JOURNAL OF BACTERIOLOGY,
0021-9193/00/$04.00⫹0Jan. 2000, p. 233–235 Vol. 182, No. 1
Copy igh © 2000, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Me aboli e Rep ession and Induce Exclusion in he P oline
U iliza ion Gene Clus e o Aspe gillus nidulans
BEATRIZ CUBERO,† DENNIS GO
´MEZ, AND CLAUDIO SCAZZOCCHIO*
Ins i u de Ge´ne´ ique e Mic obiologie, Uni e si e´ Pa is-Sud, UMR CNRS C8621, 91405 O say Cedex, F ance
Recei ed 29 July 1999/Accep ed 8 Oc obe 1999
The clus e ed p nB,p nC, and p nD genes a e ep essed by he simul aneous p esence o glucose and
ammonium. A de ep essed mu a ion inac i a ing a C eA-binding si e ac s in cis only on he pe mease gene
(p nB) while de ep ession o p nD and p nC is la gely he esul o e e sal o induce exclusion.
All genes in ol ed in he u iliza ion o p oline in Aspe gillus
nidulans a e clus e ed in ch omosome VII (Fig. 1). p nB en-
codes he speci ic p oline pe mease, p nD encodes p oline
oxidase, and p nC encodes L-⌬
1
-py oline ca boxyla e dehyd o-
genase (11). p nA encodes a Zn-binuclea clus e ansc ip-
ional ac i a o media ing p oline induc ion (5, 14). p nX is a
gene o unknown unc ion whose inac i a ion does no a ec
p oline u iliza ion (8).
The s uc u al genes o he p oline u iliza ion clus e a e
subjec o speci ic induc ion by p oline and o me aboli e e-
p ession. Ca bon ep ession is media ed by C eA, a nega i e-
ac ing zinc inge p o ein (4, 7). Ni ogen de ep ession is
media ed by A eA, a posi i e-ac ing ansc ip ion ac o be-
longing o he GATA amily (1, 12). I was shown many yea s
ago ha hese genes a e ep essed signi ican ly only when bo h
ep essing ca bon and ni ogen sou ces a e p esen , i.e., in he
simul aneous p esence o ammonium and glucose (8). We ha e
shown ha his p ocess ope a es a he le el o he s eady s a e
o he cogna e mRNAs (5, 16) (Fig. 2A). O he da a indica e
ha he ep ession p ocess ope a es a he le el o ansc ip-
ion a he han a he le el o mRNA s abili y (6). We ha e
shown ha ep ession ope a es di ec ly on he p nB gene and
ha he exp ession o he p nA gene is no a ec ed by ep es-
sion (5, 6, 16). A model accoun ing o hese indings has been
p esen ed (9). Mu a ion in any o bo h o wo speci ic C eA
binding si es (p n
d
), loca ed be ween he p nD and p nB in e -
genic egion, esul s in de ep ession o p nB,p nC, and p nD
exp ession (5, 6, 16) (Fig. 2B).
A. nidulans s ains lacking a eA canno u ilize p oline as a
ni ogen sou ce in he p esence o glucose as he sole ca bon
sou ce. The p n
d
mu a ions supp ess his pheno ype. A s and
collabo a o s ha e aken ad an age o his pheno ype o show
ha a mu a ion in wha we now know o be one o he wo
physiologically essen ial C eA-binding si es (6) is cis dominan
and ans ecessi e in ela ion o p nB bu is cis and ans
dominan in ela ion o p nC and p nD (3). The cis- ans es
was ca ied ou by checking he g ow h on p oline o a ho-
mozygous a eA diploid mu an ca ying a p n
d
mu a ion placed
ei he cis o ans o mu a ions in each o he s uc u al genes.
The e a e wo ways o explain he esul s o he cis- ans es .
The i s explana ion is ha he e is no di ec ep ession o
p nD and/o p nC, bu ha he appa en ep ession o hese
genes is he esul o induce exclusion due o he ep ession o
he p nB-encoded pe mease by glucose, and hei appa en
de ep ession is he esul o e e sal o induce exclusion,
ac ing ia he de ep ession o p nB. The second is ha he
C eA-binding si es in he p nD-p nB in e genic egion di ec ly
a ec he exp ession o p nD and/o p nC, bu ha he only
limi ing s ep o g ow h in p oline unde ep essing condi ions
is he exp ession o he pe mease coded by he p nB gene. In
his no e, we demons a e ha induce exclusion is la gely and
pe haps exclusi ely esponsible o p nD and, o a lesse ex en ,
p nC ep ession.
The s a egy we ha e used o his aim is shown in Fig. 1. In
a s ain ca ying an in e nal dele ion o p nB,p nB337 esul ing
in a null pheno ype o p oline up ake (17), we ha e inse ed a
agmen o he p n clus e in ans. This agmen includes he
whole wild- ype p nB gene, he en i e p nB-p nD in e genic
egion, and also a sho (902-bp) segmen o he p nD gene.
Two s ains we e cons uc ed, one ca ies he wild- ype e-
p essible p omo e , he o he ca ies a p omo e con aining a
mu a ion in one o he essen ial C eA-binding si es, p n
d
22 (6,
16). This mu a ion changes he canonical C eA-binding si e
5⬘CTGGGG in o 5⬘CTGAGG. This base pai change is su i-
cien o p e en all binding o his si e o C eA in i o (6).
uaZ11 is a I/VIII ch omosomal ansloca ion, which spli s he
uaZ gene (13). This null mu a ion esul s in he inabili y o
u ilize u ic acid as he sole ni ogen sou ce. T ans o man s able
o u ilize u ic acid can be selec ed by ans o ming uaZ11
s ains wi h a plasmid con aining he 3⬘uaZ moie y, including
an o e lap wi h he 5⬘moie y. Thus, ans o ming sequences
a e always a ge ed o he 5⬘moie y o he uaZ gene. The p n
ansgenes we e in eg a ed adjacen o he uaZ locus by ans-
o ming a p nB337 uaZ11 s ain (see he legend o Fig. 1 o
he comple e geno ype) wi h plasmids pXCw and pXCp n
d
22
(Fig. 1). We can hus di ec ly in es iga e he le els o exp es-
sion o he p nD and p nC genes in ans o he p omo e
egion d i ing he ac i e p nB gene.
The esul s o his in es iga ion a e shown in Fig. 2B. (i) In
spi e o he p esence o he comple e p nD-p nB in e genic
egion, he p nB ansgene is exp essed less han he co e-
sponding sequences a he p n locus. This emains unex-
plained. Ne e heless, he a io be ween de ep essed and e-
p essed le els in he wild- ype p omo e and he a ios be ween
he le els o he wild ype and hose o he p n
d
22 mu an a e
he same o he gene in he clus e and o he ansgene. (ii)
The p n
d
22 mu a ion in he esiden clus e esul s in de ep es-
sion o p nB,p nC, and p nD, as p e iously desc ibed by
Sophianopoulou e al. (16). The same e ec is seen on he p nB
ansgene. (iii) In he de ep essed mu an , he s eady-s a e
le els o p nB a e highe han in he nonde ep essed (wild- ype
* Co esponding au ho . Mailing add ess: Ins i u de Ge´ne´ ique e
Mic obiologie, Uni e si e´ Pa is-Sud, Baˆ imen 409, UMR CNRS
C8621, 91405 O say Cedex, F ance. Phone: 33-01-69-15-63-56. Fax:
33-01-69-15-57-08. E-mail: [email p o ec ed].
† P esen add ess: Depa amen o de Gene ica, Uni e sidad de
Se illa, Se illa 41080, Spain.
233
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p omo e ) s ain e en unde condi ions o de ep ession o he
la e . I could be said ha he p n
d
22 mu a ion esul s also in
an “up-p omo e ” e ec . This is ue bo h o he ansgene
and o he esiden locus (16). (i ) We know om classical
gene ic es s ha p n
d
mu a ions a e cis dominan and ans
ecessi e in ela ion o p nB (3). Howe e , he le el o he
sho p nB message ansc ibed om he esiden gene ca y-
ing a dele ion mu a ion is no ep essed when in ans wi h he
p n
d
22 mu a ion. This could be expec ed i o e exp ession o
he p nB ansgene esul ed in induce accumula ion and i his
accumula ion could pa ially bypass ca bon and ni ogen me-
aboli e ep ession. Al e na i ely o addi ionally, he small e-
sidual message may be mo e s able han he wild- ype p nB
message. ( ) The c ucial obse a ion is ha he in oduc ion o
he p n
d
22 mu a ion esul s in comple e de ep ession o p nD
in ans and a conside able de ep ession o p nC in ans. The
mRNA o he unca ed p nD ansgene ollows he same e-
p ession pa e n as he wild- ype mRNA om he esiden
clus e . The conclusion is ha induce exclusion accoun s o
he ca bon and ni ogen ca aboli e ep ession o p nD and
p nC. ( i) Fo p nC, howe e , de ep ession in ans seems less
p onounced han de ep ession in cis. No he n blo s p o ide
semiquan i a i e es ima es, and we would no wish o claim
ha his small di e ence is signi ican . Howe e , a clea e ec
a a dis ance o sequences in he p nD-p nB in e genic egion
on he exp ession o p nC was demons a ed long ago wi h
classical gene ics (2). These esul s ha e now been con i med
a he le el o he exp ession o he p nC mRNA (15) (D.
Go´mez and C. Scazzocchio, unpublished esul s). The egion
in ol ed has been mapped (D. Go´mez and C. Scazzocchio,
unpublished esul s) and coincides wi h a egion in ol ed in
he in eg a ion o ca bon and ni ogen me aboli e ep ession
(9). This may sugges ha while induce exclusion is he main
pa ame e in ol ed in he ep ession o p nD, a mode a e
FIG. 1. (a) Schema ic ep esen a ion o p n clus e . The di ec ion o ansc ip ion o he genes (acco ding o e e ences 8, 9, 10, and 16) is indica ed by a ows. (b)
S a egy used o he cons uc ion o s ains CS-2289-1 and CS-2289-2. S ain uaZ11 p nB337 pabaA1 iboB2 yA2 was ans o med wi h he plasmids pXCw and
pXCp n
d
22. Bo h plasmids con ain he 3⬘end o he uaZ gene and he XhoI-ClaI egion o he p n clus e . pXCw con ains a wild- ype egion, and pXCp n
d
22 con ains
a egion ca ying a mu a ion in an essen ial C eA-binding si e (6). (c) Schema ic ep esen a ion o s ains CS-2289-1 and CS-2289-2. Fo each s ain, he uppe scheme
shows he esiden p n clus e , and he lowe scheme shows he p n sequences in eg a ed a he uaZ locus. The XhoI-ClaI agmen o p n clus e in eg a ed a he uaZ
locus con ains a unca ed copy o p nD gene (p nD ), he p nD-B in e genic egion, he p nB gene, and he p nB-C in e genic egion. The esiden p n locus ca ies
he p nB337 dele ion. The C eA-binding si es ac i e in ep ession a e ep esen ed as o als: da ks o als o wild- ype si es and a whi e o al o he p n
d
-22 mu an si e.
234 NOTES J. BACTERIOL.
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cis-ac ing e ec o he p nD-p nB in e genic egion migh also
be exe ed on he exp ession o p nC.
This wo k was suppo ed by Eu opean Union g an s BIO2-CT93-
0147 and BIO-CT96-0535. D.G. ecei ed a esea ch s uden ship om
he Minis e` e de l’Educa ion Na ional, de l’Enseignemen Supe ieu e
de la Reche che o he F ench Go e nmen .
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FIG. 2. No he n blo analysis o p n genes ansc ip ion. (A) Exp ession o he p nB,p nD, and p nC genes in a wild- ype s ain g own unde di e en condi ions.
Noninduced le els and all possible combina ions o ca bon and ni ogen ep essing condi ions a e shown. (B) mRNA le els o he p n genes in a s ain ca ying p n
d
22
and in s ains CS-2289-1 and CS-2289-2. Only wo condi ions a e shown: induced non ep ession (UF) and induced double ep ession (NG). mRNAs ex ac ed om
a wild- ype s ain g own unde iden ical condi ions a e also shown. Less RNA, as shown by he acnA panels, has been loaded o he la e , bu he s ong ep ession
a o ded by he simul aneous p esence o glucose and ammonium is clea ly isible and can be compa ed wi h ha o he equi alen mRNAs o panel A. Mycelia we e
g own o 8ha 37°C in 0.1% uc ose and 5 mM u ea and hen we e ei he le noninduced (NI) o induced wi h 20 mM L-p oline (UF). Simul aneously, hey ei he
we e le non ep essed (UF) o we e glucose ep essed (1% glucose, 5 mM u ea) (UG), ni ogen ep essed [20 mM diammonium D-(⫹)- a a e, 0.1% uc ose] (NF),
o ca bon and ni ogen ep essed [1% glucose, 20 mM diammonium D-(⫹)- a a e] (NG) o 2ha 37°C. The memb anes ha e been p obed o p nB,p nD,p nC,
and acnA (ac in), he la e as a con ol o RNA loading. The me hods used o RNA p epa a ion we e hose o Gonza´lez e al. (9).
VOL. 182, 2000 NOTES 235
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