isolation and use of a homologous histone h4 …in the present paper, we describe the isolation and...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Nov. 2000, p. 4655–4661 Vol. 66, No. 11

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Isolation and Use of a Homologous Histone H4 Promoter and aRibosomal DNA Region in a Transformation Vector

for the Oil-Producing Fungus Mortierella alpinaDONALD A. MACKENZIE,* PRASERT WONGWATHANARAT,† ANDREW T. CARTER,

AND DAVID B. ARCHER‡

Institute of Food Research, Norwich Research Park, Colney, Norwich,Norfolk, NR4 7UA, United Kingdom

Received 6 April 2000/Accepted 29 August 2000

Mortierella alpina was transformed successfully to hygromycin B resistance by using a homologous histoneH4 promoter to drive gene expression and a homologous ribosomal DNA region to promote chromosomalintegration. This is the first description of transformation in this commercially important oleaginous organ-ism. Two pairs of histone H3 and H4 genes were isolated from this fungus. Each pair consisted of one histoneH3 gene and one histone H4 gene, transcribed divergently from an intergenic promoter region. The pairs ofencoded histone H3 or H4 proteins were identical in amino acid sequence. At the DNA level, each histone H3or H4 open reading frame showed 97 to 99% identity to its counterpart but the noncoding regions had littlesequence identity. Unlike the histone genes from other filamentous fungi, all four M. alpina genes lacked in-trons. During normal vegetative growth, transcripts from the two histone H4 genes were produced at approx-imately the same level, indicating that either histone H4 promoter could be used in transformation vectors. Thegeneration of stable, hygromycin B-resistant transformants required the incorporation of a homologous ribo-somal DNA region into the transformation vector to promote chromosomal integration.

The filamentous fungus Mortierella alpina produces up to50% of its biomass as triacyglycerol, which is rich in long-chainpolyunsaturated fatty acids (7, 26). These fatty acids are im-portant both nutritionally and pharmacologically, and there ismuch interest in developing microbial processes for their pro-duction (19, 26). To date, manipulation of M. alpina to produceoils with different fatty acid contents has been carried out bystrain mutagenesis (7). The recent isolation of several fattyacid desaturase genes from this fungus has presented the op-portunity of using recombinant methods to modify the fattyacid composition of its oil (14, 22, 27, 38). To achieve this goal,a DNA transformation system must be developed for M. al-pina, because there are no reports of transformation in thisorganism.

Efficient transformation vectors usually contain a homolo-gous promoter to drive expression of the selection marker. Inthe case of the fungus Phanerochaete chrysosporium (12) and inTetrahymena thermophila (17), dominant antibiotic resistancemarkers have been expressed using a strong, homologous his-tone H4 promoter. Histone H3 and H4 promoters have alsobeen used to express reporter genes in yeast and plants (3, 11).Additionally, a number of fungal histone genes have beencharacterized (10, 21, 28, 39). Most histone genes are highlyexpressed, and their regulation is tightly coupled to DNA syn-thesis during the cell cycle (3, 32). The use of a histone pro-

moter to express selection markers should, however, presentno problems in fungal cultures which normally grow asynchro-nously.

In the present paper, we describe the isolation and char-acterization of two pairs of histone H3 and H4 genes fromM. alpina and the use of one of the histone H4 promoters in atransformation vector to drive expression of the hygromycin Bresistance gene. We also report the need to include a homol-ogous ribosomal DNA (rDNA) region in the vector to promotechromosomal integration for the generation of stable transfor-mants. This is the first reported case of transformation in thiscommercially important fungus.

MATERIALS AND METHODS

Strains, media, and culture conditions. Most of the work described in thispaper was carried out with M. alpina strains CBS 224.37 and CBS 528.72 (ATCC32222), which were obtained from the Centraalbureau voor Schimmelcultures(CBS), Baarn, The Netherlands. Other strains used were CBS 210.32, CBS250.53, and CBS 527.72, all from the CBS culture collection, and CCF 2639, fromthe Culture Collection of Fungi, Charles University, Prague, Czech Republic,which was kindly supplied by R. Herbert, University of Dundee, Dundee, Scot-land. Two media used for culturing M. alpina were potato dextrose broth (PDB;Difco, Detroit, Mich.) and S2GYE (5% [wt/vol] glucose, 0.5% [wt/vol] yeastextract [Oxoid, Basingstoke, United Kingdom], 0.18% [wt/vol] NH4SO4, 0.02%[wt/vol] MgSO4 z 7H2O, 0.0001% [wt/vol] FeCl3 z 6H2O, 0.1% [vol/vol] traceelements [Fisher Scientific UK, Loughborough, United Kingdom], 10 mMK2HPO4–NaH2PO4 [pH 7.0]), and growth conditions were as described previ-ously (38). Streptomyces sp. strain no. 6 (16) was obtained from the NationalCollection of Industrial Bacteria, Aberdeen, United Kingdom, and grown in achitin-chitosan-containing minimal medium to produce “streptozyme” as de-scribed previously (34).

Isolation and analysis of the histone H3–H4 genes. Degenerate primers H3,59-YTSMGSGAYAAYATHCA-39 (192-fold degeneracy), and H2, 59-ARSGCRTASACSACRTC-39 (64-fold degeneracy), were designed after aligning the his-tone H4 protein sequences of Aspergillus nidulans (P23750 and P23751) (10),Neurospora crassa (P04914) (39), P. chrysosporium (P35058), Saccharomyces cer-evisiae (P02309) (28), and Schizosaccharomyces pombe (P09322) (21). They bindto regions of the histone H4 gene which encode LRDNIQ and DVVYAL,respectively, and amplify a 206-bp fragment, H3H2. Standard PCR conditionswere used at an annealing temperature of 50°C. The H3H2 fragment was clonedinto vector pGEM-T (Promega, Madison, Wis.) and sequenced. 32P-labeled

* Corresponding author. Mailing address: Institute of Food Re-search, Norwich Research Park, Colney, Norwich, Norfolk, NR4 7UA,United Kingdom. Phone: 44 1603 255255. Fax: 44 1603 507723. E-mail:[email protected].

† Present address: Department of Biotechnology, Faculty of Scienceand Technology, Thammasat University, Rangsit Center, Patumthanee12121, Thailand.

‡ Present address: School of Life and Environmental Sciences, Uni-versity of Nottingham, University Park, Nottingham NG7 2RD, UnitedKingdom.

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H3H2 was used subsequently to probe Southern blots and to screen a BamHIgenomic DNA library from M. alpina strain CBS 528.72, which had been con-structed as described previously (38). Positive pBK-CMV (Stratagene, La Jolla,Calif.) phagemid clones were excised in vivo, and their inserts were sequenced.Total RNA was isolated from fungal mycelia using TRIzol (Life Technologies,Rockville, Md.) according to the manufacturer’s instructions.

Poly(A)-containing RNA (0.5 to 5.0 mg) for Northern analysis was enrichedfrom 500 mg of total RNA using Oligotex mRNA minicolumns (QIAGENGmbH, Hilden, Germany). In this case, total RNA was isolated from freeze-ground mycelia using a method for extracting RNA from yeast cells (24). Totalor poly(A)-enriched RNA samples were denatured at 55°C for 15 min in GFMbuffer (0.55 M glyoxal, 39% [vol/vol] deionized formamide, 60 mM morpho-linepropanesulfonic acid [MOPS], 1.5 mM sodium acetate, 0.3 mM EDTA [pH7.0]) prior to electrophoresis through 1% (wt/vol) agarose gels with 0.24- to9.5-kb RNA molecular weight (MW) markers (Life Technologies). The histoneH4 gene-specific probes P4.1 (91 bp) and P4.2 (194 bp) were amplified by PCRat annealing temperatures of 60 and 58°C, respectively, from genomic clonesusing primers H4.15 (59-TCAGCCGCACTCGCAGCTGC-39) and H4.10 (59-AGTGTCAAAGAGGGTTCTAT-39) for P4.1 and primers H4.25 (59-GACTTGCCCATCGTCGTCCT-39) and H4.20 (59-GCATTGCTGCGAGGACAATT-39)for P4.2. Signals on Northern blots were detected using a Fuji BAS 1500-phos-phorimager.

For reverse transcription-PCR (RT-PCR), approximately 0.1 mg of poly(A)-containing RNA which had been purified twice through Oligotex mRNA mini-columns (QIAGEN GmbH) was used as a template in a cDNA first-strandsynthesis reaction (Amersham Pharmacia Biotech, Uppsala, Sweden) with ananchored oligo(dT)18 primer, CN95 [59-CTTCTGGATGTGCGTACTCGAGCT(T)18-39], according to the manufacturer’s instructions. PCR was then carriedout with the following gene-specific primers: forward primers H4.11 (59-ATTTCAAAAAACAGAAAAAC-39), H4.12 (59-CAGCCCAAGAAAAAAAATAC-39), H4.13 (59-CGCATCCCGCAAACACACAC-39), and H4.14 (59-TCACCCAACACTCTCTCAAC-39) with reverse primer H4.10 for histone H4.1 and for-ward primers H4.21 (59-TGTGTGGGCTCGTCTGGAAT-39), H4.22 (59-GCCCCTCCCCGACAACACAT-39), H4.23 (59-AGGAAAAGAAAAGCACAAAC-39), and H4.24 (59-ACACACACACTCACACTCAC-39) with reverse primerH4.20 for histone H4.2. Primers H4.11 to H4.14 and H4.21 to H4.24 anneal toregions upstream of the respective ATG start codons, while primers H4.10 andH4.20 anneal to the 39 untranslated region (39-UTR). PCR with these primerswas carried out at an annealing temperature of 50°C.

Vector construction and transformation of M. alpina. Vectors pAN7-1 (25)and pAN-CCG were kindly supplied by P. Punt, TNO, Zeist, The Netherlands,and J. Springer, ATO-DLO, Wageningen, The Netherlands, respectively. pAN7-1contained the A. nidulans glyceraldehyde-39-phosphate dehydrogenase (gpdA)promoter, the Escherichia coli hygromycin B resistance gene (hpt), and the A.nidulans N-(59-phosphoribosyl)anthranilate isomerase (trpC) transcription ter-minator. In plasmid pAN-CCG, the Cryptococcus curvatus gpdA promoter droveexpression of a modified hygromycin B resistance gene (hptmod) and was derivedfrom vector pANH2-1 (33). A 1-kb fragment containing the M. alpina histoneH4.1 promoter region was amplified from a CBS 528.72 histone H3.1–H4.1genomic clone using forward primer SHG1 (59-AAGAATTCAAGCGAAAGAGAGATATGAAACA-39) and reverse primer SHG2 (59-AACCATGGATTGTTGAGAGAGTGTTGGGTG-39) at an annealing temperature of 56°C. PrimerSHG1 annealed at position 2999 to 2977 with respect to the histone H4.1 ATGstart codon (11), while primer SHG2 annealed at position 22 to 223. Theseprimers contained EcoRI and NcoI restriction sites (underlined), respectively,which were subsequently used in replacing the C. curvatus gpdA promoter EcoRI-NcoI fragment of pAN-CCG with the M. alpina histone H4.1 promoter toproduce vector pAN-MAH. An rDNA region of approximately 1 kb, containingpart of the 18S rRNA gene, was amplified from M. alpina CBS 528.72 genomicDNA using forward primer P1190 (59-CAATTGGAGGGCAAGTCTGG-39)and reverse primer M3490 (59-TCAGTGTAGCGCGCGTGCGG-39) at an an-nealing temperature of 54°C. Another reverse primer, ITS4 (59-TCCTCCGCTTATTGATATG-39), was used with forward primer P3490, whose sequence wascomplementary to that of M3490, to amplify the rDNA region downstream ofP1190–M3490. Primers P1190, M3490, and ITS4 were originally designed fromthe S. cerevisiae 18S rRNA gene sequence (6, 15) and were kindly supplied byS. James, Institute of Food Research (IFR), Norwich, United Kingdom. The931-bp P1190–M3490 18S rDNA fragment was cloned into pCR 2.1 (Invitrogen,Carlsbad, Calif.) and subsequently inserted as a 1,041-bp XbaI-HindIII fragmentinto pAN-MAH to create vector pD4 (Fig. 1).

Protoplasts of M. alpina CBS 224.37 were produced by treating 4- to 6-day-oldPDB-grown mycelium with “streptozyme” from Streptomyces sp. strain no. 6 (34).Approximately 10 g (wet weight) of mycelium in 15 ml of protoplasting buffer(1 M sorbitol–10 mM NaH2PO4-Na2HPO4 [pH 6.5]) was incubated with 4 ml offilter-sterilized “streptozyme” in 20 mM NaH2PO4–Na2HPO4, pH 6.5, for 1 to2 h with gentle shaking (60 to 80 rpm) at 25°C. Protoplasts were harvestedthrough sterile polyallomer wool and concentrated by centrifugation at 1,000 3g for 10 min at 4°C. After a wash in 50 ml of ice-cold STC buffer (1 M sorbitol,10 mM Tris-HCl, 50 mM CaCl2 [pH 7.5]), the protoplasts were resuspended in1 ml of the same buffer. A total of 2 3 107 protoplasts in 100 ml of STC bufferwere incubated with 5 mg of vector DNA for 25 min at room temperature. A1.25-ml volume of PEG–CaCl2 (60% [wt/vol] polyethylene glycol with an MW of

4,000 to 6,000, 10 mM Tris-HCl, 50 mM CaCl2 [pH 7.5]) was added gradually,and the mixture was incubated for a further 20 min at room temperature. PEGwas diluted by the addition of 10 volumes of STC buffer, and protoplasts wereharvested by centrifugation. Protoplasts were resuspended in 1 ml of STC buffer,and 200 ml of this mixture was embedded in 12 ml of molten (50°C) potatodextrose agar (PDA) containing 1 M sorbitol. Following 2 days’ incubation at12°C, a 12-ml top layer of PDA–1 M sorbitol, containing 200 mg of hygromycinB ml21, was poured onto each plate, and incubation was continued at 25°C for5 to 7 days. Putative transformants were transferred onto PDA plates containing300 to 1,000 mg of hygromycin B ml21.

Transformants were checked by PCR using the forward primer HYGR1 (59-AGCGAGAGCCTGACCTATTG-39) and the reverse primer HYGR2 (59-TCGAAGTAGCGCGTCTGCTG-39) at an annealing temperature of 58°C, whichgenerate an internal hptmod fragment of 500 bp. Genomic DNA was isolatedfrom transformants using the modified QIAGEN method described previously(38). The chromosomal rDNA site of integration of plasmid pD4 was verifiedusing the vector-specific forward primer RDNA1 (59-ACAGGTACACTTGTTTAGAG-39), which anneals just upstream of the XbaI site in the A. nidulans trpCterminator region, and reverse primer RDNA2 (59-CGCTGCGTTCTTCATCGATG-39). RDNA2 anneals to the 5.8S rRNA gene, which lies downstream of the18S rDNA region and which is absent in pD4. Primers RDNA1 and RDNA2were used at an annealing temperature of 54°C and were expected to generate afragment of 1,569 bp with CBS 224.37 transformants.

Nucleotide sequence accession numbers. The following sequences have beensubmitted to the EMBL database: 18S–5.8S rDNA regions from M. alpina strainsCBS 528.72 (AJ271629) and CBS 224.37 (AJ271630), and histone H3.1–H4.1genes (AJ249812) and histone H3.2–H4.2 genes (AJ249813) from M. alpinastrain CBS 528.72.

RESULTS

Vectors containing heterologous promoters failed to trans-form M. alpina. Six M. alpina strains were screened for sensi-tivity to hygromycin B (100 to 1,000 mg ml21), but all exceptCBS 224.37 were resistant to the antibiotic. This strain wassensitive to 100 to 200 mg of hygromycin B ml21 and wastherefore chosen as the most suitable transformation host.Initial experiments using vectors pAN7-1 and pAN-CCG, inwhich expression of the hygromycin B resistance gene wasdriven by heterologous gpdA promoters from A. nidulans and

FIG. 1. Map of the M. alpina transformation vector pD4. The 1-kb EcoRI-NcoI M. alpina histone H4.1 promoter fragment from strain CBS 528.72 alsocontains the histone H3.1 promoter and ORF. The hygromycin B resistance geneis the modified version (hptmod) which lacks the internal EcoRI and NcoI sitesthat are present in the wild-type gene of pAN7-1 (33). The 700-bp BamHI-XbaIfragment contains the A. nidulans trpC transcription terminator region (trpCt).The positions of the two rDNA primers P1190 and M3490, which were used togenerate the M. alpina 18S rDNA fragment, are indicated as P and M, respec-tively. bla, ampicillin resistance gene. Restriction sites: B, BamHI; E, EcoRI; H,HindIII; N, NcoI; S, SspI; X, XbaI.

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C. curvatus, proved unsuccessful (Table 1). The use of a ho-mologous M. alpina promoter was therefore investigated, andthe histone H4 promoter was chosen because of previous suc-cesses in other organisms (12, 17).

Isolation and characterization of the histone H3–H4 genepairs from M. alpina CBS 528.72. Degenerate oligonucleotideprimers were designed from highly conserved regions of fungalhistone H4 proteins where sequence degeneracy was mini-mized. A 206-bp DNA fragment (H3H2) was amplified fromM. alpina CBS 528.72 genomic DNA, the predicted translationproduct of which had about 94% amino acid identity to anumber of histone H4 proteins. This fragment was used toprobe a Southern blot of genomic DNA from CBS 528.72which had been digested with a range of restriction enzymes.The BamHI digest gave two strongly hybridizing bands of ap-proximately 7.2 and 4.3 kb and a fainter band of about 9 to 11kb (data not shown). Subsequently, a BamHI genomic DNAlibrary of CBS 528.72 was screened with probe H3H2, andpositive clones were shown to contain one of two inserts. Onsequencing, these were found to be 7,139 and 4,257 bp, respec-tively, and encoded pairs of histone H3 and histone H4 pro-teins with high amino acid identity (92 to 95%) to histone H3and H4 proteins from other organisms (Fig. 2). Unlike allother histone genes isolated so far from filamentous fungi, thefour M. alpina genes lacked introns.

The two genomic clones did not contain other histone genes,such as those encoding histone H2A or H2B. The 7.2-kb his-

tone H3.1–H4.1 clone did, however, contain two other genes,one encoding a putative 60S ribosomal protein (rpl27A; EMBLaccession number AJ249749) located downstream of the his-tone H3.1 gene, and the other encoding a putative thioredoxinII-like protein (EMBL accession number AJ249750) locateddownstream of the histone H4.1 gene. Each gene pair wasseparated by a promoter region from which the genes weredivergently transcribed. This intergenic region was 532 bp forthe H3.1–H4.1 gene pair and 646 bp for the H3.2–H4.2 genepair. The pairs of encoded histone H3 or H4 proteins wereidentical in amino acid sequence, and their open readingframes (ORFs) shared 93 and 97% DNA sequence identity,respectively. The promoters, 59-UTRs, and 39-UTRs of eachpair of histone H3 or H4 genes showed much less DNA iden-tity, although there were small stretches of 60 to 85 nucleotides(nt) which were about 75 to 80% identical. RT-PCR analysis oftranscripts from the two histone H4 genes with gene-specificprimer sets, H4.10 to H4.14 and H4.20 to H4.24, indicated thatthe putative transcription start point for each gene was 50 to 70nt upstream of the ATG start codon (data not shown).

Expression of the two histone H4 genes in M. alpina. Twohistone H4 gene-specific probes, P4.1 and P4.2, were synthe-sized from the 39-UTRs of the respective histone H4.1 andH4.2 genes for use in Northern analysis of strain CBS 528.72.Probes P4.1 and P4.2 were 91 and 194 bp, respectively, in sizeand 75% identical at the DNA level over a 50-bp region. In dotblots, each probe did not cross-hybridize significantly with theopposing histone H4 gene, thus confirming their specificity(data not shown). In Fig. 3A and B, the gene-specific probesdetected histone H4 transcripts which differed slightly in size.From analyzing the two genomic DNA sequences and estimat-ing the approximate lengths of the 59- and 39-UTRs, the ex-pected transcript sizes for histone H4.1 and H4.2 were 600 and640 nt, respectively. This agreed with the sizes of the two tran-scripts measured on Northern blots. When the experiment wasrepeated using poly(A)-enriched RNA and the H3H2 probe,which had 99 to 100% DNA identity with the histone H4.1 andH4.2 ORFs, both transcripts could be visualized as two closelymigrating bands of approximately equal intensity (Fig. 3C). Inaddition, probe H3H2 hybridized to the same two transcriptsfrom a number of other M. alpina strains, including CBS 224.37(P. Wongwathanarat and A. T. Carter, unpublished data).

FIG. 2. Organization of the two pairs of histone H3–H4 genes in M. alpinaCBS 528.72. Heavy arrows indicate the position and direction of each ORF. Thesizes of the promoter regions in nucleotides are given in italics. A, the consensuspoly(A) addition signal AATAAA; B, BamHI site.

FIG. 3. Transcription of the two histone H4 genes from M. alpina CBS528.72. (A and B) Total RNA (20 mg per lane), which had been isolated fromPDB-grown cultures harvested on the days indicated, was probed with the his-tone H4.1 and H4.2 gene-specific 39-UTR probes P4.1 and P4.2, respectively. (C)Cultures were grown in S2GYE broth, and poly(A)-enriched RNA (approxi-mately 0.5 mg per lane) was probed with the H3H2 histone H4 fragment, whichhybridizes to both transcripts. The lower panels show the ethidium bromide-stained gels prior to Northern blotting.

TABLE 1. Transformation frequencies of M. alpina CBS 224.37and transformant stability

VectorNo. of primary trans-

formants per 5 mgof vector DNAa

Maximum hygromycin Bresistance level

(mg ml21)

% Stabletransfor-mantsb

pAN7-1 0 —c —0 — —0

pAN-CCG 0 — —0 — —0 — —

pAN-MAH 0 — —2 350 00 — —

pD4 8 1,000 7510 1,000 70

a Transformants from two or three independent experiments were selectedinitially on 100 mg of hygromycin B ml21, except for pAN-MAH (350 mg ml21).

b Determined following first subculturing on 300 to 1,000 mg of hygromycin Bml21.

c —, not applicable.

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Transformation of M. alpina strain CBS 224.37. Replace-ment of the C. curvatus gpdA promoter in pAN-CCG with thehomologous M. alpina histone H4.1 promoter in vector pAN-MAH allowed transient growth of two transformants at anantibiotic concentration of 350 mg ml21 (Table 1). PCR withgenomic DNA from these transformants, using the hpt-specificprimers HYGR1 and HYGR2, confirmed the presence of thehptmod gene, but subsequent subculturing on media containinghygromycin B showed that they had become sensitive to theantibiotic. This was confirmed by failure to amplify the hptmod

fragment from genomic DNA of these transformants grownwithout hygromycin B selection. The stability of transformantswas improved greatly by incorporating a homologous M. alpinarDNA region into pAN-MAH to create plasmid pD4 (Fig. 1).A transformation frequency of 1 to 2 transformants z mg ofvector DNA21 was obtained with pD4, and the majority ofthese transformants remained resistant to up to 1 mg of hy-gromycin B ml21 after several subculturings in the presence ofthe antibiotic (Table 1). A proportion of pD4 transformants(25 to 30%) still displayed an unstable phenotype under anti-biotic selection.

The presence of the hptmod gene in pD4 transformants wasconfirmed by PCR with primers HYGR1 and HYGR2 and bySouthern blotting (Fig. 4). When probed with the HYGR1–HYGR2 hptmod fragment, SspI genomic DNA digests fromindependent, stable transformants gave very similar, but notidentical, hybridization patterns (Fig. 4A). SspI cuts vectorpD4 once, near the bla gene (Fig. 1), and does not cut in the1,899-bp P1190–ITS4 rDNA region of CBS 224.37. The twostrongly hybridizing bands of approximately 6.4 and 11.2 kbsuggested that two copies of pD4 had integrated in tandeminto the chromosome in each transformant (Fig. 4C and D).Probing ClaI digests with the hptmod fragment gave a single,diffuse signal at about 20 kb in each transformant tested (datanot shown). ClaI does not cut in pD4 but does cut the P1190–ITS4 rDNA region once in the 5.8S rRNA gene (within primerRDNA2). The untransformed host strain CBS 224.37 dis-played no positive signals in PCR with primers HYGR1 andHYGR2 or when probed for the presence of the hptmod gene.All transformants and the untransformed control gave an iden-tical pattern after probing with the P1190–M3490 rDNA frag-ment (Fig. 4B). For each stable transformant, PCR with thevector-specific primer RDNA1 and the chromosome-specificprimer RDNA2 generated a fragment of about 1,600 bp, which,on sequencing, confirmed that pD4 had indeed integrated into atleast one of the rDNA loci (Fig. 4D).

DISCUSSION

M. alpina is a commercially important producer of polyun-saturated fatty acid-rich oil, and in this paper we have de-scribed the first successful genetic transformation of this zygo-mycete. Mucor circinelloides was the first member of this fungalgroup to be transformed (35), and this was followed by similarreports for a number of other zygomycetes (4, 29, 30). Hightransformation frequencies of up to 104 transformants z mg ofvector DNA21, which are associated with the presence of aplasmid sequence promoting autonomous replication, havebeen reported for M. circinelloides (2, 35), but a number ofintegration vectors have also been described (1, 5, 36). In thepresent study, the transformation frequency of M. alpina waslow, and there was no evidence for efficient, extrachromosomalplasmid replication. Indeed, plasmid pAN-MAH only con-ferred transient antibiotic resistance as a result of failing eitherto replicate extrachromosomally or to integrate into the chro-mosome. When a homologous rDNA region was incorporated

into the vector, integration into the chromosome was pro-moted and resulted in stable propagation of the hygromycinresistance phenotype in most cases. The presence of the rDNAfragment in the vector would be expected to increase thechance of homologous integration into the chromosome be-cause, as in other fungi (9, 23), there are probably 150 to 200tandemly repeated copies of the rDNA locus per haploid ge-nome in M. alpina, with each copy representing a potentialintegration target.

In S. cerevisiae, rDNA-containing plasmids integrate withonly low copy numbers, characteristic of standard yeast inte-grating vectors, unless the selection marker used has a defec-tive promoter (20). Even in these yeast transformants withamplified, integrated vector copies, the number of differentrDNA integration sites is low. In the present study, the rDNA-containing vector pD4 integrated only at a few chromosomalsites in M. alpina, as determined by Southern blotting, and atleast one of these was an rDNA locus. The rDNA site ofintegration was confirmed by sequencing the PCR productobtained with primers RDNA1 and RDNA2, which was gen-erated only in the stable transformants. Probing Southern blotswith the P1190–M3490 18S rDNA fragment could not distin-guish between pD4 transformants and the untransformed con-trol, but this was most likely due to the much higher copynumber of endogenous rDNA repeats swamping out the sig-nal(s) from the 18S rDNA region of the integrated vector. Thepresence of extrachromosomally replicating plasmid could notbe detected in the pD4 transformants when DNA digested withClaI, which does not cut in the vector, was probed with thehptmod fragment.

This is one of the few examples in which an rDNA fragmenthas been used to target vector integration in a filamentousfungus. In A. nidulans, incorporation of an rDNA region intoplasmids resulted in homologous integration of a proportion ofvector molecules at the rDNA locus, but the overall transfor-mation frequency was unaffected (31, 37). A proportion of theM. alpina pD4 transformants (25 to 30%) were unstable, how-ever, and became sensitive to hygromycin due to the failureof plasmid integration or to the loss or rearrangement of theintegrated vector, which is a common occurrence in otherzygomycete transformation systems (5, 40). The rearrange-ment of some integrated pD4 molecules may also explainthe varied pattern of faintly hybridizing bands observed in theSouthern analysis of stable transformants (Fig. 4A).

Two pairs of histone H3 and H4 genes were isolated fromM. alpina. Pairs of particular histone genes are common infungi, but the pairs tend not to be linked as in higher organisms(10, 21; N. J. Belshaw, M. J. C. Alcocer, C. S. M. Furniss, andD. B. Archer, submitted for publication). Exceptions are thehistone H2A.2–H2B.2 (HTA2-HTB2) and histone H3.1–H4.1(HHT1–HHF1) gene pairs of S. cerevisiae (28), which are lo-cated on either side of the centromere on chromosome II butare separated by 18.5 kb. Unlike all other histone genes iso-lated from filamentous fungi, the four M. alpina genes lackedintrons (Fig. 5). This is more like the structural organization ofhistone genes from yeasts and higher organisms, which alsolack introns. All other genes described in M. alpina, however,do contain introns (8, 38), some of which are quite large (18;D. A. MacKenzie and A. T. Carter, unpublished data). Thesignificance, if any, of the lack of introns in the M. alpinahistone genes has yet to be determined. All four genes con-tained the consensus poly(A) addition signal AATAAA (13)approximately 100 to 200 nt downstream from the translationstop codon, indicating that these transcripts are probably poly-adenylated, in common with all other histone mRNAs fromfungi.



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Analysis of the two intergenic promoter regions for the pres-ence of putative regulatory sequences revealed that both con-tained AT-rich stretches, which are common in other histonegene promoters, and a number of putative TATA boxes, whichare found in some filamentous fungal promoters (13). Tran-scripts from the two histone H4 genes were produced at ap-proximately the same level during vegetative growth, indicating

that either histone H4 promoter could be used in transforma-tion vectors. The presence of the histone H3.1 promoter andORF in vector pD4 appeared not to affect the efficiency of hptexpression from the histone H4.1 promoter in M. alpina trans-formants. This was in contrast to the situation in P. chrysospo-rium, where integrating transformation vectors containing thehistone H3 gene in addition to the histone H4 promoter were

FIG. 4. (A and B) Southern blots showing the presence of integrated pD4 intransformants of M. alpina CBS 224.37, probed with hptmod fragment HYGR1–HYGR2 (A) and rDNA regions, probed with the 18S rDNA fragment P1190–M3490 (B). Genomic DNA (approximately 5 mg) from independent, stabletransformants 45 to 50 from one transformation experiment and the untrans-formed control (lanes C) was digested with SspI. Phosphorimage exposures forpanels A and B, 1 h and 10 min, respectively. (C) Diagram of a single-crossoverintegration event in the 18S rDNA region between the circular vector pD4 andthe chromosomal locus of CBS 224.37 (not to scale). Annealing positions of thethree rDNA primers, P1190, M3490 and ITS4, and of the two PCR primers,RDNA1 and RDNA2, are indicated. The chromosomal rDNA locus representedshows part of one rDNA repeat unit containing the 39 end of the 18S rDNAregion (9 18S), the two internal transcribed sequences (ITS1 and ITS2), thecomplete 5.8S rDNA region, and the extreme 59 end of the 26S rDNA region(26S 9). (D) Diagram showing the outcome of integration of one or two copies ofpD4 into the 18S rDNA region of CBS 224.37 (not to scale). Annealing positionsof the two PCR primers, RDNA1 and RDNA2, are indicated. Double-headed ar-rows indicate predicted SspI Southern fragments hybridizing to the hptmod probe.



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unable to express antibiotic resistance, probably by a mecha-nism involving DNA methylation (12).

In conclusion, we have shown that M. alpina can be geneti-cally transformed to hygromycin B resistance. This now offersthe prospect of using recombinant methods to modify the fattyacid composition of the oil from this commercially importantorganism, by overexpressing or deleting genes involved in thebiosynthetic pathway.

ACKNOWLEDGMENTS

This work was supported by the Biotechnology and Biological Sci-ences Research Council, by the BBSRC Cell Engineering Link Pro-gramme, and by a studentship from the Thai Government to P.W.

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isolation and use of a homologous histone h4 …in the present paper, we describe the isolation and...

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