ary origins and relatedness of chalcone and stil- bene synthases. Studies of two different stilbene
synthases of Pinus syl6estris showed that they had higher homology to chalcone synthase of P.
syl6estris, than they did to the stilbene synthases of other plants [9,10]. Also, a consensus phylogenetic
tree of chalcone and stilbene synthase proteins showed that stilbene synthases did not form a
separate cluster but rather grouped with the chal- cone synthases of related families [11]. Since that
report, there have been additional reported se- quences, including chalcone synthase sequences
from Vitis 6inifera and a new stilbene synthase from V. riparia reported here, that were used in
our analyses.
Thus far, stilbene synthase genes have been cloned from a very limited number of plants,
including two varieties of cultivated grapes V. 6
inifera, peanut Arachis hypogaea and two spe- cies of pines P. strobus and P. syl6estris
[7,9,10,12 – 15]. The goal of this research was to re-examine the relatedness of stilbene synthases
and chalcone synthases and to include a newly cloned stilbene synthase gene obtained from an-
other species of Vitis, V. riparia. In our analyses, several different tree construction procedures in-
volving distance, parsimony and maximum likeli- hood methods were used, and the results were
compared.
2. Materials and methods
DNA from leaves of V. 6inifera cv. Optima and V. riparia cv. Gloire de Montpellier was extracted
according to Lodhi et al. [16]. Twenty-five nanograms of extracted DNA was used for PCR.
Amplification was done in a total volume of 25 ml using
the primers
STSYF 5-TGGGTA
TCAATGGTCTTCAG and
STSYR 5-
CACTTAATTTGTCACCAATAGGA. These primers were designed based on relatively highly
conserved nucleotide sequences at the start and stop codons, respectively, of stilbene synthases of
V. 6inifera ‘Optima’ and V. 6inifera ‘Labrusca a Foglia Frastagliata’, and on highly conserved se-
quences of the stilbene synthases of P. strobus and P. syl6estris corresponding to the 6 – 8 nucleotides
near the 3 end of the primers. The amplification reaction was composed of 200 mM dNTPs, 0.5 mM
primers, 0.03 Uml of Taq DNA polymerase Gibco BRL, Gaithersburg, MD, 1.5 mM MgCl
2
and 1 × Taq DNA polymerase buffer. The cycling parameters were: 94°C for 5 min followed by 30
cycles at 94°C for 45 s, 50°C for 45 s and 72°C for 45 s, and a final extension time at 72°C for 7 min.
The PCR fragment was cloned into T-vectors constructed from pBluescriptII KS Stratagene,
LaJolla, CA according to Hadjeb and Berkowitz [17]. Restriction enzymes and related reagents
were purchased from Pharmacia Biotech Inc., and were used according to the manufacturer’s instruc-
tions. Plasmid DNA was introduced into com- petent Escherichia coli by transformation [18].
Sequencing was done with an ABI Prism model 377 DNA sequencer Applied Biosystems Inc.,
Foster City, CA.
2
.
1
. Sequence comparisons Amino acid sequence comparisons were made
between 17 chalcone synthases and seven stilbene synthases including our sequence from V. riparia
Table 1. The sequences were aligned with
CLUSTAL-W
[19] using default parameters. Includ- ing gaps, a 410-amino acid length was aligned.
Phylogenetic trees were then constructed using distance, parsimony and maximum likelihood
methods. For all three methods, the aligned se- quences were first subjected to bootstrapping using
the program
SEQBOOT
in the
PHYLIP
package [20]
available from
http:evolution.genetics. washington.eduphylip.html.
Genetic distances
within 1000 bootstrap replicates were calculated with the
PHYLIP
program
PROTDIST
using a Day- hoff PAM matrix. The distance matrices were then
analyzed with three different procedures: 1 with the
PHYLIP
program
NEIGHBOR
using the neigh- bor-joining algorithm; 2 with the
PHYLIP
pro- gram
NEIGHBOR
using the UPGMA algorithm; and 3 with the
PHYLIP
program
FITCH
. A parsi- mony method implemented in the
PHYLIP
program
PROTPARS
was used also to examine 1000 boot- strap replicates of the aligned sequences. The
aligned sequences were also analyzed using 50 bootstrap replicates with a maximum likelihood
method in the program
PROTML
part of a com- puter package called
MOLPHY
which was compiled for PC by Russell Malmberg from the Pascal
program written by Jun Adachi and Masami Hasegawa, available from http:dogwood.botany.
uga.edumalmbergsoftware.html.
The multiple data sets from the five procedures of the three methods were then separately analyzed
with the
PHYLIP
program
CONSENSE
to obtain bootstrap values reflecting the consistency of tree
branching patterns, and dendrograms were pro- duced with the
PHYLIP
program
DRAWGRAM
. The chalcone synthase-like protein sequence from
Streptomyces griseus, a prokaryote, was used to root all trees.
3. Results
