Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5′-monophosphate decar☐ylase: use of pyrG for homologous transformation

Gene, 116 (1992) 59-67 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

59

GENE 06349

Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase: use of pyrG for homologous transformation (Recombinant DNA; Zygomycetes; filamentous fungi; transcript mapping; protein homologies; intron positions)

Ernesto P. Benito, Jose M. Diaz-Minguez*, Enrique A. Iturriaga*, Victoria Campuzano and Arturo P. Eslava Area de Gendtica. Departamento de Microbioiogia y Gendtica, Facultad de Biologia. Universidad de Salamanca, 37008 Salamanca (Spain) Received by J.R. Kinghorn: 7 July 1991; Revised/Accepted: 2 December/3 December 1991; Received at publishers: 7 January 1992

SUMMARY

A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene ofMucor circineiloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe. The complete nucleotide sequence of the M. circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis. The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P. blakesleeanus. Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart. The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes. Northern-blot hybridization revealed the pyrG transcript to be approx. 1.0 kb. The M. circinelloidespyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coil when inserted between bacterial expression signals. Additionally, the genomic clone complemented the M. circinelloides pyrG4 mutation. When an M. circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per #g DNA and per 106 viable protoplasts.

INTRODUCTION

The final enzymatic step in the de novo biosynthesis of UMP is catalyzed by the enzyme orotidine-5'-monophosCorrespondence to: Dr. A.P. Eslava, Area de Genftica, Departamento de Microbiologia y Gen6tica, Facultad de Biologia, Universidad de Salamanca, 37008 Salamanca (Spain) Tel. (34-23)294462; Fax (34-23)294513. * Present adresses: (J.M.D.-M.) Department of Plant Pathology, 334 Plant Science Building, Corneli University, Ithaca, NY 14853-5908 (U.S.A.) Tel. (607)255-3243: (E.A.I.) John lnnes Institute, Colney Lane, Norwich NR4 7UH (U.K.)Tel. (44-0603)52571. Abbreviations: aa, amino acid(s); AMV, avian myeloblastosis virus; Ap, ampicillin; ARS, autonomous replication sequence; bp, base pair(s); dd,

phate decarboxylase (OMPD; EC 4.1.1.23) which converts OMP to UMP. The coding gene for this enzyme has been cloned and characterized from a variety of organisms from E. coil to Homo sapiens. The enzyme sequence appears to double-distilled water; dNTP, deoxyribonucleoside triphosphate; 5-FOA, 5-fluoro-orotic acid; kb, kilobase(s) or 1000bp; lacZpo, lac promoteroperator; MMLV, Moloney murine leukemia virus; M., Mucor; nt, nucleotide(s); NTG, N-methyI-N'-nitro.N-nitrosoguanidine; oligo, oligodeoxyribonucleotide; OMP, orotidine-5'-monophosphate; OMPD, OMP decarboxylase; ORF, open reading frame; P., Phycomyces; PA.'polyacrylamide; PCR, polymerase chain reaction; pyrG, gene coding OMPD; a, resistance/resistant; RT, reverse transcriptase; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCi/0.015 M Na~.citrate pH 7.6; tsp, transcription start point(s); u, unit(s): ::, novel joint (insertion or fusion).

60 be well conserved during evolution, possessing stretches of highly conserved aa (Radford and Dix, 1988). Such similaxity has allowed the isolation of the OMPD-coding gene in a number of genetically poorly characterized fungi (Wilson et at., 1988; Vian and Peflalva, 1990; Smith et al., 1991; Diaz-Minguez et at., 1990). In addition, OMPD-deficient mutants have been isolated in a number of fungi by positive selection (Boeke et at., 1984), and the corresponding O M P D coding gene has been used successfully as a selectable marker in the development of efficient genetic transformation of either homologous (Van Hartingsveldt et al., 1987; Oakley et al., 1987; Ruiter-Jacobs et al., 1989; Gruber et ai., 1990b) or heterologous systems (Mattern et at., 1987; Gruber et al., 1990a). To date, only the leuA gene of M. circinelloides, which efficiently transforms the corresponding M. circineiloides ieuA 1 mutant (Van Heeswijck and Roncero, 1984) by autonomous replication (Van Heeswijck, 1986) has been isolated and characterized (Roncero et al., 1989). Recently, integrative transformation by homologous recombination in M. circinelioides has been reported (Arnau et al., 1991). We are interested in the isolation of the genes governing the biosynthesis of carotenoids in P. blakesleeanus and M. circinelloides. Several mutants disturbed in the carotenoids' biosynthetic pathway in both organisms are available. Unfortunately, these mutant strains do not carry a second mutation in a selectable marker. We focused on the M. circinelloides transformation system because of the high transformation frequency achieved in this system when compared with that reported for P. blakesleeames (Sufirez and Eslava, 1988). Moreover, these fungi are two closely related genera and it is believable that they share a common genetic architecture. The P. blakesleeamts leul gene, which is homologous to the M. circinelh~ides ieuA gene can be expressed in the corresponding M. circinelloides mutant (E.A.I,, J.M.D.-M., E.P.B., M.!, Alvarez and A.P.E., submitted for publication), and the P. blakesleeamis pyrG gene can also be e×p~¢ssed ir, M. circinelloides (A.P.E., unpt~blished). Since mutants in the leuA gene cannot be positively selected, it is convenient to develop a transformation system based in the pyrG gene, Therefore, we have cloned and characterized the M, circinelioides pyrG gene and used it in transformat',on studies.

RESULT' AND DISCUSSION

B

J ! i

H

H

Ac

I

I

I

A

Ac

B

I

,J

I....

........

0.2~;

i'

H

B

I

I

H ~

HJ X

I,

,

t

i ~__

~

--

,,e--,

S

i

A

~q

.

.

.

.I

.

.

---

6.1k~ Fig. 1. Restriction map of the M. circineih~idespyrG gene containing DNA fragment and sequencing strategy. Preliminary experiments had shown strong hybridization signals when M. circinelloidesf, hlsilanicus CBS277.49 genomic DNA was cleaved, and was probed with the prrG gene of

P. blakesleeanusunder low-stringencyconditions (hybridizationwas overnight in 6 x SSC/5 x Denhardt's solution/0.5% SDS at 54:C with a final wash in 2 x SSC/0.1'!o SDS at the same temperature). A 3.2-kb BamHl fragment and a 0.5-kb Hindlll fragment cross-hybridized to a 215-bp Hindll-Accl DNA fragmentderived from the P. hh~kesleetmus1o'rG gene eDNA which includes the nt coding sequence for the consensus aa sequence Phe,Glu-Asp-Arg-Lys-Phe found in all the OMPD genes sequenced so far. Consequently, a partial genomic bank of M. circinelhfides DNA was constructed by cloning BarnHI-generated fragments of around 3-4 kb in size in the BamHl site of pBluescriptKS+ and transformed into E. coli. Three thousand colonies were plated and duplicate filters were probed with the same probe in the same conditions leading to the isolationof two independentclones carryinga common 3.2-kb BarnH ! insert. One of these plasmids, pEPMI, was chosen for further analysis and used to generate the restriction map shown in the upper part of the figure, The lines indicate the restriction fragments in pEPM! which hybridized to the P. blakesleeam~spyrG gene-derived probe allowingto locate the M. circineliokk,s IO'rGgene in the 1.8-kb BamHI-A vall fragment of pEPM 1. To confirm that the DNA cloned was a true M. circinelhddes DNA fragment, the hybridizationpatterns obtained with M. circinelhddes genomic DNA and pEPMI restrictions after probing with the 0.57-kb Hindlil-Sall fragmentofpEPM 1 under high-stringencyconditions(Sambrook et al., 1989) were compared. This 3,2-kb BamH! fragment was present in both genomic digests and cloned genomic DNA, and both include the 1.8-kb BamHI-Avall fragment as expected. To determine which was the sense strand in pEPMI and to assess whether the complete pyrGgene had beencloned,both strands of the 1.8-kbBarnH!-,4va!I fragment were sequenced, in the lower part of the figure,the detailed map of the 1.8-kb BamHI-Avall fragment subcloned for sequencingand the sequencing strategy are pres,,'nted. The position and direction of transcription of the gent are indicated by an open arrow. Arrows show the sequenced fragments and the direction and the extent of sequencing. Blackened squares with arrows, segments sequenced using phage M 13 universal primers: blackened circles with arrows, segments sequenced using specific oligos as primers. Abbreviations for restriction endonucleases are: A, `4vail, Ac, Accl; B, BamHl: H, Hindlll; Hi, Hindll; S, Sail; X, XmaIIl.

(a) Sequence ~etd sttt~c~,':,e of the pyrG gene The putative J~. cir~,!nelloides pyrG gene on the 1.8-kb BamHI-Ava!" f r ~ m e n ' (Fig. 1) was subcloned and sequenced. Initially, the 0.5-kb llindIII fragment which hybridized to the probe was sequenced and its six-frame translation was compared with the deduced aa sequence of

the probe. An O R F that was clearly homologous to the corresponding region of the P. blakesleeanus O M P D was observed. Fig. 2 shows the complete nt sequence of the 1.8-kb B a m H l - A v a i l fragment in p E P M I . The analysis of this sequence revealed that the structural region of the

61 -605

GGATCCACCG

GGCAGAATGA TrAACAAGGA AAA'rCrAGGA

TGTrGTrrGT

GTrrATCAAC

-535

CCTrCrGGTG

GGATATI'rCA

CATrACATIT

GACACACCAC AAACAGATGA

-465

AAATGTACAT

CAGAAAACCG TCCAGCrACC

ACCACAAAAA

CACACAACAC CCATrGTACA

TrrCCGACGA

-395

(~AcrCrA'IT

'I'I'rCACAACC

CATi'rGGTAC

ATrGTI'GCC.A

TCI'rCTVrGA

CGCATCACTT

-325 G G A G T I ' r G A A

ACrAAG'ITCA

CACA'rCrACA

TrCGGAAACr

-ZS5 CGAAG'IG'I'rC

AGGCGTIGGA TITGGGTGAT

Ti'I3"CGAATG

GGATGGAAGC ATCYITI'fGA "l t~GAGCrACT V GAAAG'rGAGT GGGTCAAAAA GGTGTACAAT

-185

"rcccrGCf'IT

CrGGAAGCIT

-I 15

GTCTITrATC

ACCrATAGAG GAAGAAGATC C G T G A ( ' i T r A

-45

"rCATITAATC

cA'r(~vl'(~'~(Tr

A T G G A T r G G A ATCI'ATrAAG

"I'GATAATCrG CGI"ATGAGffr

TCTITAG'rAA

TAAACAAA'FF

TITCCrI'cTr

TCCAT~AC"T

~V "rcI'rCATCAG

TATrAGACCG

CAAACCATCr

GTrCCAAGCr

AGAAAGAAAA A A C A T r r r c A

C~.a~AC

I I

AI"G AAC A c r "I'AC A A G A c r TAT AGC GAA c G ' r GGA CAA CAG CAT CCC AAT GCC TGT G c r CGC M~I Asn Thr Tyr I.~ Thr Tyr .~k:r Glu Arg Gly Gin Gin Ilis Pro Asn Ala Cys Ala Arg

61 21

"ICT (71"I"Trr (;AA T I G ATG flAG CGT M C G/~% "rcT AAC Ti'G TCT G T r G c r f l i t GAT o T r ACT ,~r Ixu Phc Glu Lee Met C;ht Arg Asn Glu Scr Ash Leu Scr Val Ala Val Asp Val Thr

121 41

ACA ..~%G A~% GAA "I'IA T I G T('|" A T r (iCl" GAT G c r GTG GGC CCC T I T G T r TGT GTG CI'C AAG "rhr I.)~, I.)~ Glu I,eu I,eu Se. lie Ala Asp Ala Val Gly Pro Phe Val C)~ Val Leu Lys

I~1

GTAAAT('AAA ,~%AAAAAAAG CG'rIAC~%ANI GACATrAGCC: CCITGATI'AA AAGC~%TACAA CAAG

245 61

A('A CAT ATI" (iA(" ATI" G'I'G GAA GAC " l T r GAC CAC GAT ' l ' r G ( r r r G c r CAA T r G GAG C_,AAT r A 'l]tr Ills IIc Asp lie Val (;lu Asp Phe Asp Ihs Asp Leu Val Ala Gin I.eu Glu Gin Leu

.~t5 81

G(~AAAIiAAG CAT (iAC T l ' r TI'G ATC "ITI" GAA (iAT CGT AAA T I T G U r G A T A T r Ala I.)s !.).~ Ihs Asp Phe Leu lie Phe Glu Asp Arg Lys Phe Ala Asp lie

361 q8

G ( ' r I ' I ' G A I T I GAAtYI'I'GA(iC 'I'(irf~sAAAAG A(i(iATATrGA T I ' F C C G T r G C TAG

G

G'rAA

GC AAC A C t Gly Ash Thr

422 101

G'IC AAG ('AT ('AA T A T G C C AAT (HIT A T C 'rAC A A G A'IT G C C "rcr T G G T c r C A T A T r A C A AA'r

4~2 121

(i(~ C A C ACI" (;1"1' c('r GGA (iAA GGT A T r ATC AAG (IGT c l ' r GGC GAG G T r GGT C I T c c r e r r Ala IIns "i~r Val Pro Gly (ilu (ily lie fie l.ys (ily l.eu Gly Glu Val Gly Leu Pro Leu

~42 Ill

G(iC CGT (i(i'l CI'G C I T TI'(i T r G GCi' GAG ATG TCA TCI" AAG G G T GCC T r G ACC AAG GGT AGC

~'J{12 161

TAC ACA T C C GAG "r('r G T r (;AA A T G G c r CG'r C G T A A T t~'iGG A T TI~ G T r TI'I" G G T TI'C A T F Tyr Thr Scr Gin ,Sk:r Val Glu Me! Ala Arg Arg Asn I.',~ Asp Phe Val Phe Gly Phe lie

f~',2 INI

(iC'l' CAA CAC t%A(i ATG A A C (iAA C'AC (iAC G A T G A G fiAT "ITC G']'rG T C A T G TCX? CCT G G T G T r Ala (ihl Ills I)~ Mcl Asn (ilu lhs Asp Asp Glu Asp Phc Val Va[ Met Scr Pro Gly Val

722 21H

G(iT (7I'CCiAT (i'I'CAAG GCiA (IAT (i(iT'l'rG(i{i'l"C'AA (~,% T A C A G A A C C c c r C A C G A A G T C A'vr (ily I~u Asp Val I],~ Gly Asp (ily l~u (ily Gin Gln Tyr Arg Thr Pro Ills Glu Val lie

7~2 221

(i'r(i GAG A(iT (i(iA (iGA G A T A T C A'rc A T I G T C GGC: C G C G G T AI"C T A T G G C A A C C C A G A C C.AA Val Glu Set Gly Gly Asp lie lie lie %,. ,~ly Arg Gly lie Tyr Gly Asn Pro Asp Gin

842 241

G'I'FG A G GC'r CAA G C C AAG ('GT r A T CGC CAA GCI" GGC TGG GA'F G('C TAC c l ' r GAA AGA GTG Val (;lu Ala (ilu Ala I)~ Arg "l'yr Arg (iln Ala Gly 'lip Asp Ale "l'yr IA:u Glu Arg Val

'R)2 261

(!(;'r(TI'C (2~C AA(i AAA ' i ' M J,, Arg Leu Ill.', I.ys I.~

q20 '~l

I~¢~)

Val

I.)~

Ihs

(;1~ Tyr

(ily Arg GI.~ Icu

Ala Asn Gly

Leu I.eu I~u

IIc

Tyr

I.)~

Ala Glu Me! .~r

lie

Scr

P~er "rrp Set

I.ys Gly

Ills

Ala Leu ] ~ r

lie

Thr Ash

I,ys Gly

Set

ACAI (i'l ( i C I G

"l'(;A'l(i(Tl'l(i

ATCATI'GTIT

ATI'IGAAM'r

TACCACGACiG GCCCI'GTI'I'G

A(i'I('(~'ICA

GC(~CA'rAAG

AGAATGCITG

AAATrCCrGC

TGTrCAACAC

GATI'CA(iGAC ACrA'I'i (iAT(I

GGA(i'I('AA(iG CAGGT(TITrC

AC(i(:G'rAAAC

TCTI'CATCAA

AGITGGATGT

ACACGCTrGG

CAAGCATGTC

GTCCCAATrA

NITrA'I(iTCG

11.3{) ATCdlAGAGGG CC('( i'rGATGG A(IG(i(i(~AGAA G G G A G G A G G C 1200

Ala

ACGCCA'I'GGA AGAAGGGATG 'I'I'CCFI'GATC "I'CAG(~GCAT

AAGAAAAG,AA .'AAAITAGAT

CGGATGGACC

Fig. 2. The nt sequence ol' tile 1.8-kb Bamltl-..I vail li'agment containing the M. ,'irc#wlloides pyrG gent and deduced aa sequence of the encoded protcin. A CAAT box and AATAAA motif are indicated by parallel lines. The 5' and 3' intron consensus sequences are underlined, and the putative 3' internal splice signal sequence and their repeats upstream from the 5' end of each intron are overlined. A blackened arrowhead marks the position of the major t.7, and an open arrowhead shows the locations of the three minor t.7~ detected. The nt sequence is numbered from the ATG start codon. The GenBank accession No. is M69112. The deduced aa sequence is also numbered. The nt sequence reactions were performed by the enzymatic dldeoxy chain-termination method (Sanger et al., 1977). Restriction fragments were subcloned into pBluescriptKS+ and K S - vectors (Stratagene, La Jolla, CA) for single-stranded DNA preparation. Sequencing was performed using the T7 DNA polymerase (Pharmacia) and phage M I3 universal primers (occasionally, specific oligos were used as primers in the sequencing reactions) according to the manufacture,'s recommendations. The reaction products were separated on 6", PA/8 M urea gels.

M. circ#leiloidespj'rG gene comprises 919 bp and consists of three exons separated by two intervening sequences of 64 and 57 bp, respectively, showing 77% homology at the nt level with the P. blakesleeamts pyrG gene. A decrease is observed on the G +C content of intron 1 (29%) and intron 2 (3690) when compared with the G+C content of the coding sequence (45 ~,,). The removal of these two introns

gives a 798-bp ORF encoding a putative polypeptide of 265 aa with a molecular size of 29.5 kDa. (b) Transcript analysis A single pyrG gene transcript approx. 1 kb in size was detected in RNA extracted from mycelium grown in minimal medium (Lasker and Borgia, 1980) (Fig. 4).

62

A

B L

1

2

L

1

2

1.6-

1.0-

,

..........

;

.

.

.

.

.

•

.

0.6-

Fig. 3. Determination of the presence of introns in the pyrG gene. (Panel A) Comparison of the sizes of the pyrG genomic DNA and eDNA corresponding to the coding region. PCR (3 FI) was carried out with genomic DNA (lane 1) or eDNA (lane 2) and using oligo 1 as an upstream primer and oligo 2 as the downstream primer. Products were loaded and electrophoresed on a 1% agarose gel. Lane L shows the 100-bp ladder (Pharmacia) as a size marker. The difference between the genomic copy and the eDNA copy PCR-derived products corresponds to the total length of the two introns. (Panel B) Comparison of the sizes of the pyrG gen¢ genomic DNA and eDNA corresponding to the 5' upstream untranslated sequence plus the first 164 nt of the coding region. Lanes were loaded as in panel A but PCR amplifications were carried out using oligos 3 and 4 as upstream and downstream primers, respectively. Methods. Synthesis of eDNA and amplification of the pyrG genomic and eDNA copies were pe.rformed using the Gene Amp RNA PCR Kit from Perkin EImer/Cetus according to manufacturer's specifications with minor modifications, Total RNA was extracted following the method described by Choi et al. (1988) and used to synthesize the eDNA. Two #l of total RNA (1 Fg) was mixed with I FI of oligo(dT) (50 raM) and 1 FI of ddH:O. The mixture was incubated for 5 min at 68 ~C and cooled on ice. Four FI of MgCI: (25 mM)/ 2 pl of 10 x PCR buffer (4 FI; 500 mM KCI/100 mM Tris.HCI pH 8,3 at 20 °C)/8 FI of dNTPs (2,5 mM each)/1 ~d RNase inhibitor (20 u per pi)/ 1 pl MMLV RT (50 u per FI) were added. The reaction was incubated at 42°C for 15 min, 99°C for 5 rain and 5°C for 5 min in a Perkin Elmer/ Cetus DNA Thermal Cycler. PCR amplifications (Saiki et al, 1988) of the genomic pyrG copy or eDNA copy were carried out in 100 #l final volume reactions containing 2 mM MgCI,/10 mM Tris,HCI pH 8,3 at 20°C/ 50raM KCI/0.2mM each dNTP/0.15FM upstream primer/0,15FM downstream primer/100 ng of genomic DNA or 2 FI of the eDNA mixtare, The reactions were overlaid with light mineral oil and heated for 2 min at 95°C. AmpliTaq DNA polymerase (2.5 u) was added and the reaction mixtures were subjected to 35 cycles of denaturing at 95 °C for 0,5 min, annealing at 55°C for 0,5 min and polymerization at 72°C for 1 min in a DNA Thermal Cycler. A final polymerization step at 72 °C for 5 min was carried out to ensure that all PCR products were completely

Fig. 4. Northern hybridization analysis of total RNA isolated from M. circinelloides. 60 Fg of total RNA was electrophoresed through a 1.4% agarose gel containing formaldehyde, transferred to a nylon membrane (Hybond-N, Amersham) and hybridized to the 3:P-labelled 570-bp HindIlI-Sall DNA fragment derived from pEPMI according to Sambrook et al, (1989). Numbers on the left correspond to positions of RNA size markers in kb (Boehringer Mannheim).

Several tsp were observed by primer extension. A major tsp was mapped to 123 nt upstream fi'om the ATG start codon. This implies a long 5'-untranslated sequence in the pyrG mRNA when compared with the leuA mRNA of M. circinelloides which shows a 16-20 nt long 5'-untranslated sequence (Roncero et al., 1989). Three minor tsp were located at nt positions - 122, -214 and -326 (Fig. 5). The tsp mapped by primer extension analysis are correctly located only if no intron is present within the 5'-untranslated region. In filamentous fungi, the presence of an intron has been determined in the upstream untran,,lated transcribed region of the Aspergillus nidulans gpdA gene (Punt et al.,

double-stranded. The primers used for PCR amplifications were: oligo I:5'-AATGGATCCATGAACACTTACAAGACTTAT (nt position -9to +21); oligo 2: 5°-GACGGATCCTTATTTCTTGTGGAGACG CAC (complementary to nt positions + 898 to + 928); oligo 3: 5 ' - T G GATCCTGAAACTAAGTTCACACATCT (nt position -326 to -299) and oligo 4: 5'-AAGGATCCCACAGCATCAGCAATAGACAA (complementary to nt positions + 136 to + 164). All the primers were designed containing a BamHl site at the 5 ° end to facilitate the cloning of the amplified products. The PCR products were extracted with phenolchloroform, digested with BamHI and cloned into the BamHI site of pBluescriptKS+. The regions of interest of the genomic and cDNAamplified copies were sequenced and compared to locate the M. circinelloides pyrG gene introns.

63 1

2

3

AGCT

nt -326

..:B

31

59

G T A G A A G

C A T C T T C

S'

~-

-214

~

- 123 - 1 2 2

3'

Fig. 5. Determination of the t.~l, of the M. ,'ircinelloides pyrG gene. The products of the primer extension reactions carried out in the absence of template (lane i), with 30 pg of yeast tRNA (lane 2) or with 30/ag oftotal M. circineiloides RNA (lane 3) were run in parallel with the sequencing reactions (lanes A, G, C and T) obtained when the same oligo was used as primer and the pyrG DNA strand corresponding to the RNA transcript was used as template. Arrows indicate the extended detected products (large arrowhead, major tsp; small arrow, minor tsp). Labelling of the primer and primer-extension analysis were performed according to Sambrook et al. (1989). A 20-mer synthetic primer 5'-GGCATTGGGATGCTGTTGTC complementary to nt position + 32 to + 5 ! ofthe pyrG gene sequence was 5' end labelled with 32p with polynucleotide kinase (Boehringer Mannheim). About 3 ng of the labelled primer (5 x 105 cpm) were mixed with 30/~g ofM. circineiioides total RNA, desiccated under vacuum and resuspended in 30/~1 of hybridization buffer (80% formamide/0.4 M NaCl/40 mM PIPES pH 6.6/1 mM EDTA). The volume was heated at 85 °C for 10 rain and transferred to 28 ~C allowing the annealing for 12 h. The primer-RNA hybrid was ethanol-precipitated, resuspended in 20 pl of RT buffer [50 mM Tris.HCI pH 7.6/60 mM KCI/10 mM MgCl2/l mM each dNTP/I mM dithiotreitol/! u per pl of placental RNase inhibitor/ 1.5 u per/~1 of AMV RT (Pharmacia)], and incubated at 37 °C for 2 h. The reaction was stopped by the addition of I ld of 0.5 M EDTA, extracted with phenol-chloroform, ethanol-precipitated and resuspended in formamide loading buffer (90°0 formamide/10mM EDTA/0.1% xylene cyanol/0.1% bromophenol blue). Control reactions were performed in the same conditions using the same amount of primer with or without 30/~g of yeast tRNA. The extended products were separated on a 6,°,0 PA/8 M urea gel.

1988) and of the egl3 gene of Trichoderma reesei (Saloheimo et al., 1988). To study this possibility in the M. circinelloides pyrG gene, the sequence between the minor tsp located at

nt position -326 and nt + 161 of the coding region was amplified by PCR using as template either the genomic DNA copy or the eDNA copy. The data presented in Fig. 3B show that the sizes of these two aml: ,ified products are identical, indicating that no introns are present within the M. circinelloides pyrG 5'-untranslated region. (c) The 5'- and 3'-flanking regions The 605 nt upstream from the ATG start codon and the 330 nt downstream from the TAA stop codon were sequenced (Fig. 2). The G+C content of the upstream region was significantly lower (36%) than the G+C content of the coding region (46%). A CAAT box was detected between nt positions -189 and -186 (5'-CAAT), 65 nt upstream from the major tsp. A consensus TATA box was located at nt positions -102 to -99, but its relative location with respect to the major tsp, 21 nt downstream from it, probably renders it nonfunctional. However, a sequence resembling a TATA box was identified between nt positions - 146 and -141 (5'-TTATTA) at 18 nt upstream from the major tsp. It is noteworthy that the ieuA gene of M. circineiioides possesses a CAAT box but no TATA sequence in the 5'untranslated region (Roncero et al., 1989). The 3'-noncoding flanking region includes the consensus polyadenylation signal sequence AATAAA 58 nt downstream from the stop codon TAA (5'-AATAAA, nt + ~,79 to + 983). This sequence appears almost invariably in the 3' region of eukaryotic genes (Proudfoot and Brownlee, 1976) but is usually lacking in filamentous fungi genes, particularly in Ascomycetes and Basidiomycetes (Ballance, 1986; Rambosek and Leach, 1987). Interestingly, the pyrG as well as the M. circinelloides leuA (Roncero et al., 1989) genes, and trpl (Choi et al., 1988), pyrG (Diaz-Minguez etai., 1990) and leul (Iturriaga etal., 1990) genes of P. blakesleeanus (another Zygomycete) contain the consensus polyadenylation signal sequence AATAAA. (d) Protein size and codon usage The predicted M. circinelloides polypeptide of 265 aa is similar in size to other OMPD proteins from most other fungi except those from Neurospora crassa (Newbury et al., 1986), Cephalosporium acremonium (Vian and Peflalva, 1989) and T. reesei (Smith et al., 1991) which have an additional 80-90 aa in the N-terminal portion of the protein. Analysis of codon usage of the coding sequence shows a third position preference for pyrimidines over purines, wherever possible, and conforms therefore to the general tendency in filamentous fungi genes (Rambosek and Leach, 1987). In contrast, there is an absence of 12 codons: CTA (Leu), ATA (lie), GTA (Val), TCG (Ser), CCG (Pro), ACG (Thr), GCG (Ala), TGC (Cys), CGA (Arg), CGG (Arg), AGG (Arg) and GGC (Gly). Such codons are also absent or rare ( < 0.03 %) in the M. circinelloides leuA gene.

64 fungi introns and include consensus splice sequences (Ballance, 1986; Gurr et al., 1987; Oakley et al., 1987) (Fig. 2). The positions of both M. circinelloidespyrG gene introns are identical to those reported for the two introns of the pyrG gene from the Zygomycete P. blakesleeam~s (DiazMinguez et al., 1990) and the URA1 gene from the Basidiomycete Schizophyllum commune (Froeliger et al., 1989). It would be interesting to know the number and location of possible introns in the homologous gene from the Basidiomycete Ustilago maydis, but only the cDNA sequence has been reported (Kronstad et al., 1989).

(e) Introns The presence of introns was considered because of the presence of several stop codons in the ORF initially detected for the N-terminal region of the putative protein. Moreover, a second ORF was observed which matched the C-terminal region of the P. blakesleeanus OMPD. To confirm the presence of introns in the M. circinelloides pyrG gene, both the genomic copy and the corresponding cDNA were amplified by PCR, and the sizes of the products compared (Fig. 3A). Their precise location and length were determined by sequencing both PCR products. Analysis and comparison of the genomic DNA and cDNA sequences confirmed the presence of two introns, 64 and 57 bp in length, the first one located between nt positions + 181 and + 244 and the second between + 357 and + 413 (Fig. 2). Both M. circinelloides pyrG gene introns show a similar structure to that commonly observed in filamentous

(f) Homology with other O M P D sequences The deduced aa sequence of the M. circinelloides OM PD shows a high level of similarity with other fungal O M P D

sequences (Fig. 6) supporting the analysis performed by Radford and Dix (1988). When this sequence is compared

Ncir

1 M. . . . N T - - - ¥--K-TYSER GQQHPNACAR SL . . . . FELM ERNESN~VA VDVTTKKELL SIADAVGPFV CVLKTHIDIV~ . . . . . . . EDFD-HD-LV

Pblk

I 1

MML--NT--MTAAH

¥--K-SYTER AEQHPNACAR KLTYGQR A A R P T N P A A K

SL .... FELM E R K K T N L S V A A L .... LETM E Q K K S N L S V S

VDVTTKKELL SIADSVGPYV V D V V K S A D L L AIVDTVGPYI

CVLKTHIDIV CLIKTHVDW

I 1 1

MSSKSQ MSTSQETQPH MS

LTYTAR A S K H P N A L A K WSLKQSFAER VE--S-S-TH KATYKER A A T H P S P V A A

R L .... FEIA E A K K T N V T V S PLTSYLFRLM EVKQSNLCLS KL .... FNIM H E K Q T N L C A S

A D V T T T K E L L DLADRLGP¥I ADVEHARDLL ALADKVGPSI L D V R T T K E L L ELVEALGPKI

A V I K T H I D I L SDFS-DE-TI WLKTHYDLI TGWDYHPHTG CLLKTHVDIL TDFS-MEGTV

Sco~

~nla Nora Soe~

•

o

. . . . . . . . . .

. . . . . ~ . . .

..

•

o.

.o

. o ~ e o . o

•

o r .

. i

..

o.

Molt Pblk Scom

75 AQ--T:Ni~.QI.,AK]L,~'iDFLIFEDR KFADIGNTVK HQYANGIYKI AS-WSHITNA HTVPGEGIIK GLGEVGL---

Ani~ Nora Sce~

77 EG--LKALAQ K H D F L I F E D R K F I D I G N T V Q KQYHRGTLRI - S E W A H I I N C SILPGEGIVE A L A Q T A S A - -

77 77

87 75

VQ--LEALAK TK--LQALAE

TGAKLAALAR KP--LKALSA *

*

KHDFLIFEDR KHDFLIFEDR

KFADIGNTVK KFADIGNTVA

KHGFLIFEDR KKVDIGSTVQ KYNFLLFEDR K F A D I G N T V K *

**

****

*

***

**

HQYEKGVYKI LQ¥SSGVHKI

KQYTAGTARI LQ¥SAGVYRI **

*

*

AS-WSNITNA AS-WSHITNA

V-EWAHITNA A-EWADITNA *

*

.

HTVPGEGIIK GLGEVGL--HPVPGPSIIS G L A S V G Q - - -

DIHAGEAMVS HGWGPGIVS

*

.

.

.

.

.

.

....... PLG ....... PLG

-PDFSYGP-E A M A Q A A Q K - - -( ...... )- - P G I E E A P L D GLKQAAEE . . . . . . VTKE-P

*

Scom

144 144

RGLLLLAEMS RGLLLLAEMS

SKGALTKGS¥ T T E S V E M A R R TKGSLATGA¥ T E A A V Q M A R E

Ania Nora Seer

150 250 145

RGLLILAEMT RGLLZLAQMS RGLLMLAELS

SKGSLATGQ¥ T T S S V D Y A R K YKNFVMGFVS TR ...... S- -L .... GEVQ S E V S S P S D E E SKGCLMDGR¥ T W E C V K A A R K N K G F V M G Y V A Q Q N L N G I T K E A L A P S Y E D - - G E S T T - - - E E CKGSLSTGE¥ T K G T V D I A K S D K D E V I G F I A QR ...... D- -M .... G G R D EG¥ .......

Mcir Pblk SCO~

203

--DVKG~:~I.~ QQYRTPHEVI V~-SGGDIIIV GI~GIYGNP-D Q--V-RAQ---A- I~RQAGI~IDA YL~-RV-RLH KK

205 213

--DIKGDGLG --DVKGDGMG

QQYRTPHEVI QQYRTPKQW

Ania Ncra Scer

219 335 207

--SSKGDKLG EEAPQGDGLG --DDKGDALG

QQYQTPASAI G - R G A D F I I A QQYNTPDNLV NIKGTD£AIV QQYRTVDDVV S-TGSDIIIV

VESGCDVIIV QEDGCDVIIV

EDFD-KD-LA EDFD-SS-LV

N K D F V F G F I A QH . . . . . K- -M .... N E Y P DE D ..... FVVM T P G - - V G L - N R G F V I G F I A QR ...... R- -M .... DGIG A P P G V N V G D E D ..... F L V L T P G - - V G L - -

GRGIYGKP-D GRGIYGK--D

E--V-EAQ---SPSKV-EEIRRQA-

KRYREAGWNA ERYQAAGWAA

G R G I Y A A P - D P - - V - Q A A ..... Q Q Y Q K E G W E A GRGIITAA-D P--PAEAE---R¥ RR--KA-WKA G R G L F A K G R D A - K V - E G E . . . . . . RYRKAGWEA

D ..... FVVF T T G - - V N I - EAQADNFIHM TPGCKLPPPG D .......W L I M T P G - - V G L - -

Y L E R V - R M H KA YTERV-NAL V

265 267 278

YLARV-GGN YQDRRERLA ¥LRRC-GQQ N

277 397 267

Fig. 6. Deduced aa sequence of the OMPD of M. circi, elloides (MOO')and comparison with the OMPD sequences of P. blakesleeanus (Pbik), S. commune (Scorn), A. ,iger (Anig), N. crassa (Nora) and S. cerevisiae (Seer). Small gaps were introduced into the sequences to achieve optimal alignment and are represented by dashes. The double dashed line represents an 87-aa insert which is present only in the N. crassa sequencc. Dots indicate the aa appearing in equivalent positions in the M. circinelloides, P. blakesleeamt~ and S. commune OMPD sequences, while asterisks indicate equivalent positions in the six sequences. Black arrowheads mark the positions of the first and second introns of the M. circmelloides, P. blakesleemms and S. comm,ne OMPD coding sequences which are exactly coincident in these three organisms. The downward arrow shows the position of the only intron present within the pyrG gene of A. niger. The five regions of highly conserved aa found in all fungal OMPD sequences are underlined.

65 with those ofP. blakesleeanus (Diaz-Minguez et al., 1990), S. commune (Froeliger etal., 1989), Saccharomyces cerevisiae (Rose et al., 1984), Aspergiilus niger (Wilson et al., 1988) and N. crassa (Newbury et al., 1986), the homology percentages are 96~o, 76%, 71°/0, 67% and 58%, respectively. Therefore, the highest level of similarity is between the M. circinelloides and P. blakesleeanus sequences, supporting the evidence of close relationship between these two Zygomycetes (Fig. 6). (g) Functional complementation of an Escherichia coli pyrF- mutation r tasmids containing the genomic copy of the M. circinelloides pyrG gene were unable to complement the pyrF::Mu-1 mutation ofE. coli DB6656 (pyrF::Mu-1, trpam, lacZ.,m, hsdR) (Bach et al., 1979). However, when a pyrG eDNA copy was cloned in the BamHI site of pBluescriptKS+ and in the sense orientation relative to the iacZpo region, the resulting plasmid, pEPM 143, efficiently transformed the pyrF::Mu-! mutant to uracil prototrophy. As expected, pyrG eDNA cloned in reverse orientation in pEPMI41, was unable to complement this mutation. (h) Homologous transformation It has been previously reported that a 4.4-kb Pstl M. circineiloides genomic DNA fragment which includes the M. circinelloides ieuA gene and an A R S efficiently transforms an M. circinelloides leuA 1 mutant by autonomous replication (Van Heeswijck, 1986). This DNA fragment was cloned into the Pstl site of pEPM 1, and the resulting plasmid, pEPMII (Fig. 7), was used to transform the

Saci BmnH!

EcoRI

pEPMII I06O0bp

.o,iK=zH ' BamHl

Fig. 7. Restrictionmap of pEPMII. Singlelines and striped box represent vectorsequences.Open boxes and blackenedboxes representM. circinelloides genomicnt sequences.

M. circineiloides mutant strain MS 12 (leuA l, pyrG4) to leucine and uracil prototrophy. Several transformations were performed according to Van Heeswijck and Roncero (1984) and the average transformation frequency obtained in five experiments ranged from 600 to 800 transformants per #g DNA and per 106 viable protoplasts (no transformants were obtained in control transformation experiments using pBluescriptKS+ ). Fifteen transformants from independent transformation experiments were studied for mitotic stability (Fig. 8 legend). The percentage (average from the 15 transformants) of spores retaining the transforming Leu +, Ura + phenotype was 15-20% in the first generation when transformants were grown on selective medium and remained constant in the second and third generations, while these percentages were 0.5%, 0.1 °/o and 0.02% in the first, second and third generations, respectively, when transformants were grown under nonselective conditions. These results are in agreement with those reported by Van Heewijck (1986) using the same ARS. Total genomic DNA from these transformants were analyzed by Southern-blot hybridization and all gave identical signals. Therefore, one was chosen as representative (Fig. 8). When total undigested genomic DNA from the transformants were probed with pBluescriptKS+ no hybridization signal corresponding to chromosomal DNA was detected, whereas faint extrachromosomal bands were detected after long exposures of the filter (Fig. 8A, lane 3). The hybridization pattern observed was slightly different from that obtained with undigested pEPM11 DNA isolated from E. cog (Fig. 8A, lane 1). Total undigested DNA from the recipient strain gave no chromosomal or extrachromosomal hybridization signal (Fig. 8A, lane 2). When EcoRI digestions were carried out an probed with pBluescriptKS+, the 9.4-kb fragment of pEPMII, which includes vector sequences, was detected in transformant DNA but not in the genomic DNA from the untransformed strain (Fig. 8B). When the filter was reprobed with the 3.2-kb BamHI fragment which includes the M. circinelloides pyrG gene, two bands were identified: the 4.2-kb EcoRI band corresponding to the resident copy of this fragment in the recipient strain and the 9.4-kb EcoRI band corresponding to the plasmid copy (Fig. 8C). This hybridization pattern matches the expected pattern if the transforming plasmid replicates autonomously. Further evidence for the presence of free pEPM 11 molecules in M. circinelloides pyrG + transformants was obtained by using total undigested genomic DNA from these strains to transform E. coil DH5 (sutE44, hsdR 17, recA 1, endA 1, gyrA 96, thi-1, relA 1) to Ap e. The yield of E. coil Ap I~ transformants varied (0.5-2 transformants per #g DNA) between DNA preparations from different M. circinelloidespyrG ÷ transformants, but intact pEPM 11 was recovered from all the E. coli transformants analysed.

66

B

A 1

C

1 23

2 3

1 23

6.7-

m

4,3-

-

~..

~..

87-

4,3

(2) C o m p a r i s o n of the n u m b e r a n d location of introns as well as comparison of the deduced aa sequences encoded by the respective O M P D - c o d i n g genes from different fungi support the evidence of close relationship between the two Zygomycetes M. circinelioides and P. blakesleeanus, and suggest a close relationship between these two Zygomycetes and the Basidiomycete S. commune. (3) The M. circinelloides pyrG gene complements an M. circinelloides pyrG4 mutation and m a y be used as a selectable m a r k e r in transformation experiments. When an M. circinelloides A R S is included in the transforming plasmid high transformation frequencies by autonomous replication are achieved.

"

ACKNOWLEDGEM ENTS

N Fig. 8. Southern hybridization analysis of transformants. Lanes: 1, pEPM 11 (2 ng); 2, M. circilaelloidesuntransformed recipient strain genomic DNA (5/~g); 3, gonomic DNA from a representative tra~sformant obtained with pEPMII (5 #g). Samples were undigested (penel A) or digested with EcoRI (panels B and C), fractionated on a 0.5% (panel A) or 0.6°.0 (panels B and C) agarose gel and transferred to a n~,lon membrane (Hybond-N, Amersham). The filters were probed wi~h the 3~.p. labelled pBluescript KS + (panels A and B) or with the 3~-P-lab~liod3.2-kb BamHl DNA fragment which includes the M. circinelloide.~pyrG gone (panel C). Hybridizations were peri'ormed under high-stringency conditions (Sambrook et al., 1989). Exposure times were 15 h (pa:ael A, lane I and panels B and C) or 48 h (panel A, lanes Z and 3). The alkaline method was used to isolate plasmid DNA and genomic DNA was extracted according to the method of Yelton et al. (1984). The DNA fragments were radioactively labelled by the method of Feinberg and Vogelstein (1983) using the multiprime DNA Labelling System (Amersham) according to supplier's specifications. Electrophoresis procedures, transfer of DNA to nylon filters and Southern hybridizations were performed as described by Sambrook et al. (1989). In each panel the numbers on the left correspond to positions of size markers (kb). The bold arrow in panel A indicates the position of undigested genomic DNA, The M. circinelloidesmutant strain MSI2 (leuA1, pyrG4) used in transformation experiments was isolated after treatment of strain R7b (leuA !) with NTG and selection on 5-FOA. Transformants studied for mitotic stability were subjected to three successive transfers either to nonselective medium (YPG) or to selective medium (YNB) (Lasker and Borgia, 1980) and allowed to complete the full vegetative growth cycle culminating with spores formation after each transfer. The percentage of spores retaining the transforming phenotype in each generation was estimated by parallel plating of appropriate dilutions onto selective or nonselective medium. Conclusions (1) A M, circinelloides genomic D N A fragment containing the pyrG gone has been cloned by heterologous hybridization with the pyrG of P. blakesleeanus. This is the second M. circinelloides gone that has been cloned and analyzed, and the first time that introns are reported in Mucor.

(i)

We thank Dr. D. yon Wettstein for providing the plasmid pleu4, and Dr. M . I . G . Roncero for her useful suggestions and for providing the M. circinelloides mutant strain RTb (leuA 1). This work was supported by the Direcci6n General de Investigaci6n Cientifica y T~cniea of Spain ( Gr a n t PB 88-0376). J . M . D . M . , E.A.I. and V.C. held graduate fellowships from Ministerio de Educaci6n y Cieneia o f Spain and University of S a l a m a n c a .

REFERENCES Arnau, J,, Jepsen, L.P. and Stmman, P.: Integrative transformation by homologous recombination in the zygomycete Mucor circineiloides. Mol. Gen. Genet. 225 (1991) 193-198. Bach, M,L,, Lacrout¢, F. and Botstcin, D.: Evidence for transcriptional regulation of orotidine.5'-phosphate decarboxylase in yeast by hybridization ofmRNA to the yeast structural gone cloned in Escherichia coll. Prec. Natl. Acad. Sci. USA 76 (1979) 386-390. Ballanco, D.J'.: Sequences important for gone expression in filamentous fungi, Yeast 2 (1986) 229-236. Boeke, J,D., LaCroute, F. and Fink, G.R.: A positive selection for mutants lacking orotidine-5'-phosphat¢ docarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197 (1984) 345-346. Choi, H.T., Revuelta, J.L,, Sadhu, Ch. and Jayaram, M.: Structural organization of the TRPI gone of Phycomyces blakesleeanus: implications for evolutionary gone fusion in fungi. Gone 71 (1988) 85-95, Diaz-Minguez, J.M., Iturriaga, E.A,, Benito, E.P., Corrochano, L.M. and Eslava, A.P.: Isolation and molecular analysis of the orotidine-5'phosphate decarboxylase gone (pyrG) of Phrcomyces blakesleeanus. Mol. Gen. Genet. 224 (1990) 269-278. Feinberg, A,P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132 (1983) 6-13. Froeliger, E.H., Uilrich, R.C. and Novotny, C.P.: Sequence analysis of the URA1 gone encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune. Gone 83 (1989) 387-393, Gruber, F., Visser, J., Kubicek, C.P. and De Graaff, L.H.: The development of a heterologous transformation system for the cellulolytic fungus Trichodermareeseibased on a pyrG-negativemutant strain. Curr. Goner. 18 (1990)a) 71-76.

67 Gruber, F., Visser, J., Kubicek, C.P. and De Graaff, L.H.: Cloning of the Trichodenna reesei pyrG gene and its use as a homologous marker for a high-frequency ,~,ansformation system. Curr. Genet. 18 (1990b) 447-451. Gurr, S.J., Unkles, S.E. and Kinghorn, J.R.: The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, 1987, pp. 93-139. Iturriaga, E.A., Diaz-Minguez, J.M., Benito, E.P., Alvarez, M.I. and Eslava, A.P.: Nucleotide sequence of the Phyco,o'ces blakesleeatms leul gene. Nucleic Acids Res. 18 (1990)4612. Kronstad, J.W., Wang, J., Covert, S.F., Holden, D.W., McKnight, G.L. and Leong, S.A.: Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenie fungus Ustilago maydis. Gene 79 (1989) 97-106. Lasker, B.A. and Borgia, P.T.: High frequency heterocaryon formation by Mucor racemosus. J. Bacteriol. 141 (1980) 565-567. Mattern, I.E., Unkles, S., Kinghorn, J.R., Pouwels, P.H. and Van den Hondel, C.A.M.J.J.: Transformation of Aspergillus ory-ae using the A. nigerpyrG gene. Mol. Gen. Genet. 210 (1987) 460-461. Newbury, S.F., Glazebrook, J.A. and Radford, A.: Sequence analysis of the p)'r4 (orotidine 5'-P deearboxylase) gene of Neurospora crassa. Gene 43 ( ! 986) 5 !-58. Oakley, B.R., Rinehart, J.E., Mitchell, B.L., Oakley, C.E., Carmona, C.. Gray, G.L. and May, G.S.: Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans. Gene 61 (1987) 385-399. Proudfoot, N.J. and Brownlee, G.G.: 3' Non-coding region sequences in eukaryotic messenger RNA. Nature 263 (1976) 211-214. Punt, P.J., Dingemanse, M.A., Jacobs-Meijsing, B.J.M., Pouwels, P.H. and Van den Hondd, C.A.M.J.J.: Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. Gene 69 (1988)49-57. Radford, A. and Dix, N.I.M.: Comparison of the orotidine 5'monophosphate decarboxylase sequences of eight species Genome 30 (1988) 501-505. Rambos¢k, J. and Leach, J.: Recombinant DNA in filamentous fungi: progress and prospects. CRC Crit. Rev. Biotechnol. 6 (1987) 357393. Roncero, M.I.G., Jepsen, L.P., Stroman, P. and Van Heeswijck, R.: Characterization of a leuA gene and an ARS element from Mucor circinelloides. Gene 84 (1989) 335-343. Rose, M., Grisafi, P. and Botstein, D.: Structure and function of the yeast URA3 gene: expression in Escherichia coli. Gene 29 (1984) 113-124. Ruiter-Jacobs, Y.M.J.T., Broekhuijsen, M., Unkles, S.E., Campbell, E.I.,

Kinghorn, J.R., Contreras, R., Pouwels, P.H. and Van den Hondel, C.A.M.J.J.: A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus or.17ae. Curt. Genet. 16 (1989) 159-163. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn G.T., Mullis, K.B. and Erlich, H.A.: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 (1988) 487-491. Saloheimo, M., Lehtovaara, P., Penttil',t, M., Teeri, T.T., St~thlberg, J., Johansson, G., Pettersson, G., Claeyssens, M., Tomme, P. and Knowles, J.K.C.: EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gent and enzyme. Gene 63 (1988) 11-21. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A L.aboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chaintermination inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 54635467. Smith, J.L., Bayliss, F.T. and Ward, M.: Sequence ofthe cloned pyr4 gene of Trichmterma reesei and its use as a homologous selectablc marker for transformation. Curr. Genet. 19 (1991) 27-33. Su.'irez, T. and Eslava, A.P.: Transformation of Phycooo'ces with a bacterial gene for kanamycin resistance. Mol. Gen. Genet. 212 (1987) 120-123. Van Hartingsveidt, W., Mattern, I.E., Van Zeijl, C.M.J., Pouwels P.H. and Van den Hondel, C.A.M.J.J.: Development of a homologous transformation system for Aspergiilus niger based on the pyrG gene. Mol. Gen. Genet. 206 (1987)71-75. Van Heeswijck, R.: Autonomous replication of plasmids in Mucor transformants. Carlsberg Res. Commun. 51 (1986)433-443. Van Heeswijck, R. and Roncero, M.I.G.: High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res. Commun. 49 (1984) 691-702. Vian, A. and Peflalva, M.A.: Nucleotide sequence of the Cephalosporium acremonium pyr4 gene. Nucleic Acids Res. 17 (1989) 8874. Vian, A. and Peflalva, M.A.: Cloning of the PYR4 gene encoding orotidine-5'-phosphate decarboxylase in Cephalosporium acremonimn. Curr. Genet. 17 (1990) 223-227. Wilson, L.J., Carmona C.L. and Ward, M.: Sequence of the Aspergillus nigerpyrG gene. Nucleic Acids Res. 16 (1988) 2339. Yelton, M.M., Timberlake, W.E. and Van den Hondel, C.A.M.J.J.: Transtormation ofAspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81 (1984) 1470-1474.