Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene by functional complementation in Escherichia coli.

1. Carotenoid Science, Vol.11, 2007, 10-15 ISSN 1880-5671
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1Y
and characterization of β-carotene hydroxylase (crtZ) gene by functional
complementation in Escherichia coli
Arunkumar.K.Ramasamy,1,2 Seon-Kang Choi,2# V.Udayasuriyan,1* Norihiko Misawa 2
1. Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology,
Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India -641 003.
2. Laboratory of Applied Molecular Design, Marine Biotechnology Institute,
Heita, Kamaishi-shi, Iwate 026-0001, Japan.
# Present address: Gangneung Marine Bio Foundation, Block Ga-13-1, Gangneung Techno Valley,
Daejeon-dong, Gangneung City, Gangwondo,Republic of Korea
Abstract
A carotenoid biosynthesis gene cluster was cloned from an Indian isolate Pantoea sp. strain C1B1Y, and
sequenced. The carotenoid gene cluster (6290 bp) contained the following six crt genes: crtE, geranylgeranyl
diphosphate synthase (912 bp); crtX, zeaxanthin glucosyltransferase (1296 bp); crtY, lycopene cyclase (1161
bp); crtI, phytoene desaturase (1479 bp); crtB, phytoene synthase (891 bp) and crtZ, β-carotene hydroxylase
(528 bp). The nucleotide sequence of this 6.3 kb crt gene cluster showed 98% identity with that of the crt
gene cluster from Pantoea agglomerans (GenBank accession number AB076662). Whereas, the IPP
isomerase (type 2) gene, which existed between the crtE and crtX genes in the cluster of P. agglomerans
(accession M87280), was not present in the cluster from strain C1B1Y. The N terminus of the phytoene
desaturase from Pantoea sp. strain C1B1Y showed amino acid variations at positions that are known to alter
catalytic activity. Heterologous expression of crtZ from strain C1B1Y was studied by functional
complementation in E. coli.
1. Introduction hydroxylase gene (crtZ) from Pantoea sp. in tobacco
Carotenoids are a class of natural fat-soluble plants has been found to result in rapid synthesis of
pigments found in plants, algae, zeaxanthin on exposure to high intensity white light
non-photosynthetic bacteria, yeasts, and molds. These resulting in enhanced stress tolerance. [9] Transgenic
pigments are capable of quenching photosensitizers, plants with enhanced zeaxanthin content may be useful
interacting with singlet oxygen [1] and scavenging in meeting the dietary requirements of the aging
peroxy radicals. [2] Industrially, carotenoid pigments are population and would be a tool in the fight against AMD
utilized as food colorants and feed supplements. and cataract. Molecular breeding of carotenoid
Recently, carotenoids have attracted greater attention biosynthetic pathways for in vitro evolution of novel
due to their beneficial effect on human health such as pathways and products has been successful. [10] Apart
involvement in cancer prevention [3] and enhancement from combinatorial biosynthesis and directed evolution
of immune response. [4] Deficiency of vitamin-A is studies it is important to source new genes from natural
recognized as a serious public health problem, which isolates because genes with variations even in a few
contributes to a considerable proportion of blindness in amino acid residues resulting from natural selection may
children. The carotenoids lutein and zeaxanthin are exhibit altered function. [11-13] In this study, we report
found in the macula in high concentrations and play an structural analysis of a carotenoid biosynthesis gene
important role in eye health. Considerable data now cluster from an Indian isolate of Pantoea sp. strain
indicate that these components of the macular pigment C1B1Y [14] and functional analysis of β-carotene
may reduce the risk or delay onset of age-related hydroxylase (crtZ) gene in Escherichia coli.
macular degeneration (AMD) and the incidence of
cataract formation. [5-7] 2. Materials and Methods
The phytoene desaturase (crtI) gene from Recombinant DNA Techniques
Pantoea sp. has successfully been used in the functional The restriction enzymes were purchased from
expression in higher plants, e.g., by Ye et al. [8] in the New England BioLabs. DNA ligation kit was purchased
construction of Golden Rice (genetically engineered rice from Nippon Gene. DNA manipulation was conducted
to produce β-carotene). Expression of the β-carotene by the standard methods [15] or as instructed by the
*Corresponding author, e-mail address: suppliers. Plasmid DNA was prepared with the Qiaprep
udayvar@yahoo.com Plasmid Mini Kit (Qiagen). The PCR product was
10

2. Carotenoid Science, Vol.11, 2007, 10-15 ISSN 1880-5671
purified using QIAquick PCR Purification Kit (Qiagen) - Met Thr Met Ile Thr Asn Ser Met-
and the gel extraction was done using Geneclean turbo The nucleotide sequence of the inserted fragment of the
kit (QBiogene). The polymerase chain reaction (PCR) plasmid was confirmed by DNA sequencing.
was carried out by an automated thermal cycler (Takara)
with KOD plus DNA polymerase (Takara). The Culture condition of recombinant E. coli
amplification of the carotenoid gene cluster from the E. coli JM109 [15] carrying plasmid
strain C1B1Y of Pantoea sp. was carried out with KOD pACCAR16∆crtX [16] that contained the four
Dash DNA polymerase. carotenoid biosynthesis genes, crtE, crtB, crtI and crtY,
from Pantoea ananatis, was used as the host for
Amplification of the carotenoid gene cluster and crtZ producing β-carotene and to produce zeaxanthin using
from Pantoea sp. strain C1B1Y crtZ from Pantoea sp. strain C1B1Y (pUCC1B1Z). E.
The primers CEF1 (5’- coli JM109 carrying plasmid pACCAR25∆crtX [16] that
AATCATTCGACCTGCTGTGAC) and C1Zr contained the five carotenoid biosynthesis genes, crtE,
(5’-ATGTTGTGGATTTGGAATGCCCTG) were crtB, crtI, crtY and crtZ, from P. ananatis, was for
designed based on the already available crt gene cluster producing zeaxanthin. A Luria-Bertani medium [15]
sequence of Pantoea agglomerans containing appropriate antibiotics was inoculated with
(DDBJ/EMBL/GenBank accession number: AB076662) 40 µl of a fully grown culture of E. coli transformants,
to clone the crt gene cluster from bacterial isolate and incubated at 300C while shaking. The media were
C1B1Y. The carotenoid gene cluster of Pantoea sp. supplemented when required with the following
strain C1B1Y was amplified from the genomic DNA antibiotics at the indicated concentrations: ampicillin,
under the following conditions: 940C for 4 min, followed 100 µg ml; chloramphenicol, 20 to 30 µg ml. When OD
by 25 cycles of 940C for 15 sec, 650C for 30 sec, 720C at 600 nm of the culture had reached about 0.5, IPTG
for 7 min. The crtZ from Pantoea sp. strain C1B1Y was (isopropyl-1-thio-beta-D-galactoside) was added to a
amplified from the plasmid pT7C1B1Y using the final concentration of 1mM. After cultivating for 48 h,
primers PAZF (5’ the cells were harvested by centrifugation at 40C and
CGAATTCGATGTTGTGGATTTGGA - EcoRI site is stored at –700C.
shown in italics) and PAZR (5’ –
TAGAGGATCCACTTCCCGGGTG- BamHI site is HPLC analysis of carotenoid pigments accumulated in E.
shown in italics). The crtZ from Pantoea sp. strain coli.
C1B1Y was amplified under the following conditions: Frozen cells were vigorously shaken for 30
one cycle of 940C for 5min, followed by 30 cycles of minutes after adding a volume of acetone sufficient to
940C for 15 sec, 480C for 30 sec, 680C for 1 min, with extract the carotenoid pigments. The extract was
one final cycle of 680C for 2 min. centrifuged at 14,000g for 20 min and at 4°C to remove
the cell debris. The carotenoid pigments were analyzed
Cloning and sequencing by high-performance liquid chromatography (HPLC)
Blunt end conversion and cloning of DNA (Waters 2695) with photodiode array (PDA) detection
fragments were performed using the pT7Blue Perfectly (Waters 2996). The HPLC-PDA analysis was carried out
Blunt Kit (Novagen). The nucleotide sequences of the on a TSK ODS-80Ts column (4.6 x 150 nm, Tosoh).
cloned fragments were confirmed using a DNA [17] The crude extract was eluted at a rate of 1 ml/min
sequencing kit (Big dye terminator cycle sequencing with solvent A (water-methanol, 5:95, v/v) for 5 min,
ready reaction kit version 2, PerkinElmer) and a model followed by a linear gradient from solvent A to solvent
3700 DNA sequencer (PerkinElmer) according to the B (tetrahydrofuran-methanol, 3:7, v/v) for 5 minutes,
manufacturer’s instructions. solvent B alone for 8 min, and then back to solvent A.
Authentic samples of carotenoids purified from the E.
Construction of expression plasmids coli transformants expressing the crt genes derived from
The PCR products were digested with EcoRI P. ananatis. [16]
and BamHI, and then inserted into the corresponding
sites of pUC18 to construct pUCC1B1Z (for Pantoea sp. Spectral Data for the Individual Carotenoids
strain C1B1Y crtZ), where the ATG start codon of the 1. β-carotene: HPLC-PDA, retention time: 19.4
cloned gene was placed next to the EcoRI site minutes, λmax, 453, 478 nm. 2. Zeaxanthin: HPLC-PDA,
(underlined) to form the CrtZ protein fused with the retention time: 13.2 minutes, λmax 451, 478 nm
additional 7-amino-acid terminus of beta-galactosidase
(LacZ) as follows (start codon of the crt genes): Sequence analysis and GenBank accession numbers
-ATG ACC ATG ATT ACG AAT TCG ATG-
11

3. Carotenoid Science, Vol.11, 2007, 10-15 ISSN 1880-5671
The amino acid sequences having significant
similarity to crtZ gene from Pantoea sp. strain C1B1Y
were retrieved from the GenBank database with the
BLAST program [18], and aligned by using the
CLUSTAL X program. [19] The nucleotide sequence of
the crt gene cluster of Pantoea sp. strain C1B1Y is
available under the accession number AY876938.
3. Results and discussion
We had earlier reported the isolation and
characterization of a carotenoid-producing Pantoea sp.
strain C1B1Y, an Indian isolate. Nucleotide sequence
analysis of the DNA fragment (612 bp) cloned from the
crtI-crtB region of the new isolate C1B1Y showed
highest homology (98%) with that of Pantoea
agglomerans that had formerly called Erwinia herbicola
(GenBank accession number AB076662). [14] Hence,
primers CEF1 and CZR1 were designed based on the
nucleotide sequence of P. agglomerans (accession Figure. 2: Cloning of the crtZ gene from Pantoea sp.
AB076662). The 6290 bp DNA fragment was amplified strain C1B1Y. Lanes: (a) 1, 1 kb ladder; 2,
from the genomic DNA of Pantoea sp. strain C1B1Y Amplification of crtZ gene by PCR. (b) 1, 1 kb ladder; 2,
using the primers, and inserted into pT7blue, designated Gel eluted crtZ gene fragment.
as pT7-C1B1Y (Figure 1). A plasmid pUCC1B1Z for
expression analysis of crtZ from strain C1B1Y was
constructed from pT7-C1B1Y (Figure 2). phytoene desaturase; crtB, phytoene synthase and crtZ,
The nucleotide sequencing of the 6.3 kb DNA β-carotene 3,3’-hydroxylase (Figure 3). This 6.3 kb crt
fragment from Pantoea sp. strain C1B1Y confirmed the gene fragment showed 98% identity with that of P.
presence of the following crt genes: crtE, geranylgeranyl agglomerans (GenBank accession number AB076662).
diphosphate (GGPP) synthase; crtX, zeaxanthin The 528 bp crtZ gene of strain C1B1Y had 99% identity
glucosyltransferase; crtY, lycopene β-cyclase; crtI, with crtZ from P. agglomerans (accession AB076662).
Whereas, the IPP isomerase (type 2) gene, which existed
between the crtE and crtX genes in the crt gene cluster
of P. agglomerans (accession M87280), was not present
in the cluster from strain C1B1Y. The organization of
the crt gene cluster of strain C1B1Y was same as that of
the crt gene cluster of Pantoea ananatis, which formerly
called Erwinia uredovora (GenBank accession number
D90087). The deduced amino acid sequence of crtI from
Pantoea sp. strain C1B1Y showed 87% identity with
that of crtI from P. ananatis. The P. ananatis crtI gene
was shown to express functionally in higher plants, for
the first time, by adding the pea Rubisco small subunit
transit peptide sequence to the N-terminus sequence. [20,
21] This crtI gene construct has successfully been used
in the development of β-carotene-accumulating tomato
fruit [22] and rice endosperm (Golden Rice) [8].
Figure. 1: Cloning of the carotenoid gene cluster from
Pantoea sp. strain C1B1Y.Lanes: (a) 1, 1 kb ladder; 2,
Amplication of crt gene cluster by PCR with primers
CEF1 and CZR1. (b) 1, 1 kb ladder; 2, Gel eluted 6.3 kb
fragment. (c) 1, EcoRI and NdeI restriction digestion of
the positive clone plasmid, pT7-C1B1Y; 2, 1 kb ladder. Figure. 3: Organization of the crt genes in the gene
cluster of Pantoea sp. strain C1B1Y.
12

4. Carotenoid Science, Vol.11, 2007, 10-15 ISSN 1880-5671
Molecular breeding studies have revealed that expression vector (pUCC1B1Z) was constructed in a
only four amino acid residues variation (P3K, T5V, way such that the cloned gene product was fused to a
V27T and L28V) at the N-terminal region of CrtI from P. lead sequence of β-galactosidase (LacZ) under
ananatis were critical for altered catalytic activity transcription from the lac promoter by vector pUC18.
leading to the introduction of six double bonds rather The lead sequence in these plasmids, with the seven
than four into phytoene. [10] The deduced amino acid amino acid residues Met-Thr-Met-Ile-Thr-Asn-Ser
sequence from the crtI of Pantoea sp. C1B1Y reveals derived from LacZ, were to be added to the Met of the
amino acid variations at three positions (P3R, V27T, original start of the cloned gene. Such hybrid genes have
L28R) on comparison with the N terminus (residues 1- been efficiently translated using the original ribosomal
40) of CrtI from P. ananatis (Figure 4). The CrtI from binding site and start codon of the lacZ gene. [23, 24]
Pantoea sp. C1B1Y had 87% identity with that of P. The expression vector pUCC1B1Z was introduced into
ananatis and may serve as another gene source for DNA E. coli synthesizing β-carotene due to the presence of
shuffling experiments to generate new carotenoid pACCAR16∆crtX. [16] This expression analysis of crtZ
products by directed evolution. Alignment of CrtZ from Pantoea sp. strain C1B1Y by functional
proteins of Pantoea sp. showing significant homology complementation in E. coli revealed that that the crtZ
with CrtZ of C1B1Y is given in Figure 5. The deduced gene was functional. HPLC analysis of the carotenoid
amino acid sequence from the crtZ of Pantoea sp. strain extract from the recombinant E. coli (48 h culture after
C1B1Y showed 85% identity with the deduced amino IPTG induction) carrying the plasmids pUCC1B1Z and
acid sequence from crtZ of P. ananatis (GenBank pACCAR16∆crtX revealed the synthesis of zeaxanthin
accession number D90087). To analyze the functions of from β-carotene (Figure 6).
the crtZ gene of Pantoea sp. C1B1Y, the E. coli
10 20 30 40
....|....|....|....|....|....|....|....|
AY876938 1 MNRTTVIGAGFCGLALAIRLQASGVPTRLLEQRDKPGGRA 40
AB076662 1 MNRTTVIGAGFGGLALAIRLQASGVPTRLLEQRDKPGGRA 40
D90087 1 MKPTTVIGAGFGGLALAIRLQAAGIPVLLLEQRDKPGGRA 40
M90698 1 MKPTTVIGAGFGGLALAIRLQAAGIPVLLLEQRDKPGGRA 40
AY166713 1 MKPTTVIGAGFGGLALAIRLQAAGIPVLLLEQRDKPGGRA 40
M87280 1 MKKTVVIGAGFGGLALAIRLQAAGIPTVLLEQRDKPGGRA 40
M38423 1 MKKTVVIGAGFGGLALAIRLQAAGIPTVLLEQRDKPGGRA 40
Clustal Consensus *: *.****** **********:*:*. ************
Figure. 4: Alignment of the N terminus (residues 1-40) of CrtI from Pantoea sp. The amino acids at positions 3, 5, 27,
28 are shown in italics. The GenBank accession number details are as follows: Pantoea agglomerans (AB076662,
AY876938, M90698, M87280 and M38423), Pantoea ananatis (D90087) and Pantoea stewartii (AY166713).
10 20 30 40 50 60
....|....|....|....|....|....|....|....|....|....|....|....|
D90087 1 MLWIWNALIVFVTVIGMEVIAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAA 60
AY166713 1 MLWIWNALIVFVTVVGMEVVAALAHKYIMHGWGWGWHLSHHEPRKGAFEVNDLYAVVFAI 60
AB076662 1 MLWIWNALIVLVTVIGMEITAALAHRYIMHGWGWGWHLSHHEPHKGWFEVNDLYAVVFAA 60
AY876938 1 MLWIWNALIVLVTVIGMEITAALAHRYIMHGWGWGWHLSHHEPHKGWFEVNDLYAAVFAA 60
M87280 1 --MLVNSLIVILSVIAMEGIAAFTHRYIMHGWGWRWHESHHTPRKGVFELNDLFAVVFAG 58
Clustal Consensus 1 : *:***:::*:.** **::*:******** ** *** *:** **:***:*.*** 47
70 80 90 100 110 120
....|....|....|....|....|....|....|....|....|....|....|....|
D90087 61 LSILLIYLGSTGMWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAH 120
AY166713 61 VSIALIYFGSTGIWPLQWIGAGMTAYGLLYFMVHDGLVHQRWPFRYIPRKGYLKRLYMAH 120
AB076662 61 LSILLIYLGSTGVWPLQWIGAGMTLYGLLYFIVHDGLVHQRWPFRYVPRRGYLRRLYMAH 120
AY876938 61 LSILLIYLGSTGVWPLQWIGAGMTLYGLLYFIVHDGLVHQRWPFRYVPRRGYLRRLYMAH 120
M87280 59 VAIALIAVGTAGVWPLQWIGCGMTVYGLLYFLVHDGLVHQRWPFHWIPRRGYLKRLYVAH 118
Clustal Consensus 48 ::* ** .*::*:*******.*** ******:************:::**:***:***:** 102
13

5. Carotenoid Science, Vol.11, 2007, 10-15 ISSN 1880-5671
130 140 150 160 170
....|....|....|....|....|....|....|....|....|....|....|...
D90087 121 RMHHAVRGKEGCVSFGFLYAPPLSKLQATLRERHG--ARAGAARDAQGGEDEPASGK- 175
AY166713 121 RMHHAVRGKEGCVSFGFLYAPPLSKLQATLRERHA--ARSGAARDEQDGVDTSSSGK- 175
AB076662 121 RMHHAVRGKEGCVSFGFLYAPPLSKLQATLRERHG--VKRGAARDQRSVERDAPPGK- 175
AY876938 121 RMHHAVRGKEGCVSFGFLYAPPLSKLQATLRERHG--VKRGAARDQRSVERDAPPGK- 175
M87280 119 RLHHAVRGREGCVSFGFIYARKPADLQAILRERHGRPPKRDAAKDRPDAASPSSSSPE 176
Clustal Consensus 103 *:******:********:** :.*** *****. : .**:* . .... 136
Figure. 5: Alignment of CrtZ proteins of Pantoea sp. showing significant homology with CrtZ of C1B1Y. The
GenBank accession number details are as follows, P. ananatis (D90087); P. stewartii (AY166713); P. agglomerans
(AB076662 and M87280) and Pantoea sp. strain C1B1Y (AY876938).
Figure. 6: HPLC analysis of pigments extracted from the recombinant E. coli. Chromatograms were recorded as a
function of the absorbance (AU, absorbance units) at 470 nm. (a) Carotenoid extract from the recombinant E. coli
carrying plasmid pACCAR16∆crtX, (b) Carotenoid extract from the recombinant E. coli carrying plasmid
pACCAR25∆crtX, (c) Carotenoid extract of the recombinant E. coli carrying plasmids pACCAR16∆crtX and
pUCC1B1Z. Peaks: 1, β-carotene; 2, zeaxanthin.
The crt genes from Pantoea sp. strain C1B1Y carotenoid biosynthetic pathway in the Golden Rice
could be used as a source to develop transgenic crops from β-carotene to zeaxanthin using crtZ from Pantoea
that produce important carotenoids like β-carotene and sp. strain C1B1Y may be feasible. Genetically
zeaxanthin. The phytoene desaturase (crtI) gene from engineered rice with enhanced zeaxanthin content may
Pantoea sp. was used in the genetic engineering of rice be useful in meeting the dietary requirements of the
(Golden Rice; genetically engineered rice to produce β- aging population and would be a tool in the fight against
carotene) [8] and hence further extension of the AMD and cataract. Expression of a bacterial β-carotene
14

6. Carotenoid Science, Vol.11, 2007, 10-15 ISSN 1880-5671
3,3’-hydroxylase gene (crtZ) of P. ananatis (GenBank [14] A.K. Ramasamy and V. Udayasuriyan, Biotechnol.
accession number D90087) has been known to enhance 5 (2006) 79.
UV tolerance in tobacco. [9] When the production of [15] J. Sambrook et al., in Molecular cloning: A
zeaxanthin is prevented by mutation [25] or antisense laboratory manual, 2nd edn. (Cold Spring Harbor
suppression of violaxanthin de-epoxidase, [26] plants Laboratory, Cold Spring Harbor, New York, 1989).
have an increased sensitivity to light stress. It has been [16] N. Misawa et al., J. Bacteriol. 177 (1995) 6575.
suggested that zeaxanthin also has a role in protecting [17] A. Yokoyama and W. Miki, FEMS Microbiol. Lett.
thylakoid membranes specifically from the effects of 128 (1995) 139.
heat stress. [27] Davison et al. [28] concluded that [18] S.F. Altschul et al., Nucleic Acids Res. 25 (1997)
genetic manipulation of a single enzyme in carotenoid 3389.
metabolism can bring about a pronounced increase in the [19] J.D. Thompson et al., Nucleic Acids Res. 24 (1997)
stress tolerance of plants and thus represents a 4876.
potentially powerful way forward in the production of [20] N. Misawa et al., Plant J. 4 (1993) 833.
stress-tolerant crops. The crt genes from Pantoea sp. [21] N. Misawa et al., Plant J. 6 (1994) 481.
strain C1B1Y were cloned with the intention of sourcing [22] S. Romer et al., Nature Biotechnol. 18 (2000) 666.
indigenous genes for the improvement of nutritional [23] S.K. Choi et al., Mar. Biotechnol. 7 (2005) 515.
profile and stress tolerance in higher plants. [24] Y. Nishida et al., Appl.Environ. Microbiol. 71
(2005) 4286.
4. Conclusion [25] M. Havaux and K.K. Niyogi, Proc. Natl. Acad. Sci.
The crt genes cloned from the Pantoea sp. U.S.A. 96 (1999) 8762.
strain C1B1Y can be used as an additional source of [26] A.S. Verhoeven et al., Photosyn. Res. 67 (2001) 27.
carotenoid biosynthetic genes for combinatorial [27] M. Havaux, Trends Plant Sci. 3 (1998) 147.
biosynthesis or directed evolution. The crt genes from [28] P.A. Davison et al., Nature 418 (2002) 203.
Pantoea sp. strain C1B1Y can also be used for the
improvement of nutritional profile and stress tolerance in
plants through metabolic engineering.
Acknowledgements
The Jawaharlal Nehru Memorial Fund
scholarship to Arunkumar. K. Ramasamy for part of the
doctoral research at Marine Biotechnology Institute,
Japan is gratefully acknowledged. We thank Yukie
Inomata for her technical assistance.
References
[1] N.I. Krinsky, Pure Appl. Chem. 66 (1994) 1003.
[2] P.F. Conn et al., Free Radical Res. Commun. 16
(1992) 401.
[3] T.A.D. Smith, Br. J. Biomed. Sci. 55 (1998) 268.
[4] J.D. Thompson et al., Nucleic Acids Res. 24 (1997)
4876.
[5] J.D. Burke et al., J. Nutri. 135 (2005) 1208.
[6] C. Chitchumroonchokchai et al., J.Nutri. 134 (2004)
3225.
[7] R.D. Semba and G. Dagnelie, Med. Hypoth. 61
(2003) 465.
[8] X. Ye et al., Science 287 (2000) 303.
[9] T. Götz et al., Plant Mol. Biol. 50 (2002) 129.
[10] C. Schmidt-Dannert et al., Nature Biotechnol. 18
(2000) 750.
[11] E.V. Sokurenko et al., Proc. Natl. Acad. Sci. U.S.A.
95 (1998) 8922.
[12] M. de Bono et al., cell 94 (1998) 679.
[13] H.E. Hoekstra et al., Science 313(2006) 101.
15