A single gene o lycopene cyclase, phy oene
syn hase, and egula ion o ca o ene
biosyn hesis in
Phycomyces
Nabil A ach, Ra ael Fe na
´ndez-Ma ı´n, En ique Ce da
´-Olmedo*, and Ja ie A alos
Depa amen o de Gene´ ica, Uni e sidad de Se illa, 41012 Se illa, Spain
Communica ed by No man H. Giles, Uni e si y o Geo gia, A hens, GA, No embe 20, 2000 ( ecei ed o e iew Augus 15, 2000)
P e ious complemen a ion and mapping o mu a ions ha change
he usual yellow colo o he Zygomyce e Phycomyces blakesleea-
nus o whi e o ed led o he de ini ion o wo s uc u al genes o
ca o ene biosyn hesis. We ha e cloned one o hese genes, ca RA,
by aking ad an age o i s close linkage o he o he , ca B, espon-
sible o phy oene dehyd ogenase. The sequences o he wild ype
and six mu an s ha e been es ablished, compa ed wi h sequences
in o he o ganisms, and co ela ed wi h he mu an pheno ypes.
The ca RA and ca B coding sequences a e sepa a ed by 1,381
un ansla ed nucleo ides and a e di e gen ly ansc ibed. Gene
ca RA con ains sepa a e domains o wo enzymes, lycopene cy-
clase and phy oene syn hase, and egula es he o e all ac i i y o
he pa hway and i s esponse o physical and chemical s imuli om
he en i onmen . The lycopene cyclase domain o ca RA de i ed
om a duplica ion o a gene om a common ances o o ungi and
B e ibac e ium linens; he phy oene syn hase domain is simila o
he phy oene and squalene syn hases o many o ganisms; bu he
egula o y unc ions appea o be speci ic o Phycomyces.
Ca o enoids a e e a e penoid pigmen s syn hesized by pho-
osyn he ic o ganisms and many bac e ia and ungi and
acqui ed by animals wi h hei die . The a ac i e colo s and he
bene icial heal h e ec s o
␤
-ca o ene and o he ca o enoids
ha e s imula ed in e es in hei chemical and biological
p oduc ion.
The p oduc ion o
␤
-ca o ene by he Zygomyce e Phycomyces
blakesleeanus has been in es iga ed o o e hal a cen u y (1).
Th ee enzyme ac i i ies a e equi ed (Fig. 1): phy oene syn hase
joins wo molecules o ge anylge anyl py ophospha e o o m
phy oene; phy oene dehyd ogenase in oduces ou double
bonds o yield lycopene; and lycopene cyclase shapes he
␺
acyclic ends o lycopene as
␤
ings. This scheme is main ained in
all ca o enogenic o ganisms, wi h some modi ica ions. Fo ex-
ample, oxygen a oms may be in oduced in he ca o enes o o m
xan hophylls.
The yellow colo o wild- ype Phycomyces is modi ied by
mu a ions o ed, whi e, o a ious g ada ions o yellow. The ed
mu an s accumula e lycopene, and he whi e mu an s ei he
accumula e phy oene o lack all ca o enes, o a leas do no ha e
enough o a isible colo (2, 3). The gene ic analysis by
complemen a ion, ecombina ion, and e e sion o he ed and
whi e mu an s iden i ied wo closely linked genes, ca B and
ca RA (4–6). Thei p oduc s, and possibly hose o o he genes,
a e o ganized in o an enzyme complex wo king as an assembly
chain in which he ou dehyd ogena ions a e ca alyzed by ou
iden ical uni s o phy oene dehyd ogenase (7, 8) and he wo
cycliza ions a e ca alyzed by wo iden ical uni s o lycopene
cyclase (9, 10).
Gene ca RA has wo dis inc domains (5). Domain R, p oxi-
mal wi h espec o he ansla ion s a , is cha ac e ized by he
ed ca R mu an s and is esponsible o lycopene cyclase. The
dis al domain A is cha ac e ized by ca A mu an s, which no -
mally a e whi e and ha e small amoun s o
␤
-ca o ene, bu , in he
p esence o e inol, p oduce subs an ial amoun s o
␤
-ca o ene
and become yellow (11). The ca RA mu an s wi h null pheno ype
o bo h domains a e whi e and do no espond o e inol (5).
The concen a ion o
␤
-ca o ene in he cells depends on
en i onmen al ac o s. Syne gisms and di e en ial e ec s on he
mu an s allow he classi ica ion o hese ac o s in ou g oups
wi h sepa a e mechanisms o ac ion. Blue ligh inc eases he
ca o ene con en in he wild ype mo e han 10- old. This
esponse is de ec i e in mu an s o many genes (12). Sexual
ac i i y, media ed by ispo a es, inc eases he ca o ene con en
mo e han 5- old (13, 14) and e inol and dime hyl ph hala e
mo e han 40- old (11, 15). Some ‘‘insensi i e’’ mu an s we e
isola ed because o hei limi ed esponse o e inol, bu hey
u ned ou o be equally de ec i e in hei esponses o he o he
ac i a o s (11, 14, 15); he mu a ion in one o hese mu an s,
s ain S119, is e y closely linked o a ca A mu a ion; he one in
s ain S144 is unlinked (6) and de ines gene ca I. Unde s anda d
condi ions, bo h a e ligh yellow and ha e jus a li le less
␤
-ca o ene han he wild ype.
The ca o ene con en is inc eased pe manen ly by ecessi e
mu a ions a gene ca S (13), dis an om he ca B–ca RA gene
clus e on he same ch omosome (6). The ca S mu an s a e
insensi i e o e inol, bu sensi i e o ispo a es and dime hyl
ph hala e (14, 15). O he a ia ions in ca o ene con en a e
caused by mu a ions in he ca C,ca D, and ca F egula o y genes
(16–18), none o which is known o be close o he s uc u al
genes.
The gene o phy oene dehyd ogenase, ca B, was cloned (19)
because o i s simila i y o al-1, he gene om Neu ospo a c assa
wi h he same unc ion (20). We ha e cloned and sequenced gene
ca RA om Phycomyces by aking ad an age o i s close linkage
wi h ca B and ha e co ela ed he sequences o se e al Phyco-
myces mu an s wi h hei pheno ypes and wi h homologous
sequences om a ious o ganisms.
Ma e ials and Me hods
S ains and Cul u e Condi ions. The P. blakesleeanus Bg . wild- ype
NRRL1555 (used unless o he wise s a ed) and he mu an
s ains used in his wo k a e lis ed in Table 1. In he s ain
designa ions, NRRL s ands o he U.S. Depa men o Ag i-
cul u e labo a o y in Peo ia, IL; C o he collec ion o he
o me P o . Max Delb u¨ck a he Cali o nia Ins i u e o Tech-
nology (Pasadena), and S o ou collec ion. The mu an s we e
ob ained a e ea men s wi h he mu agen N-me hyl-N⬘-ni o-
N-ni osoguanidine (21). Phycomyces was cul u ed and handled
as desc ibed (22); s anda d condi ions a e 4 days on minimal aga
Da a deposi ion: The nucleo ide sequence ha con ains gene ca RA has been deposi ed in
he GenBank da abase (accession no. AJ278287).
*To whom ep in eques s should be add essed. E-mail: [email p o ec ed].
The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This
a icle mus he e o e be he eby ma ked “ad e isemen ” in acco dance wi h 18 U.S.C.
§1734 solely o indica e his ac .
A icle published online be o e p in : P oc. Na l. Acad. Sci. USA, 10.1073兾pnas.021555298.
A icle and publica ion da e a e a www.pnas.o g兾cgi兾doi兾10.1073兾pnas.021555298
PNAS
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Feb ua y 13, 2001
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ol. 98
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no. 4
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GENETICS
a 22°C. Esche ichia coli DH5
␣
was used o he mul iplica ion
o plasmids.
DNA Isola ion and Manipula ion. Genomic DNA was isola ed as
desc ibed (23). Cleane genomic DNA p epa a ions (mo e sen-
si i e o es ic ion enzymes) a e ob ained om spo angiopho es
han om mycelium (24). Fo Sou he n blo s, genomic DNA
(1–4
␮
g) was diges ed o e nigh wi h es ic ion enzymes, sep-
a a ed by elec opho esis in an aga ose gel (8 g兾li e ), ans-
e ed o a nylon memb ane, hyb idized wi h p obes labeled wi h
digoxigenin-11-dUTP, and de ec ed wi h Lumigen PPS ollow-
ing he manu ac u e ’s ecommenda ions (Roche, Mannheim,
Ge many). Phycomyces cDNA was ob ained wi h he TimeSa e
cDNA ki and mRNA om ege a i e mycelia isola ed wi h
he QuickP ep mRNA Pu i ica ion ki (bo h om Ame sham
Pha macia).
A lambda 2001 gene lib a y was cons uc ed by inse ing
Phycomyces DNA pa ially diges ed wi h Sau3A in o he BamHI
si e o he ec o and packaged in o phage pa icles wi h he
Gigapack Gold Ki (S a agene). The lib a y, consis ing o abou
7,000 independen clones, was mul iplied and sc eened o
hyb idiza ion wi h a 2.2-kb Phycomyces DNA agmen (p obe B
in Fig. 2A) ha con ains he p omo e and pa o he coding
sequence o gene ca B. P obe B was cu wi h HindIII om he
5-kb EcoRI agmen ha includes he whole ca B gene (19). A
posi i e clone con ained a 13-kb inse , a SacI si e in he inse ,
and wo o he SacI si es in he polylinke sequence, lanking he
inse ion si e. Two DNA agmen s o abou 5.7 kb and 7.8 kb,
espec i ely, we e cu om he chime ic lambda molecule by
SacI diges ion, subcloned in he SacI si e o ec o Bluesc ip
KS(⫹), and cha ac e ized by es ic ion and pa ial sequencing.
The loca ion o gene ca B was deduced by compa ison o he
es ic ion agmen s wi h hose expec ed om he known ca B
gene sequence.
A 1.4-kb EcoRV–SacI agmen o he 13-kb inse (p obe R
in Fig. 2A) was ound o hyb idize wi h a single 9-kb EcoRI–SpeI
genomic DNA agmen . A pa ial genomic lib a y o 6- o 10-kb
EcoRI–SpeI agmen s o Phycomyces genomic DNA in Blue-
sc ip KS(⫹) was cons uc ed and sc eened wi h p obe R. This
led o he isola ion o a posi i e clone wi h a 9-kb inse .
The PCR mix u es con ained abou 5 ng genomic DNA, 0.2
mM o each o he ou deoxynucleoside iphospha es, 1
␮
M
o each p ime , and 0.5
␮
l o he DNA polyme ase mix u e
Expand High Fideli y (Roche) in a inal olume o 50
␮
l. A e
o e laying hem wi h ligh mine al oil, hey we e incuba ed
once a 95°C o 3 min, 35 imes a 95°C o 1 min, 55°C o 1
min, and 68°C o 3 min, and once a 68°C o 10 min in a
GeneAmp 2400 p og ammable he mocycle (Pe kin–Elme 兾
Ce us). The cDNA o be sequenced was mul iplied wi h
p ime s 5⬘-TTCTTTATTCCTGTGACTTTCCACGATCA-3⬘
and 5⬘-GCGCGCATGCTGACTTATATGGAAGT-3⬘. Fo
sequencing he mu an alleles, his las p ime was eplaced by
5⬘-CAGTACTAAGCACCATTCAC-3⬘.
DNA was sequenced by Medigene (Ma ins ied, Ge many)
using he ABI377 au oma ic sequence (Pe kin–Elme ). Mu an
alleles we e sequenced wice om independen PCR p oduc s.
Fo o he DNA manipula ions and echnical de ails, see e . 25.
Sequence Analyses. Alignmen s we e acili a ed by he CLUSTAL W
1.6 and X 1.63B compu e p og ams (26–28). Hyd ophobici y
p o iles we e ob ained wi h he Expe P o ein Analysis Sys em
(ExPASy; Swiss Ins i u e o Bioin o ma ics, Gene a, Swi ze -
land). The sea ch o p o ease clea age si es (29) was ca ied ou
wi h he SIGNALP 1.1 (Technical Uni e si y o Denma k, Lyngby)
and he PSORT II (Ins i u e o Medical Science, Uni e si y o
Tokyo, Tokyo) compu e p og ams.
Resul s
DNA Flanking he
ca B
Gene o
Phycomyces
.The e y close linkage
o genes ca RA and ca B led us o expec bo h o be p esen
simul aneously in small DNA agmen s o he Phycomyces
genome. A Phycomyces lambda 2001 gene lib a y was sc eened
o hyb idiza ion wi h a DNA agmen (p obe B in Fig. 2A) ha
con ains gene ca B and i s p omo e . A clone wi h a 14-kb DNA
inse was isola ed and cha ac e ized o ob ain a es ic ion map
and he loca ion o he ca B gene.
A agmen om one o he ends o he 14-kb DNA inse
(p obe R in Fig. 2A) hyb idized wi h single agmen s o
Phycomyces genomic DNA cu wi h a ious es ic ion enzymes,
e en a low s ingency condi ions (Fig. 3). This indica ed ha he
Phycomyces genome con ains a single copy o he p obe R
Fig. 1. Genes, enzymes, and chemical eac ions o ca o ene biosyn hesis in
Phycomyces. Two molecules o ge anylge anyl py ophospha e a e con e ed
o one molecule o
␤
-ca o ene by phy oene syn hase (diamonds), ou copies
o phy oene dehyd ogenase (squa es), and wo copies o lycopene cyclase
(ci cles). The scheme on he le ep esen s he genes ha code o hese
enzymes. The chemical changes a e ma ked wi h a owheads.
Table 1. S ains o P. blakesleeanus used in his wo k wi h he nucleo ide changes in hei ca RA gene and he p edic ed amino acid
changes
S ain Mu a ion Main ca o ene Nucleo ide change Amino acid change
NRRL1555 none
␤
-ca o ene None None
C9 ca R21 Abundan lycopene 908 C 3T 215 P 3S
C11 ca R23 Abundan lycopene 229 G 3A77 E3K
C6 ca RA12 T aces o lycopene 400 G 3A See ex
S92 ca RA91 T aces o lycopene 177 G 3A59W3s op
C2 ca A5 Sca ce
␤
-ca o ene 1710 C 3T 481 P 3L
S119 ca A113
␤
-ca o ene 1659 T 3C 465 I 3T
See e . 21 o addi ional de ails.
1688
兩
www.pnas.o g A ach e al.
sequence. This p obe was used o isola e a 9-kb agmen o
genomic DNA, which ex ended o o e 20 kb he a ailable DNA
ha includes and su ounds gene ca B.
The whole egion was explo ed by cloning and sequencing 13
sub agmen s (Fig. 2A). S4 and S5 caugh ou a en ion and led
us o sequence comple ely a segmen o 3,100 bp, whe e a
pu a i e new gene was ound (Fig. 2B). This gene was iden i ied
as gene ca RA by sequencing he same agmen om some
mu an s ha had been used o de ine gene ca RA, as desc ibed
below.
The sequences S2, S3, and S11 had low p opo ions o GC base
pai s, as is usual in he in e genic sequences o Phycomyces (30),
and con ained abundan s op codons in all possible eading
ames. Se e al pu a i e genes seem o be p esen in he egion,
bu hey do no seem o be ela ed o ca o ene biosyn hesis. One
o hem, closely simila o
␣
-
␤
hyd olases (well known ungal
lipases), would be ansc ibed owa d he igh o e lapping
sequences S12 and S13. Ano he one, e y simila o an unknown
gene om D osophila melanogas e and, o a lowe ex en , o
genes o mucins om se e al animals, seem o be p esen in
sequence S10. Th ee addi ional ORFs we e ound in sequences
S1, S6-S7, and S8–S9.
The Wild-Type
ca RA
Sequence. Gene ca RA was composed o wo
ORFs wi h a o al o 1,806 bp, in e up ed by a 265-bp in on.
The sequence and he p ecise limi s o he in on we e de ined
by sequencing DNA agmen s ob ained by PCR om a cDNA
sample (Fig. 2B). The pu a i e Ca RA p o ein would ha e 602
aa. Codon usage in gene ca RA is simila o ha o o he
Phycomyces genes, wi h a ma ked p e e ence o py imidines,
and pa icula ly o C, in he hi d posi ion o he codons.
The pola i y p o ile o Ca RA (Fig. 4) de ined wo di e en
domains. Domain R, co e ing 40% o he p o ein om i s amino
end, would be hyd ophobic and ansmemb anal; he o he one,
domain A, co e ing he es o he ca boxyl end, would be
hyd ophilic. The on ie be ween bo h domains lies app oxi-
ma ely a he mo i AHAIV (amino acid esidues 239–243),
which is a pu a i e p o ease clea age si e ha could spli he
polypep ide be ween esidues 241 and 242.
DNA Sequences in
ca RA
Mu an s. The ca RA sequence was ob-
ained om six independen colo mu an s ha we e known o
suspec ed o ca y mu a ions in gene ca RA, acco ding o
p e ious gene ic analyses (4–6). Th ee o hese mu an s a e
albino, wo a e ed, and one is ligh yellow. All o hem ca ied
mu a ions in he newly cloned gene (Table 1).
S ains C6 and S92 a e albino ca RA mu an s, wi h e y li le
lycopene, no
␤
-ca o ene, and insensi i e o e inol. The mu a ion
ca RA91 in s ain S92 eplaced a TGA s op wi h he no mal
yp ophan a codon 59. In s ain C6, he i s base o he in on,
Fig. 2. S uc u al genes o ca o ene biosyn hesis in Phycomyces.(A) A DNA egion ha con ains genes ca B and ca RA. P obes B and R we e used o sc een
a lambda lib a y and a pa ial plasmid lib a y and isola e he 14-kb and he 9-kb agmen s, espec i ely. Sequencing eac ions a e indica ed by he hin a ows
S1 h ough S13. The g ay segmen ep esen s he DNA sequence published by Ruiz-Hidalgo e al. (19). (B) A 6494-bp DNA sequence ha con ains genes ca B and
ca RA. Ha ched segmen s ep esen in ons and whi e segmen s ep esen in e genic sequences. Thin a ows ep esen genomic sequencing eac ions, and
hicke a ows below ep esen cDNA sequencing eac ions. The a ge si es o es ic ion endonucleases a e: B, BglII; Bm, BamHI; C, ClaI; E, EcoRI; H, HindIII;
K, KpnI; P, Ps I; S, SacI; Sp, SpeI; V, EcoRV; X, XhoI; and Xb, XbaI.
Fig. 3. Hyb idiza ion o p obe R (Fig. 2A) wi h Phycomyces genomic DNA
agmen s cu wi h se e al es ic ion enzymes (abb e ia ed as in Fig. 2) and
sepa a ed by elec opho esis. The wo pic u es di e in he s ingency o he
es (washed a 65°C, Le , and 42°C, Righ ).
A ach e al. PNAS
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Feb ua y 13, 2001
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1689
GENETICS
guanine, was eplaced by adenine. Bo h changes in he sequence
o he gene amply jus i y he pheno ypes o he espec i e
mu an s.
S ains C9 and C11 a e ed ca R mu an s, de icien in lycopene
cyclase, ich in lycopene, and insensi i e o e inol. In each, an
amino acid o he R domain was eplaced by a e y di e en
amino acid (p oline by cys eine, glu ama e by lysine). C i ical
si es o lycopene cyclase ac i i y he e o e mus lie in he R
domain o he Ca RA p o ein.
The wo o he s ains had single amino acid subs i u ions in
he A domain o he Ca RA p o ein. The albino pheno ype o
s ain C2 is he esul o a p oline o leucine eplacemen . S ain
S119, isola ed because o i s weak esponse o chemical ac i a-
o s, was suspec ed o ha e a mu a ed gene ca RA only because
o igh linkage wi h he ca A5 mu a ion (6). The inding o an
isoleucine o h eonine subs i u ion in he A domain con i ms
his suspicion and jus i ies calling he mu a ion ca A113.
Sequence Compa isons. Gene ca RA om P. blakesleeanus is
simila o genes om o he ungi (Fig. 5) in he gene al s uc u e
o he p edic ed p oduc (an amino- e minal hyd ophobic do-
main and a ca boxyl- e minal hyd ophilic domain) and in he
ac ual sequence. Pa icula ly well conse ed a e he si es o
mu a ions ha we ha e analyzed, wi h he ob ious excep ion o
he one a he in on. The pu a i e p o ease clea age si e
AHAIV ha would spli he Phycomyces ansla ion p oduc
a e esidue 241 is no conse ed, bu he o he gene p oduc s
con ain al e na i e clea age si es. Fo example, he Xan hophyl-
lomyces p oduc may be spli a LSACD a e esidue 248 and he
Neu ospo a gene p oduc , a LWAVD a e esidue 190.
The Phycomyces R domain e ains he aces o an old
duplica ion. This is seen by compa ing he sequences be o e and
a e he loca ion o he in on (Fig. 6). These app oxima e
hal es a e ela ed o each o he (27 posi ions in he co espond-
ing polypep ide sequences a e occupied by iden ical amino acids)
and e en mo e so o gene p oduc C Yc om B e ibac e ium
linens (31 and 38 iden ical amino acids, espec i ely). The
Phycomyces R domain is simila o hose o o he ungi (130, 62,
and 56 iden ical amino acids wi h hose o Muco ,Xan hophyl-
lomyces, and Neu ospo a, espec i ely; Fig. 5), bu bea s li le o
no ela ionship o he lycopene cyclases om plan s.
The Phycomyces domain A coincides in 211, 120, and 100
amino acids wi h he co esponding domains o Muco ,Xan ho-
phyllomyces, and Neu ospo a, espec i ely (Fig. 5), in 74–98
amino acids wi h nine phy oene syn hases (Ag obac e ium au-
an iacum,Rhodobac e sphae oides,Synechococcus PCC7942,
Synechocys is sp, Spi ulina pla ensis,Dunaliella ba dawil,A abi-
dopsis haliana,Lycope sicon esculen um, and Zea mays) and in
69–79 amino acids wi h six squalene syn hases (Candida u ilis,
Capsicum annuum,Solanum ube osum,Zea mays,Leishmania
majo , and Homo sapiens). Phy oene and squalene syn hases
ca alyze exac ly he same chemical changes on di e en
subs a es.
Discussion
We ha e cloned a gene om he Phycomyces wild ype and six
colo mu an s o en used o in es iga e he gene ics and o he
aspec s o ca o ene biosyn hesis. The sequences o he six colo
mu an s clea ly iden i ied he gene as he ca RA gene p e iously
de ined by complemen a ion and ecombina ion (4–6). Ou
molecula cha ac e iza ion o gene ca RA con i ms he esul s o
classical gene icis s, o such an ex en ha he bes way o s a e
ou conclusions is o copy hei s (5): ‘‘The A and R unc ions a e
de e mined by con iguous DNA segmen s, co ansc ibed o a
single mRNA, and co ansla ed o a single polypep ide. The R
segmen is p oximal o he gene’s beginning (5⬘o he mRNA
and NH
2
end o he polypep ide). Pheno ype R would a ise om
missense mu a ions in he R segmen ; pheno ype AR om
nonsense o ameshi mu a ions in he R segmen .’’
In ac , he wo ca R mu an s ca y missense mu a ions in he
R domain. These a e a apa on he p ima y s uc u e, bu bo h
a ec he PLEE e apep ide ha is epea ed in he wo hal es
o he R domain (Fig. 6). The ca R21 mu an p o ein su e s om
a d as ic loss o enzyme ac i i y, bu in eg a es in he enzyme
agg ega e as e ec i ely as he wild- ype p o ein (9).
The ca RA91 mu a ion in oduces a p ema u e TGA s op
codon in he R domain and would esul in a sho , p esumably
inac i e, polypep ide wi h only 58 aa. The aces o lycopene
ound in mu an mycelia equi e some phy oene syn hase ac i -
i y, which may esul om a low na u al le el o TGA supp es-
sion. I is also possible ha squalene syn hase p ocesses phy oene
o a ce ain ex en .
The ca RA12 mu a ion in s ain C6 modi ies he i s nucle-
o ide o he c i ical GU dinucleo ide splice si e a he 5⬘end o
he in on. Because he in on con ains a s op codon in-phase
wi h he eading ame o he gene, he excision ailu e would
esul in a polypep ide wi h 149 aa, 16 o hem in a ail un ela ed
o he o iginal sequence. The aces o lycopene in he mu an
migh esul om esidual excision o he in on o om he a e,
in-phase excision o ano he nucleo ide segmen . Red e e an s
we e ob ained om s ain C6, and he mu a ion in one o hem
was e y closely linked o he o iginal mu a ion (5). This is likely
o esul om he c ea ion o a new in on excision si e, so ha
he R ac i i y is los , bu he gene is ead h ough o p oduce a
co ec phy oene syn hase.
The missense mu a ion ca A5 in s ain C2 esul s in a 10- o
15- old educ ion o he
␤
-ca o ene con en wi h espec o he
app op ia e con ols. Unde s anda d condi ions, s ain C2
con ains 4
␮
g兾g d y mass, s. 62 in he wild ype (35), and simila
educ ions a e ound unde sexual s imula ion, in he p esence o
ispo ic acids, e inol, and dime hyl ph hala e, and a e in o-
duc ion o a ca S mu a ion in he gene ic backg ound (11, 14, 15,
36). O he ca A mu an s exhibi hei own educ ion le els. This
means ha he mu a ions impai o a ce ain ex en he ca aly ic
ac i i y o he p o ein, ega dless o he egula o y in e ac ions.
The ca A5 mu a ion has o he pleio opic e ec s ha can
ha dly be asc ibed o he educed phy oene syn hase ac i i y.
This mu a ion, e en in he e oka yosis, inhibi s subs a e ans e
in he cycliza ion o lycopene o
␤
-ca o ene and ende s hese
eac ions mo e suscep ible o chemical inhibi ion (37). This
Fig. 4. Hyd ophobici y index o he p edic ed Ca RA p o ein sequence. The
e ical do ed line ma ks he loca ion o he pu a i e p o ease clea age si e
AHAIV. The si es o he mu a ions in six colo mu an s a e indica ed below.
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de ec may be ela i ely i ial, because i is co ec ed by he
me e addi ion o de e gen o he medium. Addi ionally, he
mu an lacks pho oinduc ion o ca o ene syn hesis (12) and has
a se e e pho omo phogene ic de ec (38). These wo de ec s
canno be a ibu ed o he ca o ene sho age, because hey
occu in ca A ca S double mu an s ha con ain mo e
␤
-ca o ene
han he wild ype.
Pheno ypes such as ha o s ain C2 canno esul om se e e
mu a ions in domain A ha would cause an i epa able loss o
phy oene syn hase ac i i y. Such mu an s would ha e he null
AR pheno ype. In ac , all mu an s induced by a ameshi -
inducing agen p esen ed he null pheno ype (5).
S119 s ain has he same pheno ype as S144, whose mu a ion
ca I131 a ec s an unlinked gene (6). We p opose ha mu a ion
ca A113 ma ks he binding si e o he ca I gene p oduc , which
media es he s imula o y e ec s o ligh , sexual ac i i y, e inol,
and dime hyl ph hala e. The ca S gene p oduc media es only he
e ec o e inol and i s analogues, p obably by in e ac ing wi h
he ca I gene p oduc , al hough bo h could in e ac wi h phy-
oene syn hase.
Fig. 5. Alignmen o he p o ein sequences Pb, Mc, Nc, and Xd p edic ed om he sequences o genes ca RA om Phycomyces blakesleeanus,al-2 ( e . 31;
GenBank accession no. L27652) om Neu ospo a c assa,c YB ( e . 32; accession no. AJ133646) om Xan hophyllomyces dend o hous (syn. Rhodomyces
dend o hous and Pha ia hodozyma), and ca RP ( e . 33; accession no. AJ250827) om Muco ci cinelloides. The line ma ks he pu a i e clea age si e in he
Phycomyces sequence a he app oxima e limi o he lycopene cyclase and phy oene syn hase domains. The black ci cles loca e he mu a ions in six Phycomyces
colo mu an s. The a owheads indica e he p esence o in ons in he gene sequence.
Fig. 6. Alignmen o wo pa s o he lycopene cyclase domain o he Ca RA p o ein om Phycomyces and p o eins C Yc and C Yd om B e ibac e ium linens
( e . 34; GenBank accession no. AF139916). Ca RA(1) and Ca RA(2) ep esen he 133-aa sequence ha p ecedes he in on loca ion and he nex 128 aa,
espec i ely. The black ci cles loca e missense mu a ions in ed mu an s o Phycomyces.
A ach e al. PNAS
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ol. 98
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no. 4
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GENETICS

The esul s om classical gene ics (5) sugges ed ha ‘‘ he
polypep ide is spli in o sepa a e R and A p o eins,’’ unless ‘‘ he
A unc ion is media ed by a di usible p oduc .’’ I is hus
in e es ing o no e he p esence o a pu a i e p o ease clea age
si e a abou he limi be ween domains R and A in Phycomyces
and o he ungi.
The h ee enzyme ac i i ies ha ca alyze he se en chemical
eac ions needed o he syn hesis o
␤
-ca o ene in Phycomyces
depend on he exp ession o wo adjacen genes, ca RA and ca B,
which a e ansc ibed di e gen ly. The ad an age o his se -up
would be he join egula ion o he whole pa hway om a single
p omo e egion. This a angemen is no conse ed: he ho-
mologous genes o Fusa ium ujiku oi a e adjacen , bu an-
sc ibed in he same di ec ion (M. M. P ado and J.A., unpublished
esul s); hose o Neu ospo a a e linked, bu sepa a ed by o he
genes (39).
Gene ca RA is ema kably simila (Fig. 5) o homologous
genes om ep esen a i es o h ee majo classes o Fungi,
Zygomyce es (Muco ), Ascomyce es (Neu ospo a), and Basidio-
myce es (Xan hophyllomyces). Gene al-2 om Neu ospo a, de-
ined by albino mu a ions and esponsible o phy oene syn hase
(40), was he i s o be cloned and sequenced (31). A cyclase
ac i i y has been shown o be p esen in he amino- e minal
domain o he al-2 gene p oduc by he ecen isola ion o eddish
mu an s o Neu ospo a ha ca y mu a ions in ha domain (J.A.
and T. Schmidhause , unpublished obse a ions). Ve does e al.
(32) we e he i s o show, by unc ional complemen a ion in E.
coli, ha lycopene cyclase and phy oene syn hase a e coded by
a single gene om Xan hophyllomyces, which hey called c YB.
Sequence compa isons sugges ha he lycopene cyclase do-
main o he ungal genes was o med by a duplica ion o a small
gene in a common ances o o he ungi and B e ibac e ium. The
lycopene cyclase ac i i y o his bac e ium (34) is he esul o he
join ac ion o he p oduc s o genes c Yc and c Yd, which a e
con iguous, ela ed by hei sequence, and p obably o med by
a di e en duplica ion. The phy oene syn hase domain o he
ungal genes seems o de i e om he common ances o o all
p esen phy oene syn hases and squalene syn hases.
The ac i a ion o he ca o ene pa hway by ligh and chemicals
is no uni e sal. Ligh is an ac i a o in Phycomyces and Neu o-
spo a, bu no in Xan hophyllomyces (41) o Blakeslea (42). As a
as hey ha e been es ed, he bes chemical ac i a o s o
Phycomyces ha e li le o no e ec on Ascomyce es, Basidiom-
yce es, bac e ia, and plan s. In iew o he conse a ion o he A
domain, including he amino acids ha a e changed in Phyco-
myces mu an s, i would no be su p ising i o he o ganisms used
he same p o ein domain o hei own egula o y pu poses.
We hank P o . A. P. Esla a, Uni e sidad de Salamanca, Salamanca,
Spain, o he kind gi o he Phycomyces DNA agmen ha con ains
he ca B gene, P o . L. M. Co ochano, Uni e sidad de Se illa, o he
Phycomyces cDNA, and D. Pe´ ez del Camino o echnical assis ance.
This wo k was inanced by he Eu opean Union (Con ac FAIR
PL96-1633), he Spanish Go e nmen (G an DGES PB96-1336), and
Jun a de Andalucı´a (G oup 3038).
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