids recovered from Aspergillus trans-formants. DNA from transformants HY201-9 and HY201-10 (seeFig. 3) was digested to completion with Sst I, ligated, and used totransform E. coli MC1066 to ampicillin resistance. Selected bacterialclones were then grown, and plasmid DNA was prepared by a rapidisolation procedure (19). The DNA was digested with either EcoRI(E) or EcoRI/Sal I (E+S) as indicated and fractionated by electro-phoresis in a 1% agarose gel. Identical digests of pHY201 DNA areshown for comparison. One Sal I-EcoRI fragment comigrated withthe smallest EcoRI fragment in plasmids pHY201, 9-1, 9-2, and 10-3,while the expected Sal I-Sal I fragment shown in Fig. 1 migrated toa position in the gel not shown in this figure. The sizes of molecularweight standards (HindIII fragments of XCI857) are given in kilo-bases.

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DNA, either in circular or linear form, can be used to trans-form a trpC- strain of Aspergillus to trpC' at efficienciessimilar to those observed for integrative transformation ofyeast (23) and Neurospora crassa (25, 26). The transforma-tion procedure we used is simple, can be completed in about6 hr, and yields conidiating colonies that can be transferredin about 2 days. We also adapted a rapid DNA isolation pro-cedure developed for yeast (19) for use with Aspergillus andshowed that DNA from some transformants can be used torecover pHY201 by transformation ofE. coli. Thus, it is nowpossible to transform A. nidulans and to characterize DNAfrom the transformants in less than 2 weeks.The types of integration events we observed in the 15

transformants investigated by hybridization analysis areanalogous to those originally described for yeast by Hinnenet al. (23) (Table 1). The majority (12 of 15) appeared to oc-cur at the site of the resident trpC gene in the recipientstrain. Of these, most (9 of 12) involved integration of bothAspergillus and pBR329 sequences. The remainder did notinvolve integration of pBR329 sequences and presumablywere the result of either double crossover events at the siteof the resident gene or single crossover events followed byintrachromosome recombination, eliminating the trpC- al-lele and pBR329 DNA sequences. However, we cannot for-mally exclude the possibility that these trpC+ strains wererevertants. These results are in contrast to those obtainedwith the closely related Euascomycete N. crassa, whereCase et al. (25) found that only 3 of 12 transformants ob-tained with a qa-2/pBR322 plasmid had pBR322 sequencesintegrated into the host genome.We found that representative isolates from each of the

transformation classes were mitotically stable at the level ofsensitivity of our experiments. This was true even for thosetransformants containing multiple tandem copies of trpC andpBR329 DNA. By contrast, all of the type I and type II trans-formants we tested showed some meiotic instability whenselfed. Such instability may be the result of unequal cross-ing-over in the region of the inserted DNA, giving rise todeletions and further amplifications of the inserted DNA.During the course of this work, Ballance et al. (27) report-

ed transformation of a pyrG- A. nidulans strain by using aplasmid carrying the pyr-4 gene of N. crassa. Tilburn et al.(22) also have succeeded in transforming an amdS deletionmutant by using a recombinant plasmid carrying a wild-typecopy of the amdS gene (28). In both cases, the transformingDNA was found to be integrated into the host genome.Transformation with these plasmids occurred at low fre-quencies (typically -1 transformant/,ug of DNA). We re-cently succeeded in transforming an argB- strain of A. nidu-lans with a plasmid containing an argB+ allele (29) by usingthe procedures described in this paper (unpublished results).Therefore, it appears likely that many genes besides trpCmay be suitable for the construction of integrating plasmidsfor this organism. The result with the N. crassa pyr4 gene(27) raises the possibility that such plasmids may be capableof transforming other Aspergilli and perhaps more distantlyrelated Euascomycetes, including industrially, medically,and agriculturally important species.

We are grateful to Jill Tilburn and Claudio Scazzocchio for mak-ing results available to us before their publication. We thank Dr.

Bob Goldberg for many helpful suggestions; Drs. Mike Holland andStan Artz for critical reviews of the manuscript; Novo Industries fortheir gift of Novozyme 234; and Ms. Valinda Stagner and PeggyArndt for their assistance in preparing the manuscript. This researchwas supported by grants from the National Institutes of Health (GM30349), U.S. Department of Agriculture (82-CRCR-1-1145), and Na-tional Science Foundation (PCM-8020054).

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3. Marzluf, G. A. (1977) in Regulatory Biology, eds. Copeland,J. C. & Marzluf, G. A. (Ohio State Univ. Press, Columbus,OH), pp. 196-242.

4. Cove, D. J. (1977) in Genetics and Physiology ofAspergillus,eds. Smith, J. E. & Pateman, J. A. (Academic, New York),pp. 81-95.

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