Cloning and Functional Analysis of a cDNA Encoding Ginkgo biloba Farnesyl Diphosphate Synthase

Abstract

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.

Full text

Mol. Cells, Vol. 18, No. 2, pp. 150-156
Cloning and Functional Analysis of a cDNA Encoding
Ginkgo biloba Farnesyl Diphosphate Synthase
Peng Wang
1
, Zhihua Liao
1
, Liang Guo
1
, Wenchao Li
1
, Min Chen
2
, Yan Pi
1
, Yifu Gong
3
, Xiaofen Sun
1
, and
Kexuan Tang
1,3,
*
1
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,
Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai 200433, China;
2
School of Pharmacy, Fudan University, Shanghai 200032, China;
3
Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology Research and Development Center, School of
Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, China.
(Received March 30, 2004; Accepted June 1, 2004)
Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.
5.1.10)
catalyzes the synthesis of farnesyl diphosphate,
and provides precursor for biosynthesis of sesquiter-
pene and isoprenoids containing more than 15 iso-
prene units in Ginkgo biloba. Here we report the clon-
ing, characterization and functional analysis of a new
cDNA encoding FPS from G. biloba. The full-length
cDNA (designated GbFPS) had 1731 bp with an open
reading frame of 1170 bp encoding a polypeptide of
390 amino acids. The deduced GbFPS was similar to
other known FPSs and contained all the conserved
regions of trans-prenyl chain-elongating enzymes. Struc-
tural modeling showed that GbFPS had the typical
structure of FPS, the most prominent feature of which
is the arrangement of 13 core helices around a large
central cavity. Southern blot analysis revealed a small
FPS gene family in G. biloba. Expression analysis in-
dicated that GbFPS expression was high in roots and
leaves, and low in stems. Functional complementation
of GbFPS in an FPS-deficient strain confirmed that
GbFPS mediates farnesyl diphosphate biosynthesis.
Keywords: Bilobalide; Farnesyl Diphosphate Synthase;
Ginkgo biloba; RACE; Yeast Complementation.
Introduction
Isoprenoids are the most widespread and structurally di-
verse family of natural products. They are present in all
* To whom correspondence should be addressed.
Tel: 86-21-65642772; Fax: 86-21-65643552
E-mail: kxtang1@yahoo.com
plants. Many play important roles as growth hormones,
photosynthetic pigments and membrane components, and
are also involved in defense and communication. Some
are commercially important compounds such as flavors,
fragrances, medicines, and natural rubbers.
Isoprenoids are derived from a common precursor,
isopentenyl diphosphate (IPP). The major steps involved
in isoprenoid biosynthesis are sequential condensations of
IPP with a growing allylic polyisoprenoid diphosphate.
These chain elongation reactions are catalyzed by a fam-
ily of enzymes designated prenyltransferases. Farnesyl
diphosphate synthase (FPS) is a representative of this
family (Kellogg and Poulter, 1997).
FPS catalyzes the sequential head-to-tail coupling of
DMAPP with two molecules of IPP, producing farnesyl
diphosphate (FPP), which lies at the branch point of the
pathway of isoprenoid synthesis. FPP has three functions.
First, it is the precursor of a structurally diverse class of
sesquiterpenes that are widely distributed in plant king-
dom including a diversity of functionally important com-
pounds, such as phytoalexins, antibiotic compounds that
respond to microbial challenge, and anti-feedants that dis-
courage opportunistic herbivory. In addition, sesquiter-
penes are found in essential oils and act as anticancer and
antimalarial drugs. Second, FPP is the precursor of
squalene synthase which condenses two molecules of FPP
to produce squalene, the committed precursor for triter
Abbreviations: CTAB, cetyltrimethylammonium bromide; DMAPP,
dimethylallyl diphosphate; DXP, 1-deoxyxylulose-5-phosphate;
FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase;
GbFPS, farnesyl diphosphate synthase from Ginkgo biloba; GPP,
geranyl diphosphate; IPP, isopentenyl diphosphate; ORF, open
reading frame; RACE, rapid amplification of cDNA ends.
Molecules
and
Cells
KSMCB 2004

Peng Wang et al. 151
pene synthesis. This large class of molecules includes the
brassinosteroids, certain phytoalexins, and various toxins
and components of surface waxes. Finally, FPP provides
precursors for further chain elongation in the biosynthesis
of diterpenes, tetraterpenes and polyterpenes (Croteau et
al., 2000). Because of its crucial status, FPS has been ex-
tensively and thoroughly studied. It has been purified
from a number of organisms and its three-dimensional
structure has been characterized by Tarshis et al. (1994).
cDNAs encoding FPS have been obtained from various
angiosperms, such as Arabidopsis thaliana (Cunillera et
al., 1997), Artemisia annua (Hemmerlin et al., 2003), and
Zea mays (Li and Larkins, 1996). However, as far as we
know, there has been no report of the cloning of FPS from
gymnosperms.
The gymnosperm, Ginkgo biloba, one of the oldest liv-
ing tree species in the world, is the sole extant representa-
tive of the order Ginkgoles. It thrived 125 million years
ago, and the genus has remained virtually unchanged
from that time (Biswas and Johri, 1997). G. biloba is a
great survivor in polluted environments (Kim et al., 1997),
and it is exceptionally resistant to fungal (Aoki, 1997) and
insect attack (Honda, 1997). It is now considered a me-
dicinal plant in the United States and Europe, as has been
recognized as such since ancient times in East Asia
(Hasebe, 1997). The tree produces a variety of secondary
metabolites, among which the terpene trilactones, gink-
golides and bilobalide, are the most characteristic. Gink-
golides are diterpenes that have been detected both in
roots and leaves, and the sesquiterpene bilobalide is the
major terpene trilactone in leaves and a minor component
in roots. These compounds all utilize FPP as precursor
(Carrier et al., 1998; Park et al., 2004). Ginkgolides are a
structurally unique family of diterpenoids that are highly
specific platelet-activating factor receptor antagonists
(Guinot and Braquet, 1994), and bilobalide has neuropro-
tective effects and is effective in neurodegenerative dis-
eases (Thompson, 1995). It is important to elucidate the
pathway of biosynthesis of these terpene trilactones in G.
biloba at both the genetic and enzymatic level because of
their pharmaceutical status and potential biological func-
tion. There have been a few reports of the cloning of
genes involved in the biosynthesis of isoprenes such as
ginkgolide in G. biloba, (Liao et al., 2004; Schepmann et
al., 2001) but they are far from exhaustive. Here, we re-
port the cloning and characterization of a full-length
cDNA encoding G. biloba FPS. We also studied its role
using a complementation test in a mutant yeast strain.
Materials and Methods
Plant material, yeast strains and culture methods Ginkgo
biloba was grown on the campus of Fudan University, Shanghai,
China, and fresh roots, stems and leaves were collected, frozen
immediately in liquid nitrogen and stored at −80
o
C prior to total
RNA extraction.
Saccharomyces cerevisiae strain CC25 (MATa/MATalpha,
deltaERG20/+) was obtained from the American Type Culture
Collection (ATCC number 4021258; for further details see Cu-
nillera et al., 2000; Winzeler et al., 1999 and the ATCC website:
www.atcc.org). The strain was maintained in YPD medium with
1% (w/v) yeast extract, 2% (w/v) bactopeptone and 2% (w/v)
glucose. Unless otherwise stated, the cells were grown at 28°C
in liquid culture or on 2% agar. When required, ergosterol (80
mg/ml in agar plates) was added to the growth medium.
Cloning of full-length GbFPS cDNA by rapid amplification of
cDNA ends (RACE) Total RNA was isolated from young leaves
of G. biloba with TRIzol reagent according to the manufac-
turer’s instructions (Invitrogen, USA). Single-stranded cDNAs
were reverse transcribed from 5 µg of total RNA with an oligo(dT)
primer according to the manufacturer’s protocol (PowerScript,
CLONTECH, USA). After RNaseH treatment, the single-stranded
cDNA mixtures were used as templates for polymerase chain
reaction (PCR). Two degenerate oligonucleotide primers, DFFPS
(5′-TGGTG(C/T)AT(A/T/C)GAATGGCT(T/C)CA(A/G)GC-3′)
and DRFPS (5′-TC(A/G)TCCTG(A/T/C/G)ACTTG(A/G)AA(A/G)
TA(T/G)(A/G)T(A/T/C)CCCAT-3′), encoding the highly con-
served amino acid sequences (GWCVEWLQ and MG(I/T)
YFQVQDD) of plant FPSs were synthesized and used in gradient
PCR-amplification of the core cDNA fragment. The gradient PCR
was carried out by denaturing the cDNA at 94°C for 3 min fol-
lowed by 29 cycles of amplification (94°C for 45 s, 51−59°C for
45 s, 72°C for 1 min) and extension at 72°C for 6 min. The core
fragment was amplified at the annealing temperature of 55.5°C,
and subcloned into pGEM T-easy vector (Promega, USA). Se-
quencing and a blast-n search confirmed that it was highly ho-
mologous to other plant FPS genes. The core fragment was sub-
sequently used to design and synthesize gene-specific primers to
clone the full-length cDNA by RACE.
A SMART RACE cDNA Amplification Kit (Clontech,
USA) was used to isolate the 3′- and 5′-ends of GbFPS cDNA.
First-strand 3′-RACE-ready and 5′-RACE-ready cDNA samples
from G. biloba were prepared according to the manufacturer’s
protocol (SMART RACE cDNA Amplification Kit, User
Manual, Clontech, USA) and used as templates for 3′-RACE
and 5′-RACE, respectively. The 3′-end of GbFPS cDNA was
amplified using 3′-gene-specific primers and the universal prim-
ers provided by Clontech. For the first round PCR amplification
of 3′-RACE, GBFPS3-1 (5′-GGACAATTTTGTAGCTGTCAA-
GAAC-3′) and UPM (Universal Primer Mix, provided by Clon-
tech) were used as the PCR primers (3′-RACE), and 3′-RACE-
ready cDNA was used as template. For nested PCR amplifica-
tion of 3′-RACE, GBFPS3-2 (5′-TGCAGATGGGAACCTA-
TTTTCAAG-3′) and NUP (Nested Universal Primer, provided
by Clontech) were used as PCR primers (3′-RACE), and the
first round PCR products were used as templates. The 5′-end of
GbFPS cDNA was amplified using 5′-gene-specific primers and
the universal primers provided. For the first round PCR amplifi-

152 FPS from Ginkgo biloba
cation of 5′-RACE, GBFPS5-1 (5′-TGTGAGACCCATCCATA-
ATGTCATC-3′) and UPM were used as primers (5′-RACE),
and 5′-RACE-ready cDNA, as template. For the nested PCR
amplification of 5′-RACE, GBFPS5-2 (5′-AATACAAGGAAA-
TAGGCTTGAAGCC-3′) and NUP were used as primers (5′-
RACE) and the first round PCR products, as templates. For the
first and nested PCR amplification of 3′ and 5′-ends of GbFPS
cDNA, we used an Advantage 2 PCR Kit (CLONTECH, USA)
and carried out PCR with 25 cycles of amplification (30 s at
94°C, 30 s at 68°C, 3 min at 72°C). The 3′ and 5′-RACE prod-
ucts were subcloned into pGEM T-easy vector followed by se-
quencing. By assembling the sequences of the 3′ and 5′ RACE
products and the core fragment on Contig Express (Vector NTI
Suite 6.0), we deduced the full-length cDNA sequence of
GbFPS and subsequently amplified it by PCR using primers
FGBFPS (5′-CCAATCTCTTACTAGTTCACAGATATTC-3′)
and RGBFPS (5′- TCCGCTTTCTTCAATCCAAGAAAG-3′) in
the following conditions: 3 min at 94°C followed by 29 cycles
of amplification (50 s at 94°C, 50 s at 60°C, 3 min at 72°C) and
6 min at 72°C. The amplified PCR product was purified and
cloned into pGEM T-easy vector (Promega, USA) and se-
quenced. In total, we picked three independent positive clones
and sequenced them to confirm the sequence and avoid PCR
errors. The ORF of GbFPS was predicted by ORF Finder at
NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).
Sequence analysis We analuzed the sequence of GbFPS online at
the websites http://www.ncbi.nlm.nih.gov and http://cn.expasy.org.
SubLoc 1.1 (Hua and Sun, 2001) was used to predict subcellular
localization and CLUSTRAL X (Thompson et al., 1997) for mul-
tiple alignment of GbFPS and other FPSs. Homology-based struc-
tural modeling was performed by Swiss-Model (Peitsch and Jon-
geneel, 1993; Schwede et al., 2003), and Web Lab View Lite 4.0
was used to display 3-D structures. We constructed the phyloge-
netic tree of FPSs using MEGA2 by the neighbor-joining method
with 1,000 repeats (Kumar et al., 2001).
Southern blot analyses Aliquots of DNA (20 µg/sample) were
digested overnight at 37°C with HindIII and NcoI respectively,
which does not cut within the probe region, fractionated by
0.85% agarose gel electrophoresis and transferred to a Hybond-
N
+
nylon membrane (Amersham Pharmacia, UK). The 461-bp
probe was generated by PCR using the 1173 bp coding sequence
of GbFPPS as template with primers FFPSPIN (5′-TGGTGC-
ATTGAATGGCTTCAAGCCT-3′) and RFPSPIN (5′-TCATCC-
TGTACTTGAAAATAGGTTCCCA-3′). Probe labeling (biotin),
hybridization and signal detection were performed using Gene
Images random prime labeling module and CDP-Star detection
module following the manufacturer’s instructions (Amersham
Pharmacia, UK). The film was washed under stringent condi-
tions (60°C) and signals were visualized by exposure to Fuji X-
ray film at room temperature for 1.5 h.
Semi-quantitative RT-PCR We extracted total RNA from
roots, stems and leaves by a modified CTAB method. The qual-
ity and concentration of the RNA samples were examined by
EB-stained agarose gel electrophoresis and spectrophotometric
analysis. After establishing agreement between the OD values of
the RNAs from the three tissues, we performed RT-PCR using a
One Step RT-PCR Kit (TaKaRa, Japan) with the primer pairs
RTF1 (5′-AAGCTTATGCAATTCCCTTCTCTCAGAAAA-3′)
and RTR1 (5′-AATACAAGGAAATAGGCTTGAAGCC-3′). The
procedure was: 50°C for 30 min, 94°C for 2 min followed by 20
cycles of amplification (94°C for 30 s, 60°C for 30 s, 72°C for 1
min). A fragment of 18S rRNA (108 bp) was also amplified in
the RT-PCR with primers 18SF1 (5′-ATGATAACTCGACG-
GATCGC-3′) and 18SR1 (5′-CTTGGATGTGGTAGCCGTTT-
3′), as control. The densities of the target bands were measured
with a Furi FR-200A ultraviolet analyzer (Furi Tech., China).
Functional complementation of GbFPS in yeast Saccharomy-
ces cerevisiae strain CC25 was used to test the activity of
GbFPS. A fragment containing the coding region of GbFPS was
amplified by PCR using the pair of primers (5′-AAGCTTAT-
GCAATTCCCTTCTCTCAGAAAA-3′ and 5′-GGATCCCTA-
CTTCTGTCGCTTGTATATCTTC-3′). After digesting the frag-
ment with HindIII and BamHI, the coding region was cloned
into the expression vector pYES2 (Invitrogen, USA), to yield
pYES2 + GbFPS, which was used to transform E. coli DH5α
cells. Transformants were selected on solid LB medium contain-
ing carbenicillin (125 mg/L), and the extracted plasmids were
used to transform S. cerevisiae CC25. Positive colonies were
selected by plating on YPD agar at 42°C for 16 h, followed by
37°C for 2 d.
Results and Discussion
Molecular cloning of full-length GbFPS cDNA Using
total RNA isolated from young leaves of G. biloba and
the degenerate sense primer, dffps, and anti-sense primer,
drfps, we amplified a 461-bp product by RT-PCR, ligated
it into pGEM T-easy vector and sequenced it. A BLAST-
n search of the sequence showed that the fragment was
highly homologous to FPS genes from other plant species
(data not shown). We synthesized gene-specific primers
from the sequence and used them to generate 5′-end and
3′-end DNA fragments which were subsequently ampli-
fied by RACE (see Materials and Methods). By aligning
the sequences obtained, we deduced the full-length cDNA
sequence of GbFPS (GenBank accession number AY
389818) and amplified it. The full-length cDNA of
GbFPS had 1731 bp with an ORF of 1170 bp, encoding a
polypeptide of 390 amino acids, flanked by a 172-bp 5′-
untranslated region containing a TATA box, and a 389-bp
3′-untranslated region including a poly(A) tail of 28 bp.
The predicted GbFPS protein had a calculated molecular
mass of 44.68 kDa and a theoretical pI of 5.75.
Comparison of FPS from G. biloba and those of other

Peng Wang et al. 153
Fig. 1. Alignment of the deduced amino acid sequences of GbFPS
and FPSs of other model organisms. Identical and conserved
amino acid residues are denoted by black and gray backgrounds,
respectively. The five conserved domains of prenyltransferases
are boxed and numbered. The highly conserved aspartate-rich
motifs (DDXX(XX)D) is present in domains II and V. GenBank
accession numbers: A. thaliana, AAL17614; O. sativa, T03687; H.
sapiens, NP_001995; D. melanogaster, CAA08919; S. cerevisiae,
P08524; E. coli, BAA00599.
species, and construction of a phylogenetic tree A
BLAST search of the protein database at NCBI showed
that the polypeptide sequence of G. biloba (GbFPS) had
47−76% identity and 64−88% similarity with FPS’s from
other species. The polypeptide sequence of G. biloba FPS
(GbFPS) was aligned with FPS sequences from Arabidop-
sis thaliana, Oryza sativa, Homo sapiens and Saccharo-
myces cerevisiae. The result showed that GbFPS had the
five conserved regions identified by Chen et al. (1994)
that are characteristic of prenyltransferases that synthesize
isoprenoid diphosphates with E-double bonds (Fig. 1).
The highly conserved aspartate-rich motif DDXX(XX)D
was present in domains II and V. All other amino acids
known to be important for FPS activity or substrate bind-
ing were present.
Phylogenetic tree analysis showed that GbFPS ap-
peared at the base of the clade of the plant kingdom, and
that FPSs evolved vertically from a common ancestor (Fig.
Fig. 2. Phylogenetic tree of the amino acid sequences of FPSs of
different organisms constructed by the neighbor-joining method
on MEGA 2. Bacteria-derived FPSs are indicated by ; animal-
derived FPSs by ▲; fungi-derived FPSs by ; plant-derived
FPSs by . GenBank accession numbers: Lupinus albus, P49351;
Gossypium arboreum, CAA72793; Hevea brasiliensis, AY135188;
Mentha piperita, AAK63847; Arabidopsis thaliana, AAL17614;
Capsicum annuum, CAA59170; Lycopersicon esculentum, T06272;
Malus domestica, AAM08927; Helianthus annuus, AAC78557;
Artemisia annua, P49350; Parthenium argentatum, CAA57892;
Humulus lupulus, BAB40665; Eucommia ulmoides, AB052681;
Zea mays, T03291; Oryza sativa, T03687; G. biloba, AY389818;
Mucor circinelloides, CAD42869; Saccharomyces cerevisiae,
p08524; Kluyveromyces lactis, CAA 53614; Schizosaccharomy-
ces pombe, NP593299; Gibberella fujikuroi, CAA65641; Neuro-
spora crassa, CAD21355; Drosophila melanogaster, CAA08919;
Gallus gallus, P08836; Mus musculus, AAL09445; Homo sapiens,
NP001995; Bos Taurus, AAL58886; Caenorhabditis elegans,
CAB03221; Micrococcus luteus, BAA 25265; Yersinia pestis,
NP406651; Salmonella enterica, NP806170; Escherichia coli,
BAA00599; Shigella flexneri, NP706309.
2). The rbcL and rRNA data have given similar results
(Nickrent and Soltis, 1995). As the results were based on
relatively little data and the bootstrap values were high,
there was no prior criterion to decide which datum was
the best, and additional data are needed to confirm our
results. Due to the fact that FPSs are present in a wide
variety of species and have the same evolutionary pattern
as other proteins, they may be ideal molecular markers for
evolutionary studies.
Homology modeling of GbFPS By combining the se-
quence information about GbFPS with knowledge of the
three-dimensional structure of avian FPS, we obtained a
homology model of GbFPS. This exhibited a fold com-
posed of α-helices joined by connecting loops and a spot
of sheets (Fig. 3). The fold consisted
of a large central

154 FPS from Ginkgo biloba
Fig. 3. The 3-D structure of GbFPS established by homology-
based modeling. The helix, sheet and random coil are indicated
by the column-shape, arrow plate-shape and rope-shape, respec-
tively. The two aspartate-rich substrate-binding motifs, DDIMD
and DDYLD, are indicated by balls.
cavity formed by a bundle of 13 α-helices, and was the
putative catalytic site. This finding is consistent with re-
sults obtained with other FPSs (Tarshis et al. 1994). The
two aspartate-rich DDXX(XX)D sequences that are
highly conserved
in isoprenyl diphosphate synthases were
located on opposite walls of this cavity, facing each other.
All five of the identified conserved regions are clustered
around this cavity.
Southern blot analysis To investigate the genomic or-
ganization of GbFPS, we carried out a Southern blot
analysis by digesting genomic DNA of G. biloba with
HindIII and NcoI, followed by hybridization with a 461
bp probe generated by PCR using the 1173 bp coding
sequence of GbFPS cDNA as template. As shown in Fig.
4A, more than two hybridized bands were detected in
each lane, indicating that GbFPS belongs to a small gene
family in G. biloba. This is consistent with previous re-
ports in other plant species (Cunillera et al., 1996; Hem-
merlin et al., 2003; Li and Larkins, 1996).
Prediction of the subcellular localization of GbFPS IPP
is synthesized via two pathways: the mevalonate and the
1-deoxyxylulose-5-phosphate (DXP) pathways (Kuzuyama,
2002). The mevalonate pathway is found primarily in eu-
karyotes and archaea, whereas the non-mevalonate path-
way is found primarily in prokaryotes and in the plastids
of plants. Prenyltransferases, which utilize IPP, are also
thought to be distributed in three subcellular compart-
ments: the cytosol, mitochondria and plastids (Gray, 1987;
A B
Fig. 4. Southern blot and expression analyses of GbFPS. A.
Southern blot analysis. Genomic DNA of G. biloba was di-
gested with HindIII and NcoI, separated on a 0.85% agarose gel,
blotted onto a positively charged nylon membrane and probed
with a biotin-labeled GbFPS fragment. B. Expression analysis
of GbFPS in various tissues by semi-quantitative RT-PCR. To-
tal RNA was extracted from roots, stems and leaves, and sub-
jected to semi-quantitative RT-PCR analysis. The 18S rRNA
was used for normalization of RNA loading.
Fig. 5. Functional complementation of the leaky mutant yeast
strain CC25 with plasmid pYES2 + GbFPS. Strain CC25 and
strain CC25 transformed with pYES2 + GbFPS were streaked
onto YPD plates with/without 80 mg/L ergosterol and incubated
at 42°C for 16 h followed by 37°C for 2 d.
Kleinig, 1989). However, the fact that there is crosstalk
between the various subcellular compartments involved in
isoprenoid biosynthesis (Laule et al., 2003) makes it dif-
ficult to establish the subcellular localization of prenyl-
transferases. For example, FPS in Arabidopsis is targeted
to both the cytosol and the mitochondria
(Cunillera et al.,
1997), and there are reports that FPSs are localized in the
plastid compartment (Hemmerlin et al., 2003; Sanmiya et
al., 1999). These observations may be due to the fact that
FPSs belong to a small gene family and that particular
forms are present in particular subcellular compartments

Peng Wang et al. 155
where they perform specific functions. In the present case,
prediction of the subcellular localization of GbFPS indi-
cated that it was most likely to be cytoplasmic. Taken
together with the results of the Southern blot analysis, we
may deduce that a number of genes encode FPSs in G.
biloba and that the different products occupy different
subcellular compartments, the product of the full-length
cDNA we cloned being cytoplasmic.
Semi-quantitative RT-PCR Semi-quantitative RT-PCR
was carried out to investigate the expression pattern of
GbFPS in different tissues. The result showed that there is
high GbFPS expression in roots and leaves, and low
expression in stems (Fig. 4B), reflecting the fact that the
biosynthesis of ginkgolides and bilobalide occurs in roots
and leaves (Carrier et al., 1998).
Confirmation of GbFPS activity by complementation
of the mutant yeast strain, CC25 We used the sterol-
auxotrophic yeast strain CC25 bearing a disrupted copy of
the FPS gene to confirm that the cDNA of GbFPS en-
coded the anticipated functional enzyme. CC25 (MATa/
MATalpha, deltaERG20/+) is thermo-sensitive: it bears a
leaky mutation, erg20, affecting the ability of FPS to con-
dense GPP with IPP to yield FPP and, as a result, gener-
ates an amount of geraniol that is toxic (Blanchard and
Karst, 1993), and affects viability above 36°C. This phe-
notype becomes more pronounced as the temperature is
increased and above 42°C the cells are almost unviable.
The addition of ergosterol complements the phenotype
and restores viability at non-permissive temperature. As
shown in Fig. 5, both the mutant strain and the pYES2 +
GbFPS transformants were viable when the plates were
supplemented with ergosterol (80 mg/ml) at non-permissive
temperature (42°C for 16 h and then 37°C for 2 d). In
contrast, in the absence of ergosterol, the cells were al-
most inviable whereas the pYES2 + GbFPPS transfor-
mants grew well. This result confirms that the cloned
cDNA encodes a functional GbFPS.
Acknowledgments We are grateful to Wei Cao for technical
assistance, to Juan Lin for critical reading of the manuscript,
and to Xin Li, Xiaojun Liu, and Zhonghai Chen for valuable
advice on the manuscript. This research was supported by the
China National “863” High-Tech Program, China Ministry of
Education, Shanghai Science and Technology Committee and
Shanghai Agriculture Committee.
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