- Email: [email protected]
Cloning, characterization and expression of pepF, a gene encoding a serine carboxypeptidase from Aspergillus niger
Gene, 151 (1994) 73-79 0 1994 Elsevier Science B.V. All rights reserved.
0378-l 119/94/$07.00
GENE 08380
Cloning, characterization and expression ofpep& a gene encoding a serine carboxypeptidase from Aspergillus niger (Exo-protease; proteinase F; transcriptional
regulation; carbon repression; nitrogen repression; pH regulation)
J.P.T.W. van den Hombergh”, G. Jaraib, F.P. Buxtonb and J. Vissera “Section Molecular Genetics oflndustrial Microorganisms, Wageningen Agricultural University, Dreqenlaan 2, NL-6703-HA
Wageningen, The
Netherlands: and bBiotechnology Department, Ciba-Geigy AC. CH4002 Basel, Switzerland. Tel. (41-61) 696-1661 Received by J.R. Kinghorn:
13 April 1994; Revised/Accepted:
10 June/l2
June 1994; Received at publishers:
25 August
1994
SUMMARY
We have cloned a gene (pepF) encoding a serine carboxypeptidase, proteinase F (PEPF), from Aspergillus niger. The sequences were identified in a phage h genomic DNA library using a synthetic probe based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA clones reveals that it is composed of four exons of 199,283,227 and 881 bp, interrupted by three introns of 53,69 and 59 bp. The sequence of pepF codes for a polypeptide of 530 amino acids (aa), of which the first 52 aa are not present in the mature PEPF. This region may represent a prepro sequence that is removed by proteolytic cleavage at a monobasic cleavage site (LYs~~). Northern blot analysis of total cellular RNA extracted from A. niger cells indicates that pepF is transcribed as a single 1%kb mRNA, which is regulated by nitrogen and carbon repression, specific induction and the pH of the culture medium.
INTRODUCTION
Studying proteases of Aspergilli is of considerable importance as these fungi are used for the commercial production of proteases. Problems with homologous and heterologous protein production in Aspergilli are often associated with the high level of extracellular proteases Correspondence
to: Dr.
J. Visser,
Section
Molecular
Genetics
of
Industrial Microorganisms, Wageningen Agricultural University, 6703 HA Wageningen, The Netherlands. Tel. (31-8370) 84439; Fax (31-8370) 84011; e-mail: [email protected] Abbreviations:
aa, amino
acid(s);
A., Aspergillus; An, A. niger; BSA,
bovine serum albumin; bp, base pair(s); C., Candida; CPD, carboxypeptidase; CPY, S. cerevisiae Ser-CPD Y; GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase or 1000 bp; N, A or C or G or T; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; PEPC, A. niger proteinase C; pepC, gene encoding PEPC; PEPF, A. niger proteinase F; pepF, gene encoding PEPF; R, A or G; S, G or C; S., Saccharomyces; SDS, sodium dodecyl CPD, serine-CPD; SSC, 0.15 M NaCI/O.OlS M Nascitrate C or T. SSDI 0378-1119(94)00591-5
sulfate; SerpH 7.6; Y,
in these organisms. In addition proteases have been implicated in the pathogenicity of A. fumigatus. A variety of different proteases from Aspergilli have been characterized (Ichishima, 1972; Bosmann, 1973; Chopra and Metha, 1985). In A. niger (An) a wide range of proteolytic activities comprising acidic (Krishnan and Vijayalakshmi, 1985; Takahashi et al., 1991), semialkaline and alkaline serine proteases (Sakka et al., 1985; Barthomeuf et al., 1988) and serine carboxypeptidases (Ser-CPD) (Krishnan and Vijayalakshmi, 1985; 1986; Dal Degan et al., 1992) have been described. Several proteases have been cloned from Aspergilli, including a pepsin-type acid protease from An var. awamori (Berka et al., 1990) and from An (Jarai et al., 1994b), a non-pepsin-type acid protease from An var. macrosporus (Inoue et al., 1991), and two subtilisins from An (Frederick et al., 1993; Jarai et al., 1994a). However, no genes coding for Ser-CPDtype proteases have been cloned. Ser-CPDs (peptidyl+amino acid hydrolases; EC. 3.4.16.1) are exo-peptidases that sequentially release most aa residues, including proline, from the C terminus of
74
polypeptide chains at acidic pH utilising a diisopropylphosphorofluoridate-sensitive active-site Ser residue. SerCPDs have been extensively studied as they can be used in food industry for debittering of peptide mixtures, and are very useful tools for C-terminal sequencing, and peptide synthesis. Kumagai et al. (1981) described one Ser-CPD from An and more recently Krishnan and Vijayalakshmi (1986) isolated three Ser-CPDs that are probably isoenzymes and similar to the enzyme isolated by Kumagai et al. (1981). Dal Degan et al. (1992) have purified and characterized two Ser-CPDs (CPD-I and CPD-II) from fermentation broths of An. They showed that CPDI corresponded to the already characterised Ser-CPD and that CPD-II has a different specificity. The aim of this study was to isolate and characterize a genomic DNA segment containing the pepF gene from An, encoding the CPD-II protein and to study regulation of the pepF expression.
plM635
:
plM634
Fig. 1. Partial
,
AND DISCUSSION
and pIM635)
(b) Nucleotide sequence, intron mapping and promoter analysis A total of 2.8 kb from pIM634 and the l.l-kb insert of pIM635 were sequenced on both strands by the dideoxytermination method using synthetic oligonucleotide primers (Sanger et al., 1977). The nt sequence of the pepF gene is shown in Fig. 2. The nt and the deduced aa sequence of the pepF gene indicated that the ORF is interrupted by several introns which were mapped by sequencing cDNAs created by reverse transcriptase PCR using oligos l-4 (Fig. 2). Three introns of 53, 69 and 59 bp were identified that all contain the consensus sequences for the 5’ splice donor site (5’-GTRNGT), the 3’ splice acceptor site (YAG) and the internal element (RCTRAC), possibly needed for lariat formation during
derived
of transcription
h clones containing
region of the pepF gene is shown
5’ and 3’ regions are shown as thin bars,
gaps in the coding
Orientation
DNA segment encod-
locus and two subclones
from the isolated
as filled bars, while non-coding The partial
region show the positions
is indicated
represent
plasmids:
An N400 (CBS 120.49) was used as wild-type
culture
media
vector sequences.
contained
of the introns.
with an arrow.
subclones
Shaded
Methods: (a) Strains,
per liter: 6.0 g NaNOJ1.5
shaker
and
g KH,PO,/O.S
g
1957)/l % gluand incubated
at 250 rpm. Agar was added
culi strain DHSaF’
bars in
library
strain. Minimal
KC1/0.5 g MgSO,jtrace elements (Vishniac and Santer, cose. Liquid cultures were inoculated with 10’ spores/ml
at 1.2% for
solid medium.
Escherichia
(BRL, Life Technologies.
Gaithersburg,
MD, USA) was used as host for routine plasmid propaga-
tion. E. co/i strain LE392 was used as host for h phage. pUC18 (YanischPerron
(a) Cloning of pepF Based on the N-terminal sequence (Dal Degan et al., 1992) a degenerate 48mer was designed with only the most frequently used An codons and used as probe in a series of genomic Southern analyses. The most stringent wash was found to be 4 x SSC/5 x Denhardt’s/O.l% SDS/O.l% Na2Pz07.10H,0 at 50°C which was used when probing a h genomic library. Six positive clones were identified, purified and the location of the hybridizing fragment within these h clones was established by restriction mapping, hybridization and sequencing. A 4.1-kb EcoRI fragment and a l.l-kb Sal1 fragment were subcloned into pUC18, resulting in plasmids pIM634 and pIM635, respectively, which were used for further analysis (Fig. 1).
map of the genomic
the pepF gene are shown. The coding
at 30°C on an orbital
RESULTS
map of the An genomic
restriction
ing pepF. The restriction (pIM634
:
et al., 1985) and pBluescript
used for subcloning.
The genomic
by ligating
Sau3Al-digested
partially
h replacement
vector hEMBL4
(b) Isolation, hybridization
cloning using
Sambrook 48°C
oligo
genomic
SDS/O.l%
DNA fragments
cut with BamHI (Harmsen
Hybridizations
were
into the
et al., 1990).
of the prpF gene: Plaque performed according to
were performed
overnight
at
S’-GCYCCNGTYGAGTTCTACCAGTTCCTS-
AACTACAAGACYAAGCCNGAY, taining
et al., 1988) vectors
of An N400 was constructed
and characterization nylon replicas was
et al. (1989).
with
(Short
library
4 x SSCj5 x Denhardt’s Na,P,O,.lOH,O;
using
hybridization
(Sambrook
et
buffer
al.,
con-
1989)/0.1%
the filters were washed with 4 x SSC/O.l%
SDS/O.l% NaZP,0,.10H20 at 50°C. Positive plaques, identified on duplicate replicas after autoradiography were recovered from the original plates and purified manipulations
standard
An DNA was isolated
by rescreening
at low density.
For other DNA
methods
were applied
(Sambrook
according
to de Graaff
et al. ( 1988).
et al., 1989).
splicing, although all three introns have one mismatch in this latter element. In the 5’ non-coding region no consensus TATAAAbox is present within the first 250 nt. Upstream of the ATG a TATAAA-like element is found at -85 and a perfect copy of the CAATC element at - 172 (Fig. 2). In most fungal genes the first ATG is used for start codon and a consensus has been observed in the nt sequence around it which includes an A at -3 (Gurr et al., 1987) which is also found in pepF (Fig. 2). Translation stops with a TAA stop codon, which is found in 42% of fungal genes (Unkles, 1992). No perfect AATAAA sequence, which is often found in higher eukaryotes and is sometimes observed in fungi close to the polyadenylation site (Gurr et al., 1987) is present in the pepF 3’ region. However, ATTAAA is found 315 nt after the translation stop codon (Fig. 2).
75 (c) The PEPF polypeptide
pepF expression It was of interest to determine how the expression of the pepF gene is regulated and we studied the regulation of pepF at the level of mRNA content, using Northern analysis. We have used the pepC gene, encoding a putative vacuolar subtilisin-type protease (Frederick et al., 1993) as control since we have previously shown, that the pepC gene is expressed at high levels under all growth conditions tested, with only very limited, growth-related variations (Jarai et al., 1994b). First we investigated the effect of nitrogen and carbon repression upon the expression of pepF (Fig. 4). As shown in Fig. 4A cells grown in the presence of glucose and ammonia contained no detectable pepF message. On the other hand, the absence of either the nitrogen or the carbon source resulted in elevated levels of the pepF message, indicating that both nitrogen and carbon repression affect the expression of pepF. This is supported by the fact that the promoter region of pepF contains several binding sites for both the wide domain regulatory proteins CREA and AREA involved in carbon catabolite and ammonium repression, respectively (Fig. 2); the 5’ region of pepF contains several copies of GATA in both directions. This is the recognition site for the ammoniumresponsive regulator protein AREA in A. nidulans (Kudla et al., 1990). Also two copies of the recognition sequence of the glucose-responsive CREA repressor protein of A. nidulans (5’-SYGGGG; Kulmburg et al., 1993), mediating carbon catabolite repression are present in the 5’ noncoding region of pepF. Whether any of these binding sites are involved in the observed carbon and nitrogen repression awaits further analysis. Even glycerol can effectively, although not entirely, repress the expression of pepF (Fig. 4B). Urea, as sole N source, also represses pepF expression completely (Fig. 4C). When we compared the pepF message levels in nitrogen and carbon derepressed cells grown on different pro-
76 1 121 241 361 481 601 721 841 961 1081 1 1201 19 1321 59
CAG~Tn;AACAACAAGACTAAGCGTATGTATCTCAGTI QPLNNKTK
1441 81
GffiAGATGTATPCCGGCTCC~A~AGAAGGGCA GBMYSGLVPIBKGNVSRSLFFVFQPTIGEPVDETTIWLNG
1561 121
GCC-~TAG~CCCCC~~C~~AGGAOA GPGCSSLBALSPGECRFVWQPGTYQPVBNPYSWVNLTNVL
1681 161
GGG&ATATACTGGiTCGCTAGl-&AGTTTAcAT&GCGGTATC~ACcTAACcTkl-ITTG W
-P
T
R
V
B
S
L
P
D
V
H
F
D
L
TAOGGTPGACCAACCniTC~A~~~TC~~T~C~~~~ -V D Q PVGTGPSLGVPTA
1801 178
.---
3
1921 218
CrrnCATATCCGCTGCmCCTRGATCAGAATGATACAGAAC PYISAAFLDQNDTBHFNLK
2041 239
A-cnTATonTcC~TnrrooTcnomrj4nnccTcc LAYDPCIGQPDYVQEBAPVVPFVQKNNALFNFNASFLAEL
2161 279
AGAGAGCATCCATGAGCAATGTOOATAT~AGTC~CC~GCATCC~~TC~GCCGC~~~A~~-AGCGATCC~C~~A~~A BSIHBQCGYKDP IDQYLVFPASGVQPPKAMNWSD
2281 319
TcnCATCGR'AnTnnCGCCcTCCTGGATCCCAACCCGTGC DIVNNAVLDPNPCFNPYBINBMCPILWDVLGFPTEVDYLP
2401 359
X;CGGCGCCAGCATCPAAC~~C~A~~GCGCTGATTAAOC AAPASTLTALIKRAMHAPNITWSECSVES
2521 399
GGAGGGCGA~AffCOGCCCC~TCGAG~~TCT BGDYSANPIBHVLPQVI
2641 439
TCTCrCGATCC~~~CGAATGGAAAGCA~CCCCATC~~TffiACATC~~AC~A~TA~~~G~~CA~AG~c~~~ FDTAPSTPINIDIPDLMYNEVF L s IQNMTWNGKLG
-G
A
PTCDVY
VFVGGDGGPBQ
= BGTNRVLIGNGDYSMVILTNGTL
IENGY
2761 479 2881 519 3001 3121 3241 3361 3481
CTATlTAc;ulTCAAGATAG+AAGGATGATGCcAACCAGX+ 3520
Fig. 2. The sequence 1, 2, 3 and 4 indicate
of pepF from An (GenBank accession No, X79541) and deduced aa sequence. The dashed arrows above the sequence labelled the oligos that were used for reverse transcriptase, PCR, cloning and sequencing to prove the presence of the three introns. The
consensus 5’ splice donor (GTRNGT), the 3’ splice acceptor (YAG) and the internal element TATAAA-like element, CAATC box and possible AATAAA-like element are in bold. Two Putative binding sites glucose-responsive repressor protein CREA are indicated with .--protein AREA are indicated with +. The start of the mature protein is indicated with a t.
(RCTRAC) of the three introns are underlined. Possible putative binding sites for the wide domain regulatory for the wide domain regulatory ammonium-responsive The deduced
N-terminal
sequence
of the mature
PEPF
protein compared to the sequenced N terminus (Dal Degan et al., 1992) is underlined (corresponding aa are in bold). Three putative signal-peptide cleavage sites predicted for the PEPF protein (von Heijne, 1986) are indicated with +. The three putative active-site residues (Szl*, D430 and Hso7) are double underlined. The seven putative N-linked glycosylation sites (N-X-T/S) in the mature PEPF protein are double underlined. Methods (a) Nucleotide sequence of pepF: The pepF gene was originally subcloned as a 4.1-kb EcoRI fragment into pUC18 and approx. 2.8 kb of this fragment and all of a l.l-kb Sal1 fragment, also subcloned into pUC18, were sequenced completely from both strands using the Sequenase DNA Sequencing TM Kit (Pharmacia LKB), employing synthetic oligo primers. The nt and aa Kit (US Biochemical, Cleveland, OH, USA) and the “Sequencing sequences were analyzed using the computer programmes of Devereux et al. (1984). (b) Intron mapping: An N400 conidia were inoculated into
77 1
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........................................................................................................................ ................................................ matarvs1i1lvwlaasacaegl...rlprdskfpaaqaerlirslnllpkeagptgagdvpsvapge
...
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;: : e
f g h 5 con
;: c d e f z i j con
d d
121 240 ipvedyqflnnktkpyrv~slpdvhfdlg.emyaQlvpiekgnvsrsLFFvf~tigepvdettir(LnPQCSSl~.alspgecrFvwqpgty.....qpvelYpY~~ltnvlWvDq P llerrvtlpg..1....pegvgdlg......hhaQYyrlpnthdarMFYFffEsrgk.kedPvVi~tQQPQCSS.~lavfyBnGpPtiann......mslvwMcFgNdkIsniiFvDp a ....... lpg..l....p~gvadlg......hhaQYyrlpnthdarMFYPffEsrgk.kedPvVillLtOCSS.elavfyBnGpPtiann......msln*McFgWdkIsniiFvDq P llerrvtlpg..l....pqgvgdlg......hhaQYyrlpnthdaeMFYPlfEsrgk.kedPvVi~t~PQCSS.~lavfyBnGpFtisnn......mslaw~FgWdtIsniiFvDq P iverkfvfpn..iladggptvddlg......hhaQYyklpksrgasMPYPffBsrnk.kdaPvVillLtaPOCSS.elavfyBnGpPkitsn......mslawtPeYgWdqVsnllYvDq P fwkndaienyqlrvnkikdpkilgidpnvtqytQYldved.edkhFFFWtfEsrndpakdPvI1~n00PQCSS.ltglffBlGpssigpd......lkpig~Y~WnsnatviF1Dq P fhvtdaqvpnhklrik..stpkdlgidt.vkqyeGYldwd.edkhFFYYffEsrndpkndhrIlllLnOaPQCSS.ltglffElGpssidkn......lkpvyNph~lhlanasviF1Dq P apqgaevtglpgfdgalpskhyaQYvtvdeghgrnLFYYwEserdpgkd~Vl~n~PQCSSfd.gfvyEhGpPnfesggsvkslpklhl~YaWskVstmiYlDsP ........... .......... agghaadrivrlpgqpevdfdmy~QYitvdeaagrsLFYLlqEapeea~aPlVl~nQQPQCSSvaygaseBlGaFr~prg.....aglvlWeYrWnkVanvlFlDs P P ...... ..vepsghaadriarlpgqpavdf~~QYitvdegagrsLFYLlqBapeda~aPlVl~n~PQCSSvaygaseBlGaFrvkprg.....aglvlWeYrlhlkVanvlFlDs ___--__-_______-___--_________-___Q___--______________-__________~_~~Q~~~______-_____________________~___~_________~__
360 241 vQtQPSlgvpt..........atsEeeiaeDfvkPFknWqqifg..iLn.fkiWtQBSYAQrYVPyi~aafld~dtehfnlkgalaydpcIgqfdYvqeeap~fvqk~alfnfn a tQtQFSYssd......drdt.rhdEagvsnDlydFLqvFFkkhPeFv*n..dFFItQ~SYAQhYIPaFasrVhq~kn..e.......gthINLkgFaIGNGltDpaiqykaytdYa .. tGtQFSYssd......drdt.rhdECgvsnDlydPLqvFFkkhPeFi~..dFFItGESYMhYIPaFasrVhqgnkkn..e.......gthINLkgFaIGNGltDpaipykaytdYa .. tQtQFSYssd......drdt.rhdEtgvsnDlysFLqvFFkkhPeFaLn..dFFItQESYMhYIPaFasrVhqgnkan..e.......gihINLkgFaIGNGltDpaipykaytdYa .. ~tQFSYttd......ksdi.rhdBtgvsnDlydFLqaFFaehPkLa*n..dFYItQXSYAQhYIPaFasrVhkgnkan..e.......gvhINLkgFaIGNGltDpalqypaypdYa .. vnvGFSYsgs......sgvs...ntvaagkDvynFLelFFdqfPeYvnkgqdFhIaQESYAQhYIPvFaseIlshkdrn.............fNLtsvlIGNGltDpltpynWepmac g invQYSYs.s......qsvs...ntiaagkDvyaFLqlFFknfPeYa.n.ldFhIaQ~SYAQhYIPaFaseIlthpern.............fNLtsvlIGNGltDplvqye~epmac g aGvGLSYskn......vsd.yetgDlktatDshtFLlkWqlyPeFlsn..pFYIaQ~SYAQvYVPtL~heVvkgiqgg..a.......kptINFkgYmVGNGvcDtifdgnalvpFah g aQvGFSYtnt......ssdiytsgDnrtahDsyaPLaaWFerfPhY.krr.eFYVaGXSYAGhYV P ~qlVhr....s..g.......npvINLkgFmVGNGliDdyhdyvgtfeFww n n aGvQFSYtnt ......ssdiytsgDnrtahDsyaFLakWFerfPhYkyr..dFYIaGESYAGhYVPeL~qlVhr....s..k.......npvINLkgFmVGNGliDdyhdyvgtfeFww --_Q-~___-__-____-___________-~___~_____________________Q~~~~Q_~_~______________________________________________________
480 361 sflaelesiheqcgykdffdqylvFpasgvqppkamnwsdptcdvydivnnavldpnpcfnpye~nemcpil.......................................wdvlgfpt e c .. ldmnliqkadydrinkfippcePaiklc.gtdgkascmaaymvcnsifnsimklvgtkn~dvrkece.............................................g.kl c .. lemnliqkadyerinkfippceFaiklc.gtngkascmsaymvcntifnsimklvgtkn~dvrkece.............................................g.kl .. ldmnlikksdydrinkfippceFaiklc.gtngkascmaaymvcnsifssimklvgtknyydvrkece.............................................g.kl c .. lemglitqkehdrlekivplceLsiklc.gtdgttsclasylvcnslfsgvmshaggvnyydirkkcv.............................................g.sl c eggepsvlpseecsamedslerclgliescydsqswscvpatiycnnaqlapyqrtg.rnyrdirkdce.............................................ggnl c eggepsvlepeecdgmlnllprclsliescyesgs~sc~atiycnng~gpyqktg.rnvydirtmce.............................................gssl c mglisdeiyqqastsc....hgnyWnatd.gkcdtaiski.eslisglniydilepcyhsrsikevnlqnsklpqsfkdlgttnkpfpvrtrmlgrawplrapvkagr~swqevasg . hgivsddtyrrlkdac....lhdsFihps.pacdaatdva.taeqgnidmyslytpvcni.s .................................................... hgivsddtyrrlkeac....lhdsFihps.pacdaatdva.taeqgnidmyslytpvcnits....................................................stgsy d ____._______..__.._____-___--____--____-___-____________.________-___-_______________.._-.___._-___-._--.___..___.~~~~~ ~ 600 481 vdylpaapastltalIkrAmhapnitwsecsve~vfvggdggpeqegdy~anpi~hvlpqviegtnrVlIgnQ~~iltngtllaiq~t~gklgFdtapstpinidipdlmYnevf y.dfsnlekFFgdkaVrqAigvgdie.....Fvscstsvyqaml..td.wmrnlevgipall~dginVlIYaQByDlicnwlgns~hsMewsgqkdF.a............ktaes6 f y.dfsnlekFFgdkaVrqAigvgdie.....Fv~cstsvyqaml..td.wmrnl~vgipall~dginVlIYaQEyDlicnwl~~r~hsMewsgqkdF.a............ktaess f y.dfsnlekFFgdkaVkeAigvgdle.....Fvecsttvyqaml..td.wmrnl~vgipallsdginVlIYaGByDlicnwlgnsr~hs~ewsgqkdF.v............sshesp f y.dfsnmekFLnlqsVrkslgvgdid.....Fvscstsvyqaml..vd.wmrnlevgiptlledgiallVYaCiEyDlicnwlgnsr~aMewsgkCnF.g............aakevp f yptlqdiddYLnqdyVkeAvgaevdh.....Y~~cnfdinrnf1fagd.wmkpyhtavtdllnqdlpIlWaQDkDficnwlgnkawtdvLpwkydeeF.a............sqkvrn w ysqleyidqYLnlpeVkkAlgaevde.....Yq~cnfdinrnfmfagd.wmkpyqknvidllekelpV1IYaQDkDficnwlgnqawtnrLewsgskgF.t............kapvks w cmsdevataWLdnaaVrsAihaqsvsaigpwLlc...tdklyfvhdag.~miayhknl..t.sqgyraiIFsQD~cvpftgseawtksLgygwdsWrp............witng qv pcterystaYYnrrdVqtAlhanvtgamnytWtncsdtinthwhdapr.smlpiyrel..i.aaglrIwVFsQDtDavvpltatrysigaLglatttSW~............wyddlq e pcterystaYYnrrdVqmAlhanvtgamnytWatcsdtinthwhdapr.emlpiyrel..i.aaglrIwVFsQDtDavvpltatry~igaLglptttsWyp............wydd.q e _____________-____-___-____-_________________.__Q__~__.____.___._______.___.___..___..__-..____
694 601 iengydpqggqgvmgiqhyergLmWaetfqsG~qPqfqPrvaYrhLewLlgrrdt1 ................................... 1.......vddaqagvlkshgaLsFlkVhnaG~VPmdqPkaaLeMLrrFtqgklkes~eeepattfyaai .................... 1.......vdddqagvlkshgaLsFlkVhnaQ~VPmdqPkaaL~MLrrFtqgklk .................................... .................... " ..... ..vdgaeagvlkshgpLsFlkVhnaG~~mdqPka~LeMLrrFtqgklkeewlaelpe~myaam qcvsn i.......vdgkeagllktyeqLsFlkVrdaQ~VPmdqPkaaLkMLkrWmensliedatvtvaaqggeelvaadvitssfalhenkrqqi ik' . t.....asitdevagevksykhFtYlrVfngQ~VPfdvPenaLsMvneWihggfsl ................................... . ....................................................................... ... ..kvgknaagevknykhFtFlrVfggQ~VPydqPenaLdrWisgdyky ~:....gytegye.......hgLtFatIkgaGHtVPeykPqeaFdFYsrWl~gskl v.....ggwsqvy .......kgLtLvsVrgaQHeVPlhrPrqaLiLFqqFlqgkpmpgrttn .............................. v.....ggwsqvy.......kgLtLvsVrgaQEeVPlhrPrqaLvLFQyPlqgkpmpgqcknat ............................ _~~___~___~____~~___~~____~~___GB__P~__P_.___~_______~~_______~___~.___~___~~__~___~~___~__~ ._ =
Fig. 3. The aa sequence
comparison
of PEPF
with other
Ser-CPDs.
The aa sequences
were aligned
using the program
PILEUP
available
in the
GCG package. The aligned sequences are as follows: a, An PEPF; b, wheat CPD III (Baulcombe and Barker, 1987); c, barley CPD III (Sorensen et al., 1989); d, rice CPD (Washio and Ishikawa, 1992); e, Arabidopsis thaliana CPD (Bradley, 1992: GenBank accession No. P32826); f, S. cereuisiae CPY (Valls et al., 1987); g, C. albicans CPD
Y (Munkhtar
et al., 1992); h, barley
CPD
I (Sorensen
et al., 1986); i, barley
CPD II (Sorensen
et al.,
1987); j, wheat CPD II (Breddam et al., 1987. All sequences are shown in lowercase letters. A dot indicates that there is no aa at that position. The active site residues are double underlined in the consensus sequence. Partial conservation of aa residues is indicated with capital letters. Fully conserved aa residues
are indicated
with bold capital
letters; con, consensus
sequence
of all the aa sequences
shown above.
complete YPD medium (containing 1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose) for 24 h. Mycelium was then filtered, washed with sterile water and transferred to minimal medium supplemented with 1% collagen. After a further 5-6 h of cultivation under the same conditions mycelia were harvested, washed and freeze-dried for RNA isolation. Total RNA was prepared from freeze-dried mycelia by lysis in buffer containing 10 mM Tris pH 7.5/100 mM LiCI/2% SDS followed by several phenol extractions and ethanol precipitation. Poly(A)+RNA was isolated by oligo(dT) chromatography (Aviv and Leder, 1972). The splice sites of the introns were identified by cloning and sequencing partial cDNA copies of the pepF transcript. First strand synthesis was performed by standard methods (Sambrook et al., 1989). The priming oligo for intron 1 and 2 was oligo 2 and for intron 3 oligo 4. The cDNAs were then amplified by PCR using oligos 1 and 2 for introns 1 and 2 and oligos 3 and 4 for intron 3 and cloned into pUC18. Several independent clones were sequenced for each region.
78 A
B
1
2
3
+
+
-
A
C
12
3
12
3 12
anmonia
glucore -
+
aJmanlo g1ucol5e glycerol
+
pepc
+ + -
+ +
anunonio glucose glycarol
+ -
blot analyses
of the carbon
of the pepF gene. (A) Effect of nitrogen were grown
overnight
+ +
+ +
+
ammonia g1ucoas BEA callein alamtin collagen
in complete
and nitrogen
and carbon
liquid medium
regulation
derepression.
figure and grown for an additional
of pepF by ammonium
extract/l%
stripping
and
the membranes
and urea. Experiments
(a) Growth
conditions:
An N400 (CBS 120.49) was inoculated yeast
liter:
of wild-type
YPD (1% (w/v)
(w/v) o-glucose)
shaker
and
grown
for
at 220 rpm. Mycelia were filtered,
6 g NaNO,/l.S
MgSOJlOmg
each of MnCI,,
were:
NH&l,
100 mM
glycerol. harvested
g KH,PQ,/O.S
ZnSO,
100 mM
Cells were then grown by filtration,
urea,
and FeSO,.
1% (w/v) glucose,
for an additional
freeze-dried
g KCl/O.S
g
The supplements 1% (w/v)
6-8 h. Mycelia
were
and used for RNA isolation.
(b)
RNA isolation blotting and hybridization: Total RNA and poly(A)+RNA were prepared from freeze-dried mycelia as described in the legend to Fig. 2. 10 ug of total or l-2 ug poly(A)+RNA on formaldehyde Hybond-N
gels (Sambrook
membranes
Hybridization described
agarose and
(Sambrook
washing
(Amersham)
et al., 1989)
and of the membranes
UV
was loaded transferred
to
cross-linked.
were carried
out
as
et al., 1989).
teins as sole carbon and nitrogen sources, it was clear that the expression level of pepF changed significantly (Fig. 5A). Whereas only little message could be detected in cells grown on casein (lane 4) and hardly any in cells grown on BSA (lane 3) elastin (lane 5) and collagen (lane 6) appeared to induce the expression of pepF to highly elevated levels. When we compared the transcript levels of pepF in cells which were grown at pH 8.0 to that in cells grown at pH 3.0 under both repressing and inducing conditions (Fig. 5B), we found that when cells were derepressed-induced and grown at acidic pH, high message levels were detected (lanes 3 and 5) whereas cells grown on identical derepressing-inducing media whose pH was adjusted to 8.0, showed no pepF message. (f ) Conclusions (1) The pepF
+ -
+ -
_ + -
+
3
ammonia glucoac slaatin couagm PR
P~PF
PW
PePc
PePc
blot analyses
of pepF. (A) Comparison
4 + + 3
5 + + 8
6 + 3
c *
+ 0
3
of protein
of different
induction
external
and pH regulation
protein
inducers.
Each
protein inducer was used at 1% (w/v). (B) Effect of pH. Alkaline growth conditions were achieved by continuous adjustment of the pH using NaOH.
washed three times with distilled water and transferred to minimal medium supplemented as shown in the figure. Minimal culture media per
+ + + -
12
6
Experiments
were carried
out as described
in Fig. 4.
were carried
lo6 spore/ml
into complete
(w,‘v) peptone/2%
16-24 h at 3O’C on an orbital
contained
5
(B) Carbon repression of pepF by glucose were carried out as for A. (C) Nitrogen
between the two probings. and glycerol. Experiments repression
+ + -
Fig. 5. Northern
pH 6.0, then washed
6-8 h before RNA was isolated
to the pepF and pepC probes,
out as for A. Methods:
4
Cells
three times with distilled water, divided into three aliquots and transferred into minimal medium pH 6.0 supplemented as indicated in the hybridized
3
P8PC
PePc
Fig. 4. Northern
B
gene of An has been isolated using a degenerate oligo based on the N-terminal sequence of PEPF.
(2) The pepF gene encodes a protein consisting of 530 aa, and contains three introns of 53,69 and 59 bp, which were confirmed by generating cDNA with reverse transcriptase PCR and sequencing. (3) Signal sequence cleavage prediction analysis suggests that PEPF contains a 17-aa signal peptide at its N terminus which directs transport into the endoplasmic reticulum. A second proteolytic processing event at a monobasic cleavage site (Lyss2) removes another 35-aa propeptide, thus yielding the 478-aa mature protease. (4) The deduced aa sequence of PEPF shows homology to yeast and plant Ser-CPDs. (5) Southern analysis revealed that pepF is present in a single copy in An. (6) Northern analysis showed that the pepF gene is under carbon and nitrogen catabolite repression, under pH control and is inducible by protein at the level of RNA content. However, additional regulatory mechanisms cannot be ruled out.
ACKNOWLEDGEMENT
We thank Jeroen Krijgsveld for excellent technical work.
REFERENCES Aviv, H. and Leder, P.: Purification of biologically active globin messenger RNA by chromatography on oligothymidilic acid-celhtlase. Proc. Natl. Acad. Sci. USA 69 (1972) 1408-1412. Barthomeuf, C., Pourrat, H. and Pourrat, A.: Isolation and purification of a new protease from Aspergillus niger LCF No. 9. Biotech. Tech. 2 (1988) 29-34. Baulcombe, D.C.. Barker, R.F. and Jarvis, M.G.: A gibberellin respon-
79 sive wheat gene has homology to yeast carboxypeptidase Y. J. Biol. Chem. 262 (1987) 13726613735. Berka, R.M., Ward, M., Wilson, L.J., Hayenga, K.J., Kodama, K.H., Carlomango, L.P. and Thompson, S.A.: Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 86 (1990) 153-162. Bosmann, H.B.: Protein catabolism, II. Identification of neutral and acid proteolytic enzymes in Aspergillus niger. Biochim. Biophys. Acta 293 (1973) 476-489. Breddam, K.: Serine carboxypeptidases: a review. Carl&erg Res. Commun. 51 (1986) 83-128. Breddam. K., Sorensen, S.B. and Svendsen, I.: Primary structure and enzymatic properties of carboxypeptidase 11 from wheat bran. Carlsberg Res. Commun. 52 (1987) 2977311. Chopra, S. and Metha, P.: Influence of various nitrogen and carbon sources on the production of pectinolytic, cellulytic and proteolytic enzymes by Aspergitlus niger. Folia Microbial. 30 (1985) 117-125. Dal Degan, F., Ribadeau-Dumas, B. and Breddam, K.: Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis. Appl. Environm. Microbial. 58, (1992) 21442152. de Graatf, L., Van den Broek, H. and Visser. J.: Isolation and characterization of the Aspergiilus niduians pyruvate kinase gene. Curr. Genet. 13 (1988) 315-321. Devereux, J., Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Frederick, G.D.> Rombouts, P. and Buxton. F.P.: Molecular cloning and characte~zation of pepC, a gene encoding a serine proteinase from Aspergillus niger. Gene 125 (1993) 57-64. Gurr, S.J., Unkles, SE. and Kinghorn, J.R.: The structure and organization of nuclear genes of Iilamentous fungi. In: Kinghorn, J.R. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, 1987, pp. 93..139. Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J.: Cloning and expression of a second Aspergiihs niger pectin lyase gene ($A) _ indication of a pectin lyase gene family in Aspergillus niger. Curr. Genet. 18 (1990) 161-166. Ichishima, E.: Purification and characterization of a new type acid carboxypeptidase from Aspergik~. Biochim. Biophys. Acta 258 ( 1972) 2744288. Inoue, H., Kimura, T., Makabe, 0. and Takahashi, K.: The gene and deduced protein sequences of the zymogen of Aspergiilus niger acid proteinase A. J. Biol. Chem. 266 (1991) 19484-19489. Jarai, G., Kirchherr, D. and Buxton, F.P.: Cloning and characterization of the pepll gene of Aspergillus niger which codes for a subtilisinlike protease. Gene 139 (1994a) 41-57. Jarai, G., Van den Hombegh, H. and Buxton, F.P.: Cloning and characterization of the pepE gene of Aspergiffus niger encoding a new aspartic protease and regulation of the pepE and pepC genes. Gene 145 (1994b) 171-178. Krishnan, S. and Vijayalakshmi, M.A.: Purification of an acid protease and a serine carboxypeptidase from Aspergillus niger using metalchelate affinity chromatography. J. Chromatogr. 329 (198.5) 165-170. Krishnan, S. and ~‘ijayalakshmi, M.A.: Purification and some properties from Aspergillus niger. of three serine carboxypeptidases J. Chromatogr. 315 (1986) 315-326. Kudla, B., Caddick, M.X., Langdon, T., Martini-Rossi, N.M. Bennett, CF., Sibley, S., Davies, R.W. and Arst, H.N.: The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidu-
lam. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc-finger. EMBO J. 9 (1990) 1355-1364. Kulmburg, P., Mathieu, M., Dowzer, C., Kelly, J.M. and Felenbok, B.: Specific binding sites in the al& and &A promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in A. nidulans. Mol. Microbial. 7 (1993) 847-857. Kumagai, I. and Yamasaki, M.: Enzymatic properties of an acid carboxypeptidase from A. niger var. macrosporus. Biochim. Biophys. Acta 659 (1981) 344-350. Kumagai, I. Yamasaki, M. and Ui, N.: Isolation, purification and some chemical properties of an acid carboxypeptidase from Aspergillus niger var. macrosporus. Biochim, Biophys. Acta 659 (1981) 334-343. Martin, B.M., Svendsen, I., Vishwanatha, T. and Johansen, J.T.: Amino acid sequence of carboxypeptidase Y, I. Peptides from cleavage with cyanogen bromide. Carlsberg Res. Commun. 47 ( 1982) 1-13. Munkhtar, M., Logan, D.A. and Kaufer, N.F.: The ~arboxypeptidase Y-encoding gene from Cundida ufbiccms and its transcription during yeast-to-hyphae conversion, Gene 121 (1992) 173.-177. Sakka, K., Shimada, K. and Matsushima, K.: Purification and some properties of a serine proteinase from a mutant of Aspergillus niger. 3. Ferment. Technol. 63 (1985) 4799483. Sambrook, J., Fritsch, E.F. and Mania&, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5461. Sorensen, S.B., Breddam, K. and Svendsen, I.: Primary structure of ~arboxy~ptidase I from malted barley. Car&erg Res. Commun. 5 1 (1986) 475-485. Sorensen, S.B., Svendsen, I. and Breddam, K.: Primary structure of carboxypeptidase II from malted barley. Carlsberg Res. Commun. 52 (1987) 285-295. Sorensen, S.B., Svendsen, I. and Breddam, K.: Primary structure of ~arboxypeptidase III from malted barley. Carlsberg Res. Commun. 54(1989)193-202. Short, J.M., Fernandez, J.M., Sorge, J.A. and Huse, W.D.: h ZAP: a bacteriophage expression vector with in vivo excision properties. Nucleic Acids Res. 16 (1988) 7583-7600. Takahashi, K., Tanokura, M., Inoue, H., Kojima. M., Muto, Y., Yamasaki, M., Makabe, O., Kimura, T., Takizawa, T., Hamaya, T., Suzuki, E. and Miyano, H.: Structure and function of a pepstatininsensitive acid proteinase from Aspergiilus niger var. microsporus. Adv. Exp. Mol. Biol. 306 (1991) 203-211. Unkles, SE.: Gene organization in industrial filamentous fungi. In: Kinghorn, J.R. and Turner, G. (Eds.), Applied Molecular Genetics of Industrial Fungi. Blackie. Glasgow, 1992, pp. 29-53. Valls, L.A., Hunter, C.P., Rothman, J.H. and Stevens, T.H.: Protein sorting in yeast: the localization determinant of yeast vacuoiar carboxypeptidase Y resides in the propeptide. Cell 48 (1987) 887-897. Vishniac, W. and Santer, M.: The thiobacilli. Bacterial. Rev. 21 (1957) 195-213. von Heijne, G.: A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14 (1986) 4683-4690. Washio, K. and Ishikawa, K.: Structure and expression during the germination of rice seeds of the gene for a carboxypeptidase. Plant Mol. Biol. 19 (1992) 631-640. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33 (1985) 103-l 19.
0378-l 119/94/$07.00
GENE 08380
Cloning, characterization and expression ofpep& a gene encoding a serine carboxypeptidase from Aspergillus niger (Exo-protease; proteinase F; transcriptional
regulation; carbon repression; nitrogen repression; pH regulation)
J.P.T.W. van den Hombergh”, G. Jaraib, F.P. Buxtonb and J. Vissera “Section Molecular Genetics oflndustrial Microorganisms, Wageningen Agricultural University, Dreqenlaan 2, NL-6703-HA
Wageningen, The
Netherlands: and bBiotechnology Department, Ciba-Geigy AC. CH4002 Basel, Switzerland. Tel. (41-61) 696-1661 Received by J.R. Kinghorn:
13 April 1994; Revised/Accepted:
10 June/l2
June 1994; Received at publishers:
25 August
1994
SUMMARY
We have cloned a gene (pepF) encoding a serine carboxypeptidase, proteinase F (PEPF), from Aspergillus niger. The sequences were identified in a phage h genomic DNA library using a synthetic probe based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA clones reveals that it is composed of four exons of 199,283,227 and 881 bp, interrupted by three introns of 53,69 and 59 bp. The sequence of pepF codes for a polypeptide of 530 amino acids (aa), of which the first 52 aa are not present in the mature PEPF. This region may represent a prepro sequence that is removed by proteolytic cleavage at a monobasic cleavage site (LYs~~). Northern blot analysis of total cellular RNA extracted from A. niger cells indicates that pepF is transcribed as a single 1%kb mRNA, which is regulated by nitrogen and carbon repression, specific induction and the pH of the culture medium.
INTRODUCTION
Studying proteases of Aspergilli is of considerable importance as these fungi are used for the commercial production of proteases. Problems with homologous and heterologous protein production in Aspergilli are often associated with the high level of extracellular proteases Correspondence
to: Dr.
J. Visser,
Section
Molecular
Genetics
of
Industrial Microorganisms, Wageningen Agricultural University, 6703 HA Wageningen, The Netherlands. Tel. (31-8370) 84439; Fax (31-8370) 84011; e-mail: [email protected] Abbreviations:
aa, amino
acid(s);
A., Aspergillus; An, A. niger; BSA,
bovine serum albumin; bp, base pair(s); C., Candida; CPD, carboxypeptidase; CPY, S. cerevisiae Ser-CPD Y; GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase or 1000 bp; N, A or C or G or T; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; PEPC, A. niger proteinase C; pepC, gene encoding PEPC; PEPF, A. niger proteinase F; pepF, gene encoding PEPF; R, A or G; S, G or C; S., Saccharomyces; SDS, sodium dodecyl CPD, serine-CPD; SSC, 0.15 M NaCI/O.OlS M Nascitrate C or T. SSDI 0378-1119(94)00591-5
sulfate; SerpH 7.6; Y,
in these organisms. In addition proteases have been implicated in the pathogenicity of A. fumigatus. A variety of different proteases from Aspergilli have been characterized (Ichishima, 1972; Bosmann, 1973; Chopra and Metha, 1985). In A. niger (An) a wide range of proteolytic activities comprising acidic (Krishnan and Vijayalakshmi, 1985; Takahashi et al., 1991), semialkaline and alkaline serine proteases (Sakka et al., 1985; Barthomeuf et al., 1988) and serine carboxypeptidases (Ser-CPD) (Krishnan and Vijayalakshmi, 1985; 1986; Dal Degan et al., 1992) have been described. Several proteases have been cloned from Aspergilli, including a pepsin-type acid protease from An var. awamori (Berka et al., 1990) and from An (Jarai et al., 1994b), a non-pepsin-type acid protease from An var. macrosporus (Inoue et al., 1991), and two subtilisins from An (Frederick et al., 1993; Jarai et al., 1994a). However, no genes coding for Ser-CPDtype proteases have been cloned. Ser-CPDs (peptidyl+amino acid hydrolases; EC. 3.4.16.1) are exo-peptidases that sequentially release most aa residues, including proline, from the C terminus of
74
polypeptide chains at acidic pH utilising a diisopropylphosphorofluoridate-sensitive active-site Ser residue. SerCPDs have been extensively studied as they can be used in food industry for debittering of peptide mixtures, and are very useful tools for C-terminal sequencing, and peptide synthesis. Kumagai et al. (1981) described one Ser-CPD from An and more recently Krishnan and Vijayalakshmi (1986) isolated three Ser-CPDs that are probably isoenzymes and similar to the enzyme isolated by Kumagai et al. (1981). Dal Degan et al. (1992) have purified and characterized two Ser-CPDs (CPD-I and CPD-II) from fermentation broths of An. They showed that CPDI corresponded to the already characterised Ser-CPD and that CPD-II has a different specificity. The aim of this study was to isolate and characterize a genomic DNA segment containing the pepF gene from An, encoding the CPD-II protein and to study regulation of the pepF expression.
plM635
:
plM634
Fig. 1. Partial
,
AND DISCUSSION
and pIM635)
(b) Nucleotide sequence, intron mapping and promoter analysis A total of 2.8 kb from pIM634 and the l.l-kb insert of pIM635 were sequenced on both strands by the dideoxytermination method using synthetic oligonucleotide primers (Sanger et al., 1977). The nt sequence of the pepF gene is shown in Fig. 2. The nt and the deduced aa sequence of the pepF gene indicated that the ORF is interrupted by several introns which were mapped by sequencing cDNAs created by reverse transcriptase PCR using oligos l-4 (Fig. 2). Three introns of 53, 69 and 59 bp were identified that all contain the consensus sequences for the 5’ splice donor site (5’-GTRNGT), the 3’ splice acceptor site (YAG) and the internal element (RCTRAC), possibly needed for lariat formation during
derived
of transcription
h clones containing
region of the pepF gene is shown
5’ and 3’ regions are shown as thin bars,
gaps in the coding
Orientation
DNA segment encod-
locus and two subclones
from the isolated
as filled bars, while non-coding The partial
region show the positions
is indicated
represent
plasmids:
An N400 (CBS 120.49) was used as wild-type
culture
media
vector sequences.
contained
of the introns.
with an arrow.
subclones
Shaded
Methods: (a) Strains,
per liter: 6.0 g NaNOJ1.5
shaker
and
g KH,PO,/O.S
g
1957)/l % gluand incubated
at 250 rpm. Agar was added
culi strain DHSaF’
bars in
library
strain. Minimal
KC1/0.5 g MgSO,jtrace elements (Vishniac and Santer, cose. Liquid cultures were inoculated with 10’ spores/ml
at 1.2% for
solid medium.
Escherichia
(BRL, Life Technologies.
Gaithersburg,
MD, USA) was used as host for routine plasmid propaga-
tion. E. co/i strain LE392 was used as host for h phage. pUC18 (YanischPerron
(a) Cloning of pepF Based on the N-terminal sequence (Dal Degan et al., 1992) a degenerate 48mer was designed with only the most frequently used An codons and used as probe in a series of genomic Southern analyses. The most stringent wash was found to be 4 x SSC/5 x Denhardt’s/O.l% SDS/O.l% Na2Pz07.10H,0 at 50°C which was used when probing a h genomic library. Six positive clones were identified, purified and the location of the hybridizing fragment within these h clones was established by restriction mapping, hybridization and sequencing. A 4.1-kb EcoRI fragment and a l.l-kb Sal1 fragment were subcloned into pUC18, resulting in plasmids pIM634 and pIM635, respectively, which were used for further analysis (Fig. 1).
map of the genomic
the pepF gene are shown. The coding
at 30°C on an orbital
RESULTS
map of the An genomic
restriction
ing pepF. The restriction (pIM634
:
et al., 1985) and pBluescript
used for subcloning.
The genomic
by ligating
Sau3Al-digested
partially
h replacement
vector hEMBL4
(b) Isolation, hybridization
cloning using
Sambrook 48°C
oligo
genomic
SDS/O.l%
DNA fragments
cut with BamHI (Harmsen
Hybridizations
were
into the
et al., 1990).
of the prpF gene: Plaque performed according to
were performed
overnight
at
S’-GCYCCNGTYGAGTTCTACCAGTTCCTS-
AACTACAAGACYAAGCCNGAY, taining
et al., 1988) vectors
of An N400 was constructed
and characterization nylon replicas was
et al. (1989).
with
(Short
library
4 x SSCj5 x Denhardt’s Na,P,O,.lOH,O;
using
hybridization
(Sambrook
et
buffer
al.,
con-
1989)/0.1%
the filters were washed with 4 x SSC/O.l%
SDS/O.l% NaZP,0,.10H20 at 50°C. Positive plaques, identified on duplicate replicas after autoradiography were recovered from the original plates and purified manipulations
standard
An DNA was isolated
by rescreening
at low density.
For other DNA
methods
were applied
(Sambrook
according
to de Graaff
et al. ( 1988).
et al., 1989).
splicing, although all three introns have one mismatch in this latter element. In the 5’ non-coding region no consensus TATAAAbox is present within the first 250 nt. Upstream of the ATG a TATAAA-like element is found at -85 and a perfect copy of the CAATC element at - 172 (Fig. 2). In most fungal genes the first ATG is used for start codon and a consensus has been observed in the nt sequence around it which includes an A at -3 (Gurr et al., 1987) which is also found in pepF (Fig. 2). Translation stops with a TAA stop codon, which is found in 42% of fungal genes (Unkles, 1992). No perfect AATAAA sequence, which is often found in higher eukaryotes and is sometimes observed in fungi close to the polyadenylation site (Gurr et al., 1987) is present in the pepF 3’ region. However, ATTAAA is found 315 nt after the translation stop codon (Fig. 2).
75 (c) The PEPF polypeptide
pepF expression It was of interest to determine how the expression of the pepF gene is regulated and we studied the regulation of pepF at the level of mRNA content, using Northern analysis. We have used the pepC gene, encoding a putative vacuolar subtilisin-type protease (Frederick et al., 1993) as control since we have previously shown, that the pepC gene is expressed at high levels under all growth conditions tested, with only very limited, growth-related variations (Jarai et al., 1994b). First we investigated the effect of nitrogen and carbon repression upon the expression of pepF (Fig. 4). As shown in Fig. 4A cells grown in the presence of glucose and ammonia contained no detectable pepF message. On the other hand, the absence of either the nitrogen or the carbon source resulted in elevated levels of the pepF message, indicating that both nitrogen and carbon repression affect the expression of pepF. This is supported by the fact that the promoter region of pepF contains several binding sites for both the wide domain regulatory proteins CREA and AREA involved in carbon catabolite and ammonium repression, respectively (Fig. 2); the 5’ region of pepF contains several copies of GATA in both directions. This is the recognition site for the ammoniumresponsive regulator protein AREA in A. nidulans (Kudla et al., 1990). Also two copies of the recognition sequence of the glucose-responsive CREA repressor protein of A. nidulans (5’-SYGGGG; Kulmburg et al., 1993), mediating carbon catabolite repression are present in the 5’ noncoding region of pepF. Whether any of these binding sites are involved in the observed carbon and nitrogen repression awaits further analysis. Even glycerol can effectively, although not entirely, repress the expression of pepF (Fig. 4B). Urea, as sole N source, also represses pepF expression completely (Fig. 4C). When we compared the pepF message levels in nitrogen and carbon derepressed cells grown on different pro-
76 1 121 241 361 481 601 721 841 961 1081 1 1201 19 1321 59
CAG~Tn;AACAACAAGACTAAGCGTATGTATCTCAGTI QPLNNKTK
1441 81
GffiAGATGTATPCCGGCTCC~A~AGAAGGGCA GBMYSGLVPIBKGNVSRSLFFVFQPTIGEPVDETTIWLNG
1561 121
GCC-~TAG~CCCCC~~C~~AGGAOA GPGCSSLBALSPGECRFVWQPGTYQPVBNPYSWVNLTNVL
1681 161
GGG&ATATACTGGiTCGCTAGl-&AGTTTAcAT&GCGGTATC~ACcTAACcTkl-ITTG W
-P
T
R
V
B
S
L
P
D
V
H
F
D
L
TAOGGTPGACCAACCniTC~A~~~TC~~T~C~~~~ -V D Q PVGTGPSLGVPTA
1801 178
.---
3
1921 218
CrrnCATATCCGCTGCmCCTRGATCAGAATGATACAGAAC PYISAAFLDQNDTBHFNLK
2041 239
A-cnTATonTcC~TnrrooTcnomrj4nnccTcc LAYDPCIGQPDYVQEBAPVVPFVQKNNALFNFNASFLAEL
2161 279
AGAGAGCATCCATGAGCAATGTOOATAT~AGTC~CC~GCATCC~~TC~GCCGC~~~A~~-AGCGATCC~C~~A~~A BSIHBQCGYKDP IDQYLVFPASGVQPPKAMNWSD
2281 319
TcnCATCGR'AnTnnCGCCcTCCTGGATCCCAACCCGTGC DIVNNAVLDPNPCFNPYBINBMCPILWDVLGFPTEVDYLP
2401 359
X;CGGCGCCAGCATCPAAC~~C~A~~GCGCTGATTAAOC AAPASTLTALIKRAMHAPNITWSECSVES
2521 399
GGAGGGCGA~AffCOGCCCC~TCGAG~~TCT BGDYSANPIBHVLPQVI
2641 439
TCTCrCGATCC~~~CGAATGGAAAGCA~CCCCATC~~TffiACATC~~AC~A~TA~~~G~~CA~AG~c~~~ FDTAPSTPINIDIPDLMYNEVF L s IQNMTWNGKLG
-G
A
PTCDVY
VFVGGDGGPBQ
= BGTNRVLIGNGDYSMVILTNGTL
IENGY
2761 479 2881 519 3001 3121 3241 3361 3481
CTATlTAc;ulTCAAGATAG+AAGGATGATGCcAACCAGX+ 3520
Fig. 2. The sequence 1, 2, 3 and 4 indicate
of pepF from An (GenBank accession No, X79541) and deduced aa sequence. The dashed arrows above the sequence labelled the oligos that were used for reverse transcriptase, PCR, cloning and sequencing to prove the presence of the three introns. The
consensus 5’ splice donor (GTRNGT), the 3’ splice acceptor (YAG) and the internal element TATAAA-like element, CAATC box and possible AATAAA-like element are in bold. Two Putative binding sites glucose-responsive repressor protein CREA are indicated with .--protein AREA are indicated with +. The start of the mature protein is indicated with a t.
(RCTRAC) of the three introns are underlined. Possible putative binding sites for the wide domain regulatory for the wide domain regulatory ammonium-responsive The deduced
N-terminal
sequence
of the mature
PEPF
protein compared to the sequenced N terminus (Dal Degan et al., 1992) is underlined (corresponding aa are in bold). Three putative signal-peptide cleavage sites predicted for the PEPF protein (von Heijne, 1986) are indicated with +. The three putative active-site residues (Szl*, D430 and Hso7) are double underlined. The seven putative N-linked glycosylation sites (N-X-T/S) in the mature PEPF protein are double underlined. Methods (a) Nucleotide sequence of pepF: The pepF gene was originally subcloned as a 4.1-kb EcoRI fragment into pUC18 and approx. 2.8 kb of this fragment and all of a l.l-kb Sal1 fragment, also subcloned into pUC18, were sequenced completely from both strands using the Sequenase DNA Sequencing TM Kit (Pharmacia LKB), employing synthetic oligo primers. The nt and aa Kit (US Biochemical, Cleveland, OH, USA) and the “Sequencing sequences were analyzed using the computer programmes of Devereux et al. (1984). (b) Intron mapping: An N400 conidia were inoculated into
77 1
120 ._..,_.,__.,__.._...,..,..............................I............. mlfrsllstavlbvslctdnasaakhgrfgqkardamniangs~n~vkhslk _._.__..__.._,.._..._..__....................... mattprlaslllllalcaaaagal...rlppdasfpgaqaerliralnllpgrprrglgagaedvapgq...
........................................................................................................................ ................................................ matarvs1i1lvwlaasacaegl...rlprdskfpaaqaerlirslnllpkeagptgagdvpsvapge
...
............................................. mekltflslllhfwfiastipsssfllndrtfersnlpstraeklirelnlfpqqdlnvidvadlpltaaegpg mkafts.llcglglsttlakaislqrplgldkdvllqaaekfgldld........................ldhllkeldsnvldawaqiehlypnqvmsletstkpkfpe~iktkkdw mklskstliatlaltatstnalwqnpfs.....niqqalkldlsydkltskltdtfeqgkaniistiakvmnepldgltpeikniwlemlmkfpnsitelnfk~ppk..kgkittqqf ........................................................................................................................ ........................................................................................................................ ........................................................................................................................
;: : e
f g h 5 con
;: c d e f z i j con
d d
121 240 ipvedyqflnnktkpyrv~slpdvhfdlg.emyaQlvpiekgnvsrsLFFvf~tigepvdettir(LnPQCSSl~.alspgecrFvwqpgty.....qpvelYpY~~ltnvlWvDq P llerrvtlpg..1....pegvgdlg......hhaQYyrlpnthdarMFYFffEsrgk.kedPvVi~tQQPQCSS.~lavfyBnGpPtiann......mslvwMcFgNdkIsniiFvDp a ....... lpg..l....p~gvadlg......hhaQYyrlpnthdarMFYPffEsrgk.kedPvVillLtOCSS.elavfyBnGpPtiann......msln*McFgWdkIsniiFvDq P llerrvtlpg..l....pqgvgdlg......hhaQYyrlpnthdaeMFYPlfEsrgk.kedPvVi~t~PQCSS.~lavfyBnGpFtisnn......mslaw~FgWdtIsniiFvDq P iverkfvfpn..iladggptvddlg......hhaQYyklpksrgasMPYPffBsrnk.kdaPvVillLtaPOCSS.elavfyBnGpPkitsn......mslawtPeYgWdqVsnllYvDq P fwkndaienyqlrvnkikdpkilgidpnvtqytQYldved.edkhFFFWtfEsrndpakdPvI1~n00PQCSS.ltglffBlGpssigpd......lkpig~Y~WnsnatviF1Dq P fhvtdaqvpnhklrik..stpkdlgidt.vkqyeGYldwd.edkhFFYYffEsrndpkndhrIlllLnOaPQCSS.ltglffElGpssidkn......lkpvyNph~lhlanasviF1Dq P apqgaevtglpgfdgalpskhyaQYvtvdeghgrnLFYYwEserdpgkd~Vl~n~PQCSSfd.gfvyEhGpPnfesggsvkslpklhl~YaWskVstmiYlDsP ........... .......... agghaadrivrlpgqpevdfdmy~QYitvdeaagrsLFYLlqEapeea~aPlVl~nQQPQCSSvaygaseBlGaFr~prg.....aglvlWeYrWnkVanvlFlDs P P ...... ..vepsghaadriarlpgqpavdf~~QYitvdegagrsLFYLlqBapeda~aPlVl~n~PQCSSvaygaseBlGaFrvkprg.....aglvlWeYrlhlkVanvlFlDs ___--__-_______-___--_________-___Q___--______________-__________~_~~Q~~~______-_____________________~___~_________~__
360 241 vQtQPSlgvpt..........atsEeeiaeDfvkPFknWqqifg..iLn.fkiWtQBSYAQrYVPyi~aafld~dtehfnlkgalaydpcIgqfdYvqeeap~fvqk~alfnfn a tQtQFSYssd......drdt.rhdEagvsnDlydFLqvFFkkhPeFv*n..dFFItQ~SYAQhYIPaFasrVhq~kn..e.......gthINLkgFaIGNGltDpaiqykaytdYa .. tGtQFSYssd......drdt.rhdECgvsnDlydPLqvFFkkhPeFi~..dFFItGESYMhYIPaFasrVhqgnkkn..e.......gthINLkgFaIGNGltDpaipykaytdYa .. tQtQFSYssd......drdt.rhdEtgvsnDlysFLqvFFkkhPeFaLn..dFFItQESYMhYIPaFasrVhqgnkan..e.......gihINLkgFaIGNGltDpaipykaytdYa .. ~tQFSYttd......ksdi.rhdBtgvsnDlydFLqaFFaehPkLa*n..dFYItQXSYAQhYIPaFasrVhkgnkan..e.......gvhINLkgFaIGNGltDpalqypaypdYa .. vnvGFSYsgs......sgvs...ntvaagkDvynFLelFFdqfPeYvnkgqdFhIaQESYAQhYIPvFaseIlshkdrn.............fNLtsvlIGNGltDpltpynWepmac g invQYSYs.s......qsvs...ntiaagkDvyaFLqlFFknfPeYa.n.ldFhIaQ~SYAQhYIPaFaseIlthpern.............fNLtsvlIGNGltDplvqye~epmac g aGvGLSYskn......vsd.yetgDlktatDshtFLlkWqlyPeFlsn..pFYIaQ~SYAQvYVPtL~heVvkgiqgg..a.......kptINFkgYmVGNGvcDtifdgnalvpFah g aQvGFSYtnt......ssdiytsgDnrtahDsyaPLaaWFerfPhY.krr.eFYVaGXSYAGhYV P ~qlVhr....s..g.......npvINLkgFmVGNGliDdyhdyvgtfeFww n n aGvQFSYtnt ......ssdiytsgDnrtahDsyaFLakWFerfPhYkyr..dFYIaGESYAGhYVPeL~qlVhr....s..k.......npvINLkgFmVGNGliDdyhdyvgtfeFww --_Q-~___-__-____-___________-~___~_____________________Q~~~~Q_~_~______________________________________________________
480 361 sflaelesiheqcgykdffdqylvFpasgvqppkamnwsdptcdvydivnnavldpnpcfnpye~nemcpil.......................................wdvlgfpt e c .. ldmnliqkadydrinkfippcePaiklc.gtdgkascmaaymvcnsifnsimklvgtkn~dvrkece.............................................g.kl c .. lemnliqkadyerinkfippceFaiklc.gtngkascmsaymvcntifnsimklvgtkn~dvrkece.............................................g.kl .. ldmnlikksdydrinkfippceFaiklc.gtngkascmaaymvcnsifssimklvgtknyydvrkece.............................................g.kl c .. lemglitqkehdrlekivplceLsiklc.gtdgttsclasylvcnslfsgvmshaggvnyydirkkcv.............................................g.sl c eggepsvlpseecsamedslerclgliescydsqswscvpatiycnnaqlapyqrtg.rnyrdirkdce.............................................ggnl c eggepsvlepeecdgmlnllprclsliescyesgs~sc~atiycnng~gpyqktg.rnvydirtmce.............................................gssl c mglisdeiyqqastsc....hgnyWnatd.gkcdtaiski.eslisglniydilepcyhsrsikevnlqnsklpqsfkdlgttnkpfpvrtrmlgrawplrapvkagr~swqevasg . hgivsddtyrrlkdac....lhdsFihps.pacdaatdva.taeqgnidmyslytpvcni.s .................................................... hgivsddtyrrlkeac....lhdsFihps.pacdaatdva.taeqgnidmyslytpvcnits....................................................stgsy d ____._______..__.._____-___--____--____-___-____________.________-___-_______________.._-.___._-___-._--.___..___.~~~~~ ~ 600 481 vdylpaapastltalIkrAmhapnitwsecsve~vfvggdggpeqegdy~anpi~hvlpqviegtnrVlIgnQ~~iltngtllaiq~t~gklgFdtapstpinidipdlmYnevf y.dfsnlekFFgdkaVrqAigvgdie.....Fvscstsvyqaml..td.wmrnlevgipall~dginVlIYaQByDlicnwlgns~hsMewsgqkdF.a............ktaes6 f y.dfsnlekFFgdkaVrqAigvgdie.....Fv~cstsvyqaml..td.wmrnl~vgipall~dginVlIYaQEyDlicnwl~~r~hsMewsgqkdF.a............ktaess f y.dfsnlekFFgdkaVkeAigvgdle.....Fvecsttvyqaml..td.wmrnl~vgipallsdginVlIYaGByDlicnwlgnsr~hs~ewsgqkdF.v............sshesp f y.dfsnmekFLnlqsVrkslgvgdid.....Fvscstsvyqaml..vd.wmrnlevgiptlledgiallVYaCiEyDlicnwlgnsr~aMewsgkCnF.g............aakevp f yptlqdiddYLnqdyVkeAvgaevdh.....Y~~cnfdinrnf1fagd.wmkpyhtavtdllnqdlpIlWaQDkDficnwlgnkawtdvLpwkydeeF.a............sqkvrn w ysqleyidqYLnlpeVkkAlgaevde.....Yq~cnfdinrnfmfagd.wmkpyqknvidllekelpV1IYaQDkDficnwlgnqawtnrLewsgskgF.t............kapvks w cmsdevataWLdnaaVrsAihaqsvsaigpwLlc...tdklyfvhdag.~miayhknl..t.sqgyraiIFsQD~cvpftgseawtksLgygwdsWrp............witng qv pcterystaYYnrrdVqtAlhanvtgamnytWtncsdtinthwhdapr.smlpiyrel..i.aaglrIwVFsQDtDavvpltatrysigaLglatttSW~............wyddlq e pcterystaYYnrrdVqmAlhanvtgamnytWatcsdtinthwhdapr.emlpiyrel..i.aaglrIwVFsQDtDavvpltatry~igaLglptttsWyp............wydd.q e _____________-____-___-____-_________________.__Q__~__.____.___._______.___.___..___..__-..____
694 601 iengydpqggqgvmgiqhyergLmWaetfqsG~qPqfqPrvaYrhLewLlgrrdt1 ................................... 1.......vddaqagvlkshgaLsFlkVhnaG~VPmdqPkaaLeMLrrFtqgklkes~eeepattfyaai .................... 1.......vdddqagvlkshgaLsFlkVhnaQ~VPmdqPkaaL~MLrrFtqgklk .................................... .................... " ..... ..vdgaeagvlkshgpLsFlkVhnaG~~mdqPka~LeMLrrFtqgklkeewlaelpe~myaam qcvsn i.......vdgkeagllktyeqLsFlkVrdaQ~VPmdqPkaaLkMLkrWmensliedatvtvaaqggeelvaadvitssfalhenkrqqi ik' . t.....asitdevagevksykhFtYlrVfngQ~VPfdvPenaLsMvneWihggfsl ................................... . ....................................................................... ... ..kvgknaagevknykhFtFlrVfggQ~VPydqPenaLdrWisgdyky ~:....gytegye.......hgLtFatIkgaGHtVPeykPqeaFdFYsrWl~gskl v.....ggwsqvy .......kgLtLvsVrgaQHeVPlhrPrqaLiLFqqFlqgkpmpgrttn .............................. v.....ggwsqvy.......kgLtLvsVrgaQEeVPlhrPrqaLvLFQyPlqgkpmpgqcknat ............................ _~~___~___~____~~___~~____~~___GB__P~__P_.___~_______~~_______~___~.___~___~~__~___~~___~__~ ._ =
Fig. 3. The aa sequence
comparison
of PEPF
with other
Ser-CPDs.
The aa sequences
were aligned
using the program
PILEUP
available
in the
GCG package. The aligned sequences are as follows: a, An PEPF; b, wheat CPD III (Baulcombe and Barker, 1987); c, barley CPD III (Sorensen et al., 1989); d, rice CPD (Washio and Ishikawa, 1992); e, Arabidopsis thaliana CPD (Bradley, 1992: GenBank accession No. P32826); f, S. cereuisiae CPY (Valls et al., 1987); g, C. albicans CPD
Y (Munkhtar
et al., 1992); h, barley
CPD
I (Sorensen
et al., 1986); i, barley
CPD II (Sorensen
et al.,
1987); j, wheat CPD II (Breddam et al., 1987. All sequences are shown in lowercase letters. A dot indicates that there is no aa at that position. The active site residues are double underlined in the consensus sequence. Partial conservation of aa residues is indicated with capital letters. Fully conserved aa residues
are indicated
with bold capital
letters; con, consensus
sequence
of all the aa sequences
shown above.
complete YPD medium (containing 1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose) for 24 h. Mycelium was then filtered, washed with sterile water and transferred to minimal medium supplemented with 1% collagen. After a further 5-6 h of cultivation under the same conditions mycelia were harvested, washed and freeze-dried for RNA isolation. Total RNA was prepared from freeze-dried mycelia by lysis in buffer containing 10 mM Tris pH 7.5/100 mM LiCI/2% SDS followed by several phenol extractions and ethanol precipitation. Poly(A)+RNA was isolated by oligo(dT) chromatography (Aviv and Leder, 1972). The splice sites of the introns were identified by cloning and sequencing partial cDNA copies of the pepF transcript. First strand synthesis was performed by standard methods (Sambrook et al., 1989). The priming oligo for intron 1 and 2 was oligo 2 and for intron 3 oligo 4. The cDNAs were then amplified by PCR using oligos 1 and 2 for introns 1 and 2 and oligos 3 and 4 for intron 3 and cloned into pUC18. Several independent clones were sequenced for each region.
78 A
B
1
2
3
+
+
-
A
C
12
3
12
3 12
anmonia
glucore -
+
aJmanlo g1ucol5e glycerol
+
pepc
+ + -
+ +
anunonio glucose glycarol
+ -
blot analyses
of the carbon
of the pepF gene. (A) Effect of nitrogen were grown
overnight
+ +
+ +
+
ammonia g1ucoas BEA callein alamtin collagen
in complete
and nitrogen
and carbon
liquid medium
regulation
derepression.
figure and grown for an additional
of pepF by ammonium
extract/l%
stripping
and
the membranes
and urea. Experiments
(a) Growth
conditions:
An N400 (CBS 120.49) was inoculated yeast
liter:
of wild-type
YPD (1% (w/v)
(w/v) o-glucose)
shaker
and
grown
for
at 220 rpm. Mycelia were filtered,
6 g NaNO,/l.S
MgSOJlOmg
each of MnCI,,
were:
NH&l,
100 mM
glycerol. harvested
g KH,PQ,/O.S
ZnSO,
100 mM
Cells were then grown by filtration,
urea,
and FeSO,.
1% (w/v) glucose,
for an additional
freeze-dried
g KCl/O.S
g
The supplements 1% (w/v)
6-8 h. Mycelia
were
and used for RNA isolation.
(b)
RNA isolation blotting and hybridization: Total RNA and poly(A)+RNA were prepared from freeze-dried mycelia as described in the legend to Fig. 2. 10 ug of total or l-2 ug poly(A)+RNA on formaldehyde Hybond-N
gels (Sambrook
membranes
Hybridization described
agarose and
(Sambrook
washing
(Amersham)
et al., 1989)
and of the membranes
UV
was loaded transferred
to
cross-linked.
were carried
out
as
et al., 1989).
teins as sole carbon and nitrogen sources, it was clear that the expression level of pepF changed significantly (Fig. 5A). Whereas only little message could be detected in cells grown on casein (lane 4) and hardly any in cells grown on BSA (lane 3) elastin (lane 5) and collagen (lane 6) appeared to induce the expression of pepF to highly elevated levels. When we compared the transcript levels of pepF in cells which were grown at pH 8.0 to that in cells grown at pH 3.0 under both repressing and inducing conditions (Fig. 5B), we found that when cells were derepressed-induced and grown at acidic pH, high message levels were detected (lanes 3 and 5) whereas cells grown on identical derepressing-inducing media whose pH was adjusted to 8.0, showed no pepF message. (f ) Conclusions (1) The pepF
+ -
+ -
_ + -
+
3
ammonia glucoac slaatin couagm PR
P~PF
PW
PePc
PePc
blot analyses
of pepF. (A) Comparison
4 + + 3
5 + + 8
6 + 3
c *
+ 0
3
of protein
of different
induction
external
and pH regulation
protein
inducers.
Each
protein inducer was used at 1% (w/v). (B) Effect of pH. Alkaline growth conditions were achieved by continuous adjustment of the pH using NaOH.
washed three times with distilled water and transferred to minimal medium supplemented as shown in the figure. Minimal culture media per
+ + + -
12
6
Experiments
were carried
out as described
in Fig. 4.
were carried
lo6 spore/ml
into complete
(w,‘v) peptone/2%
16-24 h at 3O’C on an orbital
contained
5
(B) Carbon repression of pepF by glucose were carried out as for A. (C) Nitrogen
between the two probings. and glycerol. Experiments repression
+ + -
Fig. 5. Northern
pH 6.0, then washed
6-8 h before RNA was isolated
to the pepF and pepC probes,
out as for A. Methods:
4
Cells
three times with distilled water, divided into three aliquots and transferred into minimal medium pH 6.0 supplemented as indicated in the hybridized
3
P8PC
PePc
Fig. 4. Northern
B
gene of An has been isolated using a degenerate oligo based on the N-terminal sequence of PEPF.
(2) The pepF gene encodes a protein consisting of 530 aa, and contains three introns of 53,69 and 59 bp, which were confirmed by generating cDNA with reverse transcriptase PCR and sequencing. (3) Signal sequence cleavage prediction analysis suggests that PEPF contains a 17-aa signal peptide at its N terminus which directs transport into the endoplasmic reticulum. A second proteolytic processing event at a monobasic cleavage site (Lyss2) removes another 35-aa propeptide, thus yielding the 478-aa mature protease. (4) The deduced aa sequence of PEPF shows homology to yeast and plant Ser-CPDs. (5) Southern analysis revealed that pepF is present in a single copy in An. (6) Northern analysis showed that the pepF gene is under carbon and nitrogen catabolite repression, under pH control and is inducible by protein at the level of RNA content. However, additional regulatory mechanisms cannot be ruled out.
ACKNOWLEDGEMENT
We thank Jeroen Krijgsveld for excellent technical work.
REFERENCES Aviv, H. and Leder, P.: Purification of biologically active globin messenger RNA by chromatography on oligothymidilic acid-celhtlase. Proc. Natl. Acad. Sci. USA 69 (1972) 1408-1412. Barthomeuf, C., Pourrat, H. and Pourrat, A.: Isolation and purification of a new protease from Aspergillus niger LCF No. 9. Biotech. Tech. 2 (1988) 29-34. Baulcombe, D.C.. Barker, R.F. and Jarvis, M.G.: A gibberellin respon-
79 sive wheat gene has homology to yeast carboxypeptidase Y. J. Biol. Chem. 262 (1987) 13726613735. Berka, R.M., Ward, M., Wilson, L.J., Hayenga, K.J., Kodama, K.H., Carlomango, L.P. and Thompson, S.A.: Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 86 (1990) 153-162. Bosmann, H.B.: Protein catabolism, II. Identification of neutral and acid proteolytic enzymes in Aspergillus niger. Biochim. Biophys. Acta 293 (1973) 476-489. Breddam, K.: Serine carboxypeptidases: a review. Carl&erg Res. Commun. 51 (1986) 83-128. Breddam. K., Sorensen, S.B. and Svendsen, I.: Primary structure and enzymatic properties of carboxypeptidase 11 from wheat bran. Carlsberg Res. Commun. 52 (1987) 2977311. Chopra, S. and Metha, P.: Influence of various nitrogen and carbon sources on the production of pectinolytic, cellulytic and proteolytic enzymes by Aspergitlus niger. Folia Microbial. 30 (1985) 117-125. Dal Degan, F., Ribadeau-Dumas, B. and Breddam, K.: Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis. Appl. Environm. Microbial. 58, (1992) 21442152. de Graatf, L., Van den Broek, H. and Visser. J.: Isolation and characterization of the Aspergiilus niduians pyruvate kinase gene. Curr. Genet. 13 (1988) 315-321. Devereux, J., Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Frederick, G.D.> Rombouts, P. and Buxton. F.P.: Molecular cloning and characte~zation of pepC, a gene encoding a serine proteinase from Aspergillus niger. Gene 125 (1993) 57-64. Gurr, S.J., Unkles, SE. and Kinghorn, J.R.: The structure and organization of nuclear genes of Iilamentous fungi. In: Kinghorn, J.R. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, 1987, pp. 93..139. Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J.: Cloning and expression of a second Aspergiihs niger pectin lyase gene ($A) _ indication of a pectin lyase gene family in Aspergillus niger. Curr. Genet. 18 (1990) 161-166. Ichishima, E.: Purification and characterization of a new type acid carboxypeptidase from Aspergik~. Biochim. Biophys. Acta 258 ( 1972) 2744288. Inoue, H., Kimura, T., Makabe, 0. and Takahashi, K.: The gene and deduced protein sequences of the zymogen of Aspergiilus niger acid proteinase A. J. Biol. Chem. 266 (1991) 19484-19489. Jarai, G., Kirchherr, D. and Buxton, F.P.: Cloning and characterization of the pepll gene of Aspergillus niger which codes for a subtilisinlike protease. Gene 139 (1994a) 41-57. Jarai, G., Van den Hombegh, H. and Buxton, F.P.: Cloning and characterization of the pepE gene of Aspergiffus niger encoding a new aspartic protease and regulation of the pepE and pepC genes. Gene 145 (1994b) 171-178. Krishnan, S. and Vijayalakshmi, M.A.: Purification of an acid protease and a serine carboxypeptidase from Aspergillus niger using metalchelate affinity chromatography. J. Chromatogr. 329 (198.5) 165-170. Krishnan, S. and ~‘ijayalakshmi, M.A.: Purification and some properties from Aspergillus niger. of three serine carboxypeptidases J. Chromatogr. 315 (1986) 315-326. Kudla, B., Caddick, M.X., Langdon, T., Martini-Rossi, N.M. Bennett, CF., Sibley, S., Davies, R.W. and Arst, H.N.: The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidu-
lam. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc-finger. EMBO J. 9 (1990) 1355-1364. Kulmburg, P., Mathieu, M., Dowzer, C., Kelly, J.M. and Felenbok, B.: Specific binding sites in the al& and &A promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in A. nidulans. Mol. Microbial. 7 (1993) 847-857. Kumagai, I. and Yamasaki, M.: Enzymatic properties of an acid carboxypeptidase from A. niger var. macrosporus. Biochim. Biophys. Acta 659 (1981) 344-350. Kumagai, I. Yamasaki, M. and Ui, N.: Isolation, purification and some chemical properties of an acid carboxypeptidase from Aspergillus niger var. macrosporus. Biochim, Biophys. Acta 659 (1981) 334-343. Martin, B.M., Svendsen, I., Vishwanatha, T. and Johansen, J.T.: Amino acid sequence of carboxypeptidase Y, I. Peptides from cleavage with cyanogen bromide. Carlsberg Res. Commun. 47 ( 1982) 1-13. Munkhtar, M., Logan, D.A. and Kaufer, N.F.: The ~arboxypeptidase Y-encoding gene from Cundida ufbiccms and its transcription during yeast-to-hyphae conversion, Gene 121 (1992) 173.-177. Sakka, K., Shimada, K. and Matsushima, K.: Purification and some properties of a serine proteinase from a mutant of Aspergillus niger. 3. Ferment. Technol. 63 (1985) 4799483. Sambrook, J., Fritsch, E.F. and Mania&, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5461. Sorensen, S.B., Breddam, K. and Svendsen, I.: Primary structure of ~arboxy~ptidase I from malted barley. Car&erg Res. Commun. 5 1 (1986) 475-485. Sorensen, S.B., Svendsen, I. and Breddam, K.: Primary structure of carboxypeptidase II from malted barley. Carlsberg Res. Commun. 52 (1987) 285-295. Sorensen, S.B., Svendsen, I. and Breddam, K.: Primary structure of ~arboxypeptidase III from malted barley. Carlsberg Res. Commun. 54(1989)193-202. Short, J.M., Fernandez, J.M., Sorge, J.A. and Huse, W.D.: h ZAP: a bacteriophage expression vector with in vivo excision properties. Nucleic Acids Res. 16 (1988) 7583-7600. Takahashi, K., Tanokura, M., Inoue, H., Kojima. M., Muto, Y., Yamasaki, M., Makabe, O., Kimura, T., Takizawa, T., Hamaya, T., Suzuki, E. and Miyano, H.: Structure and function of a pepstatininsensitive acid proteinase from Aspergiilus niger var. microsporus. Adv. Exp. Mol. Biol. 306 (1991) 203-211. Unkles, SE.: Gene organization in industrial filamentous fungi. In: Kinghorn, J.R. and Turner, G. (Eds.), Applied Molecular Genetics of Industrial Fungi. Blackie. Glasgow, 1992, pp. 29-53. Valls, L.A., Hunter, C.P., Rothman, J.H. and Stevens, T.H.: Protein sorting in yeast: the localization determinant of yeast vacuoiar carboxypeptidase Y resides in the propeptide. Cell 48 (1987) 887-897. Vishniac, W. and Santer, M.: The thiobacilli. Bacterial. Rev. 21 (1957) 195-213. von Heijne, G.: A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14 (1986) 4683-4690. Washio, K. and Ishikawa, K.: Structure and expression during the germination of rice seeds of the gene for a carboxypeptidase. Plant Mol. Biol. 19 (1992) 631-640. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33 (1985) 103-l 19.