Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements

Gene, 109 (1991) 99-105 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/91/$03.50

99

GENE 06214

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae A R S elements (Ascomycete fungi; bacterial kanamycin-resistance gene; electrophoretical karyotype; G418; protoplasts; recombinant DNA)

Martin C. Wright* and Peter Philippsen Institut j~r Mikrobiologie und Molekularbiologie, Justus-Liebig-Universitiit Giessen, 6300 Giessen (F.R.G.) Received by T.A. Bickle: 22 December 1990 Accepted: 17 September 1991 Received at publishers: 30 September 1991

SUMMARY

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coil kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2#ARS). Transformation frequencies of up to 63 transformants per #g ofplasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasrnid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiaeARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.

Ashbya gossypii is a filamentous Hemiascomycete belonging to the family Spermophtoraceae (do CarmoSousa, 1970). This fungus occurs naturally as a pathogen of cotton (Phaff and Starmer, 1987). A. gossypii is a producer of riboflavin (Demain, 1972). It has been used for biotechnological production of riboflavin (Bigelis, 1985) and for the analysis of riboflavin biosynthesis (Bacher et al.,

1983; Keller etal., 1988). So far, this fungus has been genetically uncharacterized. We are interested in the molecular biology of riboflavin synthesis. As a prerequisite for the investigation of gene function in A. gossypii, we wanted to develop a transformation system. In ret~ent years, transformation systems have been reported for several filamentous ascomycetes and related fungi impeffecti, including such weU-known species as Aspergillus n~dulans and Peniclllium chrysogenum

Correspondenceto: Dr. P. Philippsen, at his present address: Institute of Applied Microbiology, Biozentrum, University of Ba,~;el, Klingelbergstrasse 70, CH-4056 Basel (Switzerland) Tel. 41612672140; Fax416126721 18. * Present address: Basotherm GmbH, Eichendorffweg5, 7950 Biberach (F.R.G.) Tel. (49-7351)20280.

(G418); kan, Kin-resistance gene of E. coli transposon Tn903; kb, kilobase(s) or 1000bp; Km, kanamycin; Mb, 10s bp; nt, nucleotide(s); OFAGE, orthogon~.!field alternation gel electrophoresis; PEG, polyethylene glycol; PTC, (SD, SMTCI, SPEZ, ST, STC) see legend Table I; a resistance/resistant; TBE, Tris-borate-EDTA buffer; TE, IrisEDTA buffer (see Table I, footnote a).

INTRODUCTION

Abbreviations: A., Ashbya; ARS, autonomously replicating sequence; Ap, ampiciilin; bp, base pair(s); EtdBr, ethidium bromide; Gt, Geneticin

100 (Tilbur4 et al., 1983; Kolar et al., 1988; Fincham, 1989). Transfo;'mation in filamentous ascomycetes generally occurs via integration ofplasmid DNA into the host genome (Timb,~lake aad Marshall, 1989). Transformation based on a~:~.ovomously replicating plasmids like in the yeast S. cerevisiae seems to be absent or at least very rare in filamentous ascomycetes (Ballance, 1986). In this paper we report the transformation of A. gossypii to Gt resistance with S. cerevisiae/E, coil shuttle vectors containing ARS of S. cerevisiae (Newlon, 1988). The results surprisingly showed that such vectors replicate freely in

A. gossypii. A

RESULTS AND DISCUSSION

(a) Transformation of Ashbya gossypii to Gta Jiminez and Davies (1980) have shown that the bacterial kan gene of Tn903 can be expressed in S. cerevisiae and thereby confers resistance to Gt, also called G418, a Km-re!ated aminoglycoside toxic for eukaryotes. The heterologous expression of the bacterial kan gene has now been successfull~ employed for transformation of several fungal organisms (Fincham, 1989). We adapted this selection system for A. gossypii, which was inhibited at >_0.03 mg Gt/ml in liquid medium and >-0.15 mg Gt/ml in

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15.25, Pst I

o \\\ (;41e" ~ .

~V" ~.ARS

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-"- .....

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PtG~lh .......

C

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a

I

o.,,J.Z""' "Xho I

Sail

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b

~ Sail,5.2 URA3/J/~PatI, 6.15

14.5,Sail ~ 2p.ARSj~t 13.9,Sail " ~ ARSl ~t1"lJY" 12.8, EcOR I / ~ ~ (Pvu ll), 7.6 12.45, HindlIT /~T-""'~ \ 11.6,ECORI / (PvuD),9.3

B

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XhoI Fig. I. Plasmids used for transformation erA. gossypii. All plasmids contain the/can gene (G418 a) of the E. coil transposon Tng03 (Oka et al., 1981). The pBR322 sequences of all plasmids include the Ap g gene and the or/(sequence) for selection and replication in E. coli (Bolivar et al., 1977). Plasmids pAG-I and pAG-2 (A and B) contain the URA3gene orS. cerevisiae(S.c.) and pAG-3 (C) the S.c. TRPi gene. The presence of these genes can be selected for in appropriate S. cerevisiae mutants. A: pAG-I contains two copies of the S.c. chromosomal replication origin (ARSI) and two copies of the S.c. 2 micron plasmid replication origin (21z4RS). Plasmid pAG-I also contains an S.c. promoter/terminator construction (not used in the experiments described here) where the cycl- 13 promoter (blackened arrow) is linked to the CYCI terminator (hatched box) via the M 13mp8 polylinker. All functional e!e.m_e~ts~ e identical to those ofpEX4 from which pAG-I was derived (Ernst and Chan, 1985). B: pAG-2 contains the 2#AR$. C: pAG-3 contains ARSI. D: pJL3A contains no S.c. ARS and consists exclusivelyofprokaryotic sequences. P!~smids pAG-2, pAG-3 and pJL3A were constructed by introduction of the (3418R gene containing 1.7-kb Sail fragment ofpAG-I into the Sail sites ofYEp24 (~otstein et al., 1979), YRp7 (Struhl et al., 1979) and pBR322, respectively. Open headed arrows in the plasmid maps indicate direction of transcription. Clonh'g wa-_--done using standard procedures (Maniatis et al., 1982).

101

solid medium. The spontaneous mutation frequency to Gt resistance was less than 2 x 10 -7. We transformed d. gossypii mycelial protoplasts with 10-15#g of the S. cerevisiae/E, coli shuttle vector pAG-1 in the presence of PEG as described in the legend to Table I. Plasmid pAG-1 contains the kan gene of E. coil transposon Tn903 as selection marker and two copies of the S. cerevisiae autonomously replicating sequences, ARS1 and 21~ARS (Fig. 1A). Regeneration of protoplasts started approx. 10 h after plating. A regeneration frequency of 1-3 ~o (calculated upon protoplast titer after PEG treatment) was achieved routinely. For transformant selection, Gt was applied to a final concentration of approx. 0.1 mg/ml after an 18 h period of nonselective incubation to allow protoplast regeneration to begin. The Gt R colonies appeared after two to five days of further incubation on transformation plates. At 0.1 mg/ml, Gt could not completely inhibit growth of

some colonies on plates inoculated with untransformed protoplasts. These colonies could be distinguished from t r a n s f o r m a n t s by their inability to grow upon transfer to new plates with 0.3 mg Gt/ml. Subtracting the number o f such false positives, transformants were calculated to occur at frequencies o f 0 . 3 - 6 3 transformants per #g D N A and 4 - 1 0 0 transformants per 105 regenerated protoplasts (Table I). W h e n regeneration plates contained >_0.15 mg Gt/ml, the growth o f untransformed A . gossypii colonies was prevented, but no transformants were found either. The most likely explanation for this is the low activity of the prokaryotic k a n gene promoter in A . gossypii. This view was also supported by the requirement & t h e nonselective incubation period prior to application o f Gt. Without preincubation n o transformants were obtained. Similar effects have been observed previously with S. cerevisiae (Webster and Dickson, 1983).

TABLE I Results of transformation experiments with Ashbya gossypii a Plasmids:

pAG- I

pAG-2

pAG-3

pJL3A

pJL3A

linearized with Pstl S. cerevisiae ARS elements

(21AARS, A R S I )2

21t,4R$

ARM

-

-

Transformants per pg DNA (number of experiments)

14 (5)

8 (2)

4 (1)

0 (4)

0 (2)

Transformants per 105 viable protopiasts

54

31

7

0

0

" Strains and media.A, gossypiistrain ATCC!0895 was grown at 27 °C in MA2 medium (R. Kurth, personal communication), per ml: 10 mg Gibco-Peptone No. 140/1 mg Gibco-Yeast Extract/10 mg glucose/0.3mg myo-inositol (Sigma). Solid MA2 medium contained 12 mg Gibco-Agar/ml. For selective propagation of transformants, G418 (Geneticin, Gibco No. 066-01811) was added to a concentration of 60 mg/mi in liquid MA2 and 300 mg/ml in MA2 plates. The formation ofA. gossypii protoplasts and transformation is based on S. cerevisiae protocols (Beggs, 1978; Hinnen et al., 1978).The 200 ml MA2 medium in a 500 ml baffle-shake flask was inoculated with I-2 × 107 spores and incubated for 35-40 h at 27°C at 350 rpm. The myceliumwas harvested by filtration and washed in 30 ml H20. The 2-3 g of mycelium (ww) was suspended in 30 ml SD (I M sorbRol/50mM dithiothreitol) and incubated for 30 min with gentle agitation. After filtration, the myceliumwas suspended in 25 mi SPEZ (1 M sorbitoi/10 mM Na. phosphate buffer/10 mM EDTA/2 mg pet ml Zymolyase 20"1";Seikagaku Kogyo Co., Japan; pH 5.8) and inctlbated with gentle agitation at 27°C. Formation of protoplasts was followed by microscopy. After 30-45 min, the formation of protoplasts was completed. Myceliai debris was removed from the suspension by filtration through a sintered glas filter, pore size i. The protoplasts were collected by centrifugation (5 min, 700 x g), washed twice in 20 ml ST (1 M sorbitol/10 mM Tris. CI, pH 8) and resuspended in 20 ml STC (1 M sorbitol/10 mM Tris. CI/10 mM CaCI2,pH 8). After determination of the protoplast titer using a counting chamber, the protoplasts were pelleted again and suspended in STC to a final density of 4 × 10S/ml. A 100/~1of this suspension was mixed carefully with 10-15 #g of plasmid DNA (see sections a and b) dissolved in no more than 15 #! TE (10 mM Tris. CI/I mM EDTA, pH 8). After 15 rain incubation at room temperature, the suspension was mixed gently with 1 ml PTC (40% polyethylene glycol 4000/10 mM Tris. CI/10 mM CaC!2, pH 8), incubated for 15 min at room temperature and centrifuged (5 min at 1500 rpm using Heraeus biofuge A). The supernatant was carefully removed and the protoplasts were suspended in I ml SMTCI (1 M sorbitol/25% MA2 medium/0.06 mg myo-inositolper ml mixed with an equal volume of STC), followedby incubation for 3 h at 27 °C with gentle mixing every 15 min. The protoplasts were pelleted by centrifugation and resuspended in I ml SM (2 M sorbitol mixed with an equal volume of MA2 medium). This suspension was mixed with 9 ml top layer (MA2 medium/l M sorbitol/0.8% agarose at 42°C) and poured on a regeneration plate (same as top layer, instead of agarose 12 g/! Difco-agar). After 18 h incubation at 27°C, the plate was overlaid with Gt in 7 ml of 0.5% agarose (42°C) to a final concentration of 0.08-0.1 mg/ml, followed by further incubation. Gt R colonies appeared after 3-5 days. Regeneration frequencies were determined by plating appropriate dilutions of the protoplast suspension on regeneration plates. The preparation ofA. gossypii spore suspensions is based on a protocol received from R. Karth. A. gossypii mycelium from 5- to 10.days-old MA2 plates was digested in 3 ml of a 5% (w/v) solution of cellulase (Onuzaka RI0, Yakult Pharmaceutical Industry Co. Ltd., Japan) for 3 h to release the endogenously formed spores. After addition of I ml 0.03% Triton X-100 (Sigma), spores were collected by centrifugation (10 min, 700 × g), washed four times and finally suspended in 0.03~ Triton X-100.

102

(b) Evidence for autonomous replication of Saccharomyces cerevisiae A R S plasmids in Ashbya gossypii All tested pAG-1 transformants could be propagated under selective conditions and were resistant to at least 0.5 mg Gt/ml in agar plates. The initial transformants were probably heterokaryotic since A. gossypii mycelium is polynucleated and most of the protoplasts contained more than one nucleus. To assay transformant stability under nonselective conditions, we used homokaryotic transformants that we had isolated from the mononucleate spores formed by A. gossypii. The preparation of spores is described at the end of the legend to Table I. The tested strains became Gt sensitive after at maximum three rounds of nonselective incubation. To check for the presence of vector sequences in the original transformants, Sail-cleaved DNA from nine transformants was subjected to Southern analysis. Only signals identical to those of Sail-cleaved pAG-1 were detected (results not shown). This result indicated the presence of the original plasmid in the transformants. In order to check whether plasmids were present in a free form, E. coli strain HB101 was transformed with 0.15-2.5 #g of total uncleared DNA from two A. gossypii

M

1

2

3

Fig. 2. Sail pattern of plasmids from E. cog pAG-I retransformants. Plasmid DNA was isolated from Ap R E. coil HB101 (Boyer and Roulland-Dussoix, 1969) which had been transformed with 0.15-2.5 pg of total uncleaved DNA of the A. gossypii pAG-I transformants TI 1 and TIT following the transformation procedure of Mandel and Higa (1970). Piasmid DNA obtained from E. coli clones transformed with TI I DNA (lane I ) and TIT DNA (lane 7,) was assayed on a 0.85/o agarose gel after Sail cleavage (lanes I and 2). The patterns obtained were identical to the one of original pAG-I (lane 3). M, size marker with ) c i 8 5 7 DNA EcoRI-HindlII fragments and HindIII fragments; sizes of the plasmid fragments (in lanes I-3) are 8.7 kb (doublet), 1.7 kb (singlet), 0.6 kb (triplet).

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Fig. 3. Chromosomal banding pattern of A. gossypii. A. gossypii (A.g.) DNA was isolated from 0..5g mycelium from a 3-day-old culture following the S. cerevisiae protocol of Carlo and OIson (1985). The DNA was separated in a 24 h run using the OFAGE system of Cafle and Olson (1984) at 300 V with a 50 sec pulse time. The sizes of the five separated bands (see left margin) were estimated by comparison with the chromosomai bands of S. cerevisiae VB2-20A (S,c.; adapted from Mortimer and Schild, 1985).

transformants. E. coil transformants were obtained with frequencies of up to 155 transformants per #g DNA. The Sail-cleaved plasmid DNA from E. coil transformants was analysed on an agarose gel. The observed Sail fragments were identical to those of pAG-1 (Fig. 2). These results strongly indicated autonomous replication of pAG-I in A. gossypii and were consistent with the high instability of the transformants. This hypothesis could b~. confirmed by electrophoretical separation of plasmid DNA from chromosomal DNA using OFAGE (Carle and Olson, 1984). With conditions employed for the electrophoretical separation of the S, cerevisiae chromosomal DNAs, we succeeded to separate total A. gossypii DNA into five chromosomal DNA bands with sizes of approx. 1.6, 1.2, 0.97, 0.90 and 0.63 Mb (Fig. 3). IfpAG-I was truly present as an autonomously replicating plasmid, it should be possible to separate it from the chromosomal DNA bands, exploiting the different migration behaviour of circular and linear DNA in OFAGE (Hightower et al., 1987; Mathew et al., 1988). U~ing pAG-I mixed with chromosomal DNA as control, we analysed DNA of three transformants (Fig. 4A). The control plasmid had migrated to a position clearly different from those of the chromosomal DNA bands. The 1.6 and 1.2 Mb and the 0.97 and 0.90 Mb chromosomal DNA bands were not separated from each other, since the running time had to be reduced to prevent the plasmid DNA from running off the gel. The pAG-I plasmid was visualized in the transformant lanes by hybridization to a DNA probe. All transformants showed

103

4

3

1

kb

1600 1200

2

3

4

4

S... ,,.

9?0... 900 ....

630,-

RNA,,,.

A

B

C

Fig. 4. OFAGE analysis ofA. gossypiipAG-I transformants. DNA of the pAG-! transformants TII, TV2,TYI (lanes 1, 2, 3) and DNA ofuntransformed A. gossypiimixed with 0.5 #g ofp~G-! as a cont~-ol(lane 4) was separated in a 12 h OFAGE run. At~er EtdBr staining (panel A), the DNA was depurinated and denatured, blotted to nitrocellulose and hybridized with 32p-labeled pJL3A (see Fig. 1) to identify transforming sequences (panel B) (Southern, 1975; Rigby et al., 1977). Open circle pAG-I DNA used as control was detected below the 630-kb chromosomal band (Poe) in the EtdBr-stained gel. In the So,thern blot, a second, faster migrating plasmid band - probably representing a trace ofsupercoiled form - was detected (P~c). All transformants revealed signals corresponding to the major pAG-I signal (Po~). Two transformants showed an additional signal (Px) possibly reflecting an oligomeric form of the plasmid. In no case were signals overlapping with chromosomal bands. Signals observed close to the slots (S) were probably due to DNA which could not enter the gel. Panel B is combined from photos of a five-day exposure (Fuji XR X-ray film, no intensifying screen) fer lanes I, 2, 3 and the bottom of lane 4, and a l-h exposure for the other part of lane 4. The complete lane 4 after five days exposure is shown in panel C.

signals corresponding to the pAG- 1 plasmid band in the gel (Fig. 4A) and the main signal in the control lane (Fig. 4B). According to the migration behavior of plasmids of OFAGE, these signals probably represented the open circular form of pAG-1. The presumptive supercoiled form

1 2

m

3

m

4

W

5

°~'

1

~ii

2

¸,

. . . . . .

3

4

was only visible in the control lane. The low intensity of this signal and its lack in the transformant lanes was possibly due to nicking during sample preparation. The origin of the additional signal obtained with two transformants is unknown. Possibly it was caused by plasmid oligomers. Like

5

1 ,--

S

,,--

S¢

2

3

4

5

,.....

~i~i!~i~i'

A

B

C

Fig. 5. Separation ofpAG-I plasmid DNA from totalA, gossypiitransformant DNA with conventional agarose gel electrophoresis. (Panel A) 125 ng DNA of the A. gossypii pAG-I transformants TV2 (lane 1) and TVI! (lane 3) were separated in a 0.5% agarose gel together with 0.4 ng pAG-I plasmid DNA (lane 2) and 125 ng DNA from untransformed A. gossypii (lane 4). The 60 ng DNA of an integrative A. gossypii transformant (lane 5; compare section b) were used to mark the position of chromosomal DNA (chr). Transforming sequences were detected by hybridization with 32p-labeled pJL3A (see Fig. 1) and exposed for 45 min (panel B) and 16 h (panel C) to Fuji x-ray film. The positions of the open circle (oc), linear (i) and supercoiled (se) form ofpAG-I plasmid DNA are indicated. Supercoiled plasmid DNA in lanes I and 3 and linear plasmid DNA in lane 2 was visible only after 16 h exposure (C). Signals at the position of chromosemal DNA (lane 5) are absent in lanes 1-3. DNA was isolated from protoplasts formed from mycelium of 2to 5-days old 20 ml cultures using a slightly modified S. zerevisiae protocol (Struhl et al., 1979). Electrophoresis was carried out in the presence of 0.5 , g EtdBr for 22 h at 1 V/cm in TBE buffer (Maniatis et al., 1982).

104 the other signals, it is not overlapping with chromosomal DNA and therefore not due to integrated sequences. Although this result was clear evidence for the presence of free plasmid in pAG-I transformants, it could not be ruled out that the detected plasmid DNA resulted from excision of vector sequences tandemly integrated in a yet unidentified chromosome as previously demonstrated for A. nidulans by Johnstone et al. (1985). This possibility could be excluded by Southern analysis of pAG-I transformant DNA separated by conventional agarose gel electrophoresis. The open circle, linear and supercoiled form of pAG-I could be detected, whereas signals corresponding to the position of chromosomal DNA were absent (Fig. 5). Our interpretation of the obtained results is that pAG-1 is exclusively present as an autonomously replicating plasmid in A. gossypii. We have evidence that the autonomous replication is conferred by the S. cerevisiae 2#ARS and ARSI sequences in pAG-1. A. gossypii was transformed with the plasmids pAG-2, pAG-3 and pJL3A. Plasmid pAG-2 carries the 2#ARS, pAG-3 the ARS1. Plasmid pJL3A does not contain any ARS element (Fig. 1). Plasmid pAG-2 and pAG-3 yielded transformants with frequencies comparable to those with pAG- 1. No transformants were obtained with pJL3A, not even when the plasmid had been linearized prior to transformation (Table I). However, when plasmids containing homologous A. gossypii DNA are linearized within this region, integrative transformants have been obtained in some cases (Sabine Steiner, personal communication). Like homokaryotic pAG-I transformants, homokaryotic pAG-2 and pAG-3 transformants became genetitin-sensitive upon cultivation on nonselective plates. DNA was isolated from pAG-2 and pAG-3 transformants as before from pAG-1 transformants and used for Southern analysis and retransformation of £. coli. The results clearly showed that pAG-2 and pAG-3 replicate autonomously in A. gossypii, too. (¢) Conclusions (1) The filamentous hemiascomycete A. gossypii can be transformed to Gt R with plasmids containing the bacterial kan gene of Tng03 as selectable marker. For successful transformation, the presence of S. cerevisiae ARS elements were required and the corresponding plasmids were found to be replicating autonomously in A. gossypii. Although transformation based upon autonomously replicating plasraids has been reported for filamentous fungi from several classes (Glumoff et al., 1989; Tsukuda et al., 1988; Revuelta and Jayram, 1986; Stohl and Lambowitz, 1983; Stahl et al., 1982), A. gossypii is to our knowledge the first fdamentous fungus where plasmid replication is achieved by S. cerevisiae ARS elements. (2) The transformation efficiencies we have obtained so

far are low. This might be due to low expression of the bacterial kan gene. We are trying to improve the expression of the gene by using a homologous A. gossypii promoter. Additionally, the selection of Gt R transformants in general could limit the transformation efficiency. Webster and Dickson (1983) have shown that in S. cerevisiae only 1-11% of the transformation efficiency is reached when transformants are selected for G t R compared to 100% efficiency employing auxotrophic complementatiop. The authors attribute this effect to a high proportion of potential transformants which are killed by Gt before they can express the resistance gene. (3) Other dominant selection systems such as the acetamidase and nitrate reductase systems, avoid the use of toxic antibiotics. They are widely used for the transformation of filamentous fungi (Kelly and Hynes, 1985; Unkles et al., 1989). Unfortunately, they cannot be applied to A. gossypii, since these systems are based on the use of ammonium as nitrogen source by the transformants, and A. gossypii grows only very poorly on ammonium, Therefore, we are attempting to develop a selection system based on complementation of auxotrophic A. gossypii mutants. We have already isolated serine-, threonine- and uracilrequiring mutants and are searching for either homologous or heterologous genes capable of complementing these mutants.

ACKNOWLEDGEMENTS

We thank J. Lauer for her help in the construction of plasmids and for the preparation of plates and media together with K. Escher. We also thank Dr. R. Kurth for basic information on the work with A. gossypii, Dr. J. Ernst for the donation of the pEX-4 plasmid and Dr. J. Davies for the Gt used in initial experiments. Additionally, we would like to thank S. Steiner for the donation of an integrative A. gossypii transformant and D. Jager for the introduction to the OFAGE technique. The photographs were kindly prepared by E. Briegel. This work was done in partial fullfilment of the requirements for the Ph.D. thesis of M.W. and was supported by a grant of the 'Schwerpunktprogramm Gentechnologie' of the Deutsche Forschungsgemeinschaft to P.P.

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