6
02
K. Ichinose et al. / Phytochemistry 51 (1999) 599±603
Shibuya, Lee, Sankawa, & Ebizuka, 1997; You et al.,
998), together with our characterization of a speci®c
F26G (Inoue & Ebizuka, 1996a, 1996b; Inoue et al.,
996a; You et al., 1998), would lead to a better under-
standing of the whole biosynthetic pathway of steroid
saponins.
Thompson, 1980) to aord ca. 50 mg of DNA in a
volume of 25 ml. Part (1 ml) was used as a template for
PCR with the CSF26G-speci®c primers (1 mg each),
CSFG-1S (5'±ATG GCC GCT CAG TTA GGG CTT
C±3') and CSFG1689A (5'±TCA CGT CCT CAG
GAA CTT GCT±G±3') in a ®nal volume of 100 ml for
1
1
2
5 cycles of ampli®cation using a step program (1 min
at 948C, 2 min at 688C and 3 min at 728C).
3. Experimental
3
.5. Enzyme extraction and assay
3.1. Materials and general methods
Cell-free protein extracts were obtained from trans-
Seeds of Nicotiana tabacum cv. Petite Havana SR1
genic tobacco or C. speciosus plantlets as previously
described (Inoue et al., 1996b). Protein was quanti-
®ed by the method of Bradford (1976) using BSA as
a standard. F26G assay was performed according to
the method described (Inoue & Ebizuka, 1996a,
1996b). b-Glucosidase activity using p-nitrophenyl b-
D-glucoside (pNPG) as a substrate was determined
by the absorbance at 400 nm of released p-nitro-
phenol under the same assay conditions used for
F26G.
and Xanthi were obtained from JT (Japan Tobacco)
Co. Ltd, Japan. Germination of tobacco seeds and
maintenance of transgenic tobacco plantlets were per-
formed on Murashige±Skoog (MS) (Murashige &
�
1
Skoog, 1962) basal medium containing 30 g l sucrose
and 0.8% agar in a plastic culture box at 258C under
16 h light per day. In vitro cultured Costus speciosus
plantlets used have been as described previously (Inoue
et al., 1996b). Agrobacterium tumefaciens LBA4404
(
Ooms et al., 1982) and plasmid, pBI121 (Jeerson et
al., 1987) were obtained from Clontech (Palo Alto,
CA). DNA manipulations were according to standard
procedures (Sambrook, Fritsch, & Maniatis, 1989).
Electroporation was performed on a Electro Cell
Manipulator 600 (BTX Inc, San Diego, CA) following
the manufacturer's protocol.
Acknowledgements
We thank Professor T. Nohara, Faculty of
Pharmaceutical Sciences, Kumamoto University for a
generous gift of pro todioscin, and Professor K. Saito,
Faculty of Pharmaceutical Sciences, Chiba University
for helpful technical advice. Grateful acknowledgment
is made to JT Co. Ltd, Japan for providing plant ma-
terials (tobacco seeds). Financial support was received
from the Fujisawa Foundation, Osaka, Japan to K.I.
S.Y. was a recipient of Short-term Student Exchange
Promotion Program Scholarship of Association of
International Education, Japan for his study at the
University of Tokyo.
3.2. Construction of CSF26G expression vectors
CSF26G was introduced in place of the b-glucuroni-
dase (GUS) gene in the binary vector, pBI 121, as fol-
lows: (1) digestion with SmaI and SacI followed by
self-ligation, (2) treatment with XbaI and Klenow frag-
ment to aord a dephosphorylated vector (3) blunt-
end ligation with CSF26G cDNA fragment (1689 bp)
derived from pT7-CSF26G (Inoue et al., 1996a) by
digestion with XbaI and KpnI followed by Klenow
treatment.
References
3.3. Transformation of tobacco
Bradford, M. M. (1976). Anal. Biochem., 72, 248.
Corey, E. J., Matsuda, S. P. T., & Bartel, B. (1993). Proc. Natl.
Acad. Sci. USA, 90, 11628.
Mobilization of the vectors (pYSG1S or pYSG1A)
into A. tumefaciens LBA4404 was by electroporation.
Tobacco leaf discs were transformed using the
Agrobacterium binary vector method described by
Rogers et al. (1986). Transgenic plants were selected
Gru
È
nweller, S., Schro
È
Phytochemistry, 29, 2485.
der, E.,
&
Kesselmeier, J. (1990).
Inoue, K., & Ebizuka, Y. (1996a). Adv. Exp. Med. Biol., 404, 57.
Inoue, K., & Ebizuka, Y. (1996b). FEBS Lett., 378, 157.
�
1
on media containing kanamycin 100 mg l
.
Inoue, K., Kobayashi, S., Noguchi, H., Sankawa, U., & Ebizuka, Y.
(
1995). Nat. Med., 49, 336.
Inoue, K., Shibuya, M., Yamamoto, K., & Ebizuka, Y. (1996a).
FEBS Lett., 389, 273.
3.4. Genomic DNA extraction and PCR analysis
Inoue, K., Shimomura, K., Kobayashi, S., Sankawa, U., & Ebizuka,
Y. (1996b). Phytochemistry, 41, 725.
Aerial parts (ca. 0.2 g fr. wt) of the regenerated
kanamycin-resistant plantlet were used for DNA
extraction as described previously (Murray
Jeerson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). EMBO
J., 6, 3901.
&