Rapid isolation of promoter sequences by TAIL-PCR: the 5'-flanking regions of Pal and Pgi genes from yams (Dioscorea)

Abstract

Using a modified TAIL-PCR technique, the 5'-flanking regions of the phenylalanine ammonia lyase (Pal) genes of a yam species, Dioscorea bulbifera, and the phosphoglucose isomerase (Pgi) gene of D. tokoro were successfully isolated. Two novel modifications of the TAIL-PCR procedure introduced here, namely (1) the use of a battery of random 10-mers (RAPD primers) as short arbitrary primers, and (2) the use of a total of five nested, gene-specific primers, allow the rapid isolation of the 5'-flanking region of any gene from organisms with large genomes. Isolated 5'-flanking regions were fused to the gus gene, and tested for transient expression in tobacco BY2 cells. All the isolated 5'-flanking regions were shown to drive reporter gene expression. Three Pal promoters responded to salicylic acid, presumably as a result of the binding of a MYB transcriptional activator to the multiple MREs (Myb Recognition Elements) present in these regions.

Full text

ORIGINAL PAPER
R. Terauchi á G. Kahl
Rapid isolation of promoter sequences
by TAIL-PCR: the 5¢-¯anking regions
of
Pal
and
Pgi
genes from yams (
Dioscorea
)
Received: 10 October 1999 / Accepted: 31 January 2000
Abstract Using a modi®ed TAIL-PCR technique, the
5¢-¯anking regions of the phenylalanine ammonia lyase
(Pal) genes of a yam species, Dioscorea bulbifera, and the
phosphoglucose isomerase (Pgi) gene of D. tokoro were
successfully isolated. Two novel modi®cations of the
TAIL-PCR procedure introduced here, namely (1) the
use of a battery of random 10-mers (RAPD primers) as
short arbitrary primers, and (2) the use of a total of ®ve
nested, gene-speci®c primers, allow the rapid isolation of
the 5¢-¯anking region of any gene from organisms with
large genomes. Isolated 5¢-¯anking regions were fused to
the gus gene, and tested for transient expression in to-
bacco BY2 cells. All the isolated 5¢-¯ank ing regions were
shown to drive reporter gene expression. Three Pal
promoters responded to salicylic acid, presumably as a
result of the binding of a MYB transcriptional activator
to the multiple MREs (Myb Recognition Elements)
present in these regions.
Key words TAIL-PCR á Promoter á Dioscorea á PAL
gene á PGI gene
Introduction
The isolation of promoter and enhancer sequences is a
crucial step in the study of the regulation of gene ex-
pression. Flanking regions of genes, containing these
elements, have been conventionally isolated by screening
genomic libraries using cDNAs as probes. However, the
construction and screening of genomic libraries involves
time-consuming procedures. As alternatives, PCR-based
methods have increasingly been applied for this purpose.
Inverse PCR (Ochman et al. 198 8), and ligation-medi-
ated PCR (Rosenthal and Jones 1990; Devon et al. 1995;
Siebert et al. 1995; Balavoine 1996; Zhang and Chiang
1996) are the techniques most frequently used for the
isolation of ¯anking regions of genes. These methods
rely on the presence of restriction sites in the region to be
isolated, so that the fragments can be self-ligated to form
circular molecules (inverse PCR) or ligated to a DNA
cassette (ligation-mediated PCR), prior to PCR. As in-
formation on restriction sites is usually not available in
advance, there is no guarantee that digestion with a
particular restriction enzyme will be successful, thereby
necessitating trials with several dierent enzymes. Fur-
thermore, in the case of ligat ion-mediated PCR, there
exists the inherent problem of undesirable ampli®cation
of PCR products that are ¯anked by the DNA cassette
sequence at both ends, at the expense of target sequence
ampli®cation. Several commercially available kits for the
isolation of regions ¯anking a known DNA sequence try
to minimize this problem by modifying the cassette
structure (e.g. Siebert et al. 1995) , but complete pre-
vention of PCR ampli®cation of non-target sequence s is
dicult. PCR products of the target sequence may be
separated from non-target sequences by using biotiny-
lated gene-speci®c primers and streptavidin-coated
magnetic beads (Rosenthal and Jones 1990). However,
this procedure requires biotin-labeling and capture/sep-
aration of the primers, which entails further costs, takes
more time and reduces yields of target sequences.
Therefore, simpler and more reliab le techniqu es for
promoter isolation are urgently required.
The TAIL-PCR (thermal asymmetric interlaced
PCR) method developed by Liu and Whittier (1995) is
such a simple, but nevertheless ecient, technique for
genomic walking which does not require any restriction
or ligation steps. PCR is carried out with long sequence-
speci®c primers in combination with short degenerate
Mol Gen Genet (2000) 263: 554±560
Ó
Springer-Verlag 2000
Communicated by R. Hagemann
R. Terauchi á G. Kahl (&)
Plant Molecular Biology, Biocentre, University of Frankfurt,
Marie-Curie-Str. 9, 60439 Frankfurt am Main, Germany
E-mail: Kahl@em.uni-frankfurt.de
Tel.: +49-69-79829267; Fax: +49-69-79829268
R. Terauchi
Iwate Biotechnology Research Center,
Kitakami, Iwate, 024-0003 Japan
Dedicated to Professor Dr. George G. Laties (UCLA, Los Angeles,
Calif.) on the occasion of his 80th birthday

primers of arbitrary sequence. An elaborate thermal
cycling program composed of ``supercycles'', each con-
sisting of one low-stringency cycle and two high-strin-
gency cycles, allows only sequence-speci®c fragments to
be exponentially ampli®ed. This method has been suc-
cessfully used to isolate insert-end segments of P1 and
YAC clones (Liu and Whittier 1995) and ¯anking re-
gions of T-DNA inserts in Arabidopsis (Liu et al. 1995).
But to the best of our knowledge, this method has never
been employed for the isolation of ¯anking regions of
any resident genes. Here we report the successful isola-
tion of the 5¢-¯anking regions of Pal and Pgi genes of
yams using a modi®ed TAIL-PCR method. Two novel
and essential modi®cations of the standard method were
introduced for the systematic isolation of ¯anking re-
gions of resident genes of organisms with large genome
sizes: (1) use of a battery of random 10 mers originally
developed for RAPD analysis (Williams et al. 1990) as
the short arbitrary primers instead of three degenerate
16-mer primers as described in the original TAIL pro-
cedure (Liu and Whittier 1995), and (2) use of a total of
®ve nested gene-speci®c primers instead of three. By
using modi®cation (1), we were able to exploit a whole
battery of 10 mer primers from commercially available,
low-cost primer sets designed for RAPD analysis (Wil-
liams et al. 1990). The use of a large number of 10 mers
increases the probability of amplifying long target se-
quences. Modi®cation (2) was necessary to amplify tar-
get sequences from the complex genomes of the genus
Dioscorea, which are about ®ve times larger (550 Mb;
Arumuganathan and Earle 1991) than the Arabidopsis
genome, for which the original TAIL-PCR technique
was developed (Liu et al. 1995).
The Pal gene codes for phenylalanine ammonia lyase,
the enzyme that catalyzes the conversion of phenylala-
nine to transcinnamic acid in the initial step of phenyl-
propanoid biosynthesis (Hahlbrock et al. 1976;
Hahlbrock and Scheel 1989; Wanner et al. 1995). The
Pal gene was one of the ®rst plant defense genes to be
identi®ed, and was found to be induced by pathogen s
and environmental stresses (Kuhn et al. 1984; Edwards
et al. 1985; Hahlbrock et al. 1995; Logeman et al. 1995).
To obtain an elicitor-inducible promoter for the genetic
engineering of yam crops, we ha ve been trying to isolate
the 5¢-¯anking region of this gene. The Pgi gene codes
for phosphoglucose isomerase, a key enzyme in gly-
colysis, which is known to be constitutively expressed.
Molecular population genetic studies of the coding re-
gion of the Pgi from D. tokoro (Terauchi et al. 1997)
prompted us to isolate and characterize the 5¢-¯anking
region of the gene.
Materials and methods
Plant materials, genomic DNA and cDNA
Total genomic DNA was extracted from individual Dioscorea bulb-
ifera plants (cultivar sativa) and from D. tokoro using the standard
CTAB (cetlytrimethylammonium bromide) method (Murray and
Thompson 1980). DNAs were further puri®ed by CsCl ultracentrif-
ugation. Total RNA was isolated from D. bulbifera leaf tissue by a
phenol/SDS method (Palmiter 1974), and cDNA was obtained from
it by reverse transcription using Superscript II (Gibco-BRL).
Isolation of Pal coding region from D. bulbifera
Alignment of the amino acid sequences derived from phenylalanine
ammonia lyase (Pal) genes from 11 plant species (Arabidopsis
thaliana, alfalfa, avocado, Camellia, Ipomoea, parsley, Pisum,
Populus, rice, Trifolium, tobacco and tomato, obtained from the
Genbank database) identi®ed highly conserved sequence regions.
Based on the sequences of these regions, two degenerate primers
were synthesized (Fig. 1): D0 (5¢-CAYYTIGAAYGARGTIAAR-
MRIATGGT-3¢, the forward primer) and U3 (5¢-GMRCTICCRTC-
Fig. 1 Schematic outline of the
procedures used to isolate
Pal 5¢-¯anking regions from
Dioscorea bulbifera. First, a
partial coding region of Pal was
ampli®ed by two consecutive
PCRs using the exon-speci®c
primer pairs D0 and T2 (®rst
PCR), and D0 and U3 (second
PCR). On the basis of the
sequence ampli®ed, ®ve gene-
speci®c primers were synthe-
sized and used in combination
with an arbitrary primer (AP)
for TAIL-PCR to obtain the
5¢-¯anking region
555

IARDATRTGYTCCAT-3¢, the reverse primer). In addition, the
primer T2 (5¢-ACRTCYTGRTTRTGYTGYTC-3¢, reverse primer)
was synthesized according to Howles et al. (1994). PCR performed
with the primer pair D0/T2 using D. bulbifera cDNA as template
resulted in non-speci®c ampli®cation. An aliquot of this PCR
product was then used as the template for the secondary PCR with
primer pair D0/U3. The resulting discrete PCR product of the
expected size was cloned into pBluescript, and sequenced.
The TAIL-PCR procedure
In order to allow chromosome walking beyond the known Pal and
Pgi sequences into the unknown 5¢ ¯anking region, TAIL-PCR
(Liu and Whittier 1995; Liu et al. 1995) was employed with two
essential modi®cations: (1) the use of 10 mer random primers in-
stead of degenerate 16 mers as the short primer, and (2) the use of a
total of ®ve rather than three gene-speci®c primers in nested posi-
tions to ensure selection of the correct target fragments.
On the basis of the cDNA sequence of a Pal gene from D. bulb-
ifera and a genomic Pgi DNA sequence from D. tokoro (Terauchi
et al. 1997), a total of ®ve gene-speci®c primers in nested positions
close to the 5¢-end of the coding regions were designed and synthe-
sized. The primers for D. bulbifera Pal were PAL-A (5¢-CCC-
TGCTTGGTYCTCCKATGAG-3¢), PAL-B (5¢-CGCCAAAACC-
AGTAGTGACACC-3¢), PAL-C1 (5¢-TCTTCATCACCCAATCA-
CTGCT-3¢), PAL-C2 (5¢-TGGTCTGGCCTCCTCTGAGAGC-3¢)
and PAL-C3 (5¢-CTAAGCTGACAACAGGATTCCT-3¢). The
primers used for D. tokoro Pgi were PGI-A (5¢-ACAGTGGCAG-
GAAGCACTCGTTGCCGGGAATAGTC-3¢), PGI-B (5¢-GCAT
GAGATCGCGGAGATGCGTCTTCTTGATTTCT-3¢), PGI-C1-
(5¢-TTTGAGGGACGAATGGAGGAAGAG-3¢), PGI-C2 (5¢-GA-
TACATTGAGGTCCTTCCACTGC-3¢) and PGI-C3 (5¢-TTCG-
CAGATAAGCGTGGACGTAGC-3¢) (Fig. 2). These primers were
designed such that the Tm calculated according to Mazers et al.
(1991) would be higher than 62 °C for the primers used in the primary
PCR (PAL-A and PGI-A) and the secondary PCR (PAL-B and PGI-
B), and higher than 57 °C for the tertiary PCR (PAL-C1±C3 and
PGI-C1±C3). Sixteen arbitrary 10 mer primers each for Pal and Pgi
were chosen from the 10 mer primer sets available (Roth), and care
was taken to ensure that they were not predicted by the Oligo pro-
gram (National BioSciences) to form stable duplexes with either of
the two gene-speci®c primers (A and B).
Three rounds of PCR (Table 1) were carried out on a Perkin
Elmer 9600 thermal cycler using the product of the previous PCR
as template for the next, and employing a common arbitrary primer
and nested gene-speci®c primers in a consecutive manner. The
annealing temperature for the low-stringency cycle was set to
29 °C, instead of 44 °C as in the original protocol (Liu and
Whittier 1995). The primary PCR was carried out in a 20-ll volume
containing 100 ng of genomic DNA, 0.2 lM gene-speci®c primer
(Primer A), 2.0 lM 10 mer primer, 200 lM of each dNTP, 0.2 U
Taq polymerase (Gibco-BRL) and 1´ Taq polymerase buer sup-
plied with the enzyme. The secondary PCR was carried out with
Primer B in combination with the same arbitrary primer as used in
the primary PCR. The reaction solution was the same as for the
primary PCR, except that 1 ll of a 1/50 dilution of the primary
PCR product was used as template. For the tertiary PCR, three
Fig. 2A, B Localization of the ®ve gene-speci®c primers for the Pal
gene of D. bulbifera (A)andthePgi gene of D. tokoro (B)
Table 1 Reaction parameters for the TAIL-PCR used to amplify the 5¢- ¯anking regions of the Pal genes of Dioscorea bulbifera and the
Pgi gene of D. tokoro
Reaction
(primer combination)
Program
no.
Number
of cycles
Cycle (supercycle) parameters
Primary PCR
(AP/Primer A) 1 1 93 °C, 1 min; 95 °C, 1 min
2594°C, 30 s; 62 °C, 1 min; 72 °C, 2.5 min
3194°C, 30 s; 25 °C, 3 min; ramping to 72 °C over 3 min; then 72 °C, 2.5 min
41594°C, 10 s; 68 °C, 1 min; 72 °C, 2.5 min; 94 °C, 10 s; 68 °C, 1 min; 72 °C,
2.5 min; 94 °C, 10 s; 29 °C, 1 min; 72 °C, 2.5 min
5172°C, 5 min
Secondary PCR
(AP/Primer B) 6 12 94 °C, 10 s; 64 °C, 1 min; 72 °C, 2.5 min; 94 °C, 10 s; 64 °C, 1 min; 72 °C,
2.5 min; 94 °C, 10 s; 29 °C, 1 min; 72 °C, 2.5 min
5172°C, 5 min
Tertiary PCR
(AP/Primers C1±C3) 7 20 94 °C, 15 s; 29 °C, 30 s; 72 °C, 2 min
5172°C, 5 min
556

gene-speci®c primers (C1, C2 and C3) were separately used with the
common arbitrary primer. The reaction solution for the tertiary
PCR was the same as for the primary PCR except that 1 llofa1/
10 dilution of the secondary PCR product was used as template,
and the concentration of the arbitrary primer was 0.2 lM instead
of 2.0 lM.
The products of the tertiary PCR (three PCRs for each arbi-
trary primer, corresponding to the three gene-speci®c primers C1 to
C3) were separated in adjacent lanes on agarose gels to determine
whether discrete PCR products from the three gene-speci®c primers
show size dierences corresponding to the relative positions of the
nested primers. In the original protocol developed for Arabidopsis
(Liu et al. 1995), PCR products of the secondary PCR (obtained
with primer B) and tertiary PCR (obtained with primer C1) were
separated in two adjacent lanes by agarose gel electrophoresis to
detect the expected size dierence. However, this procedure was not
applicable for Dioscorea , as electrophoresis of the secondary
amplicons usually resulted in smeared patterns. Use of three (C1,
C2 and C3) instead of two primers for the tertiary PCR was also
important to allow us to discriminate the true step-wise size dif-
ferences corresponding to the dierent primer locations from spu-
rious ampli®cations.
Cloning and sequencing of the tertiary PCR products
PCR products were excised from the agarose gel and reampli®ed.
After polishing the ends with the Klenow fragment (NEB) and T4
polynucleotide kinase (NEB) in the presence of dNTPs and ATP,
they were cloned into the SmaI site of pBluescript. DNA se-
quencing was performed on an ABI 373A automated sequencer.
Transient expression assay using cultured tobacco BY2 cells
Cloned 5¢-¯anking regions of Pal (PAL-AP140, PAL-AP122 and
PAL-AP103) and Pgi (PGI-AP125) genes were PCR-ampli®ed
with primers carrying BamHI (the distal end of the 5¢-¯anking
regions) and XhoI (the proximal end) extensions at the 5¢-ends.
The products were ligated to a 2.6-kb XhoI-HindIII fragment
containing the gus (b-glucuronidase) gene and the CaMV 35S
polyadenylation signal derived from pRT101-gus (To
È
pfer et al.
1993), and cloned into the BamHI-HindIII sites of pBluescript
SK) (Stratagene), resulting in the plasmids pRG108 (PAL-
AP140), pRG109 (PAL-AP122), pRG110 (PAL-AP103) and
pRG103 (PGI2; see Fig. 5). Each of these GUS constructs was
mixed in a 1:1 ratio with plasmid pRT101-LUC (a kind gift of
Dr. C. Kirchner; To
È
pfer et al. 1993), harboring a ®re¯y lucifer-
ase gene under control of the CaMV-35S promoter and 35S
polyadenylation signal, and introduced into BY2 cells (Nicotiana
tabacum cv. Bright Yellow; Ikeda et al. 1976) by particle bom-
bardment (Biorad) following the manufacturer's instructions.
Tobacco BY2 cells maintained in the BY-2 medium containing
2,4-D (Matsuoka and Nakamura 1991) were collected on a ®lter
paper 4 days after subculturing, placed on a solid medium con-
taining 0.2% gelan gum, and bombarded with the plasmids.
After 24 h the cells were transferred to a solid BY-2 medium
containing either (1) no added ingredients, (2) a culture ®ltrate of
Botrytis cinerea, (3) N-acetylchitohexaose (0.02 mg/ml; Yamada
et al. 1993), or (4) salicylic acid (SA; 20 lM), and cultured for
another 24 h. Then cells were harvested and lysed in LC-b Pic-
aGene cell lysis buer (Wako Chemicals) by sonication. The
lysate was centrifuged, and the supernatant immediately assayed
for luciferase and b-glucuronidase activity using the PicaGene
Fig. 3 Agarose gel electropho-
resis of the tertiary PCR prod-
ucts of D. bulbifera Pal (left)
and D. tokoro Pgi (right). M1
and M2 are molecular weight
markers [HindIII digest of
k-DNA, and 100-bp ladder
(Gibco-BRL), respectively].
DNA fragments marked with
asterisks were excised from
the gel and sequenced. The
sequences of the arbitrary
10 mer primers were: 5¢-
GGTGCTCCGT-3¢ (AP103),
5¢-CGATGAGCCC-3¢
(AP122), 5¢-CTATCGCCGC-3¢
(AP138), 5¢-CGCAGACCTC-3¢
(AG140), 5¢-GTGTGCC CCA-3¢
(AP158) and 5¢-ACGGTGC-
CTG-3¢ (AP125)
557

Luciferase detection kit (Wako Chemicals) and the Gus-Light
GUS detection kit (Tropix), respectively, and an ATTO lumi-
nometer. Promoter activity was expressed as the GUS value di-
vided by the LUC value, the latter serving as a normalizing
factor to reduce the interexperimental ¯uctuation caused by
dierences in cell viability and/or eciency of plasmid delivery
by particle bombardment.
Results and discussion
Isolation of the 5¢-¯anking regions of Pal genes
from D. bulbifera
Among the 16 arbitrary primers tested in combination
with a set of Pal gene-speci®c primers (PAL-C1, PAL-
C2 and PAl-C3), ten primers resulted in ampli®cati on of
discrete PCR products. Electrophoretic patterns ob-
tained for ®ve arbitrary primers are shown in Fig. 3.
Successful walking can easily be con®rmed by the
stepwise change in the sizes of PCR products that
correspond to the relative positions of the three nested,
Pal-speci®c, primers (PAL-C1, PAL-C2 and PAL-C3).
Further con®rmation came from the observation that
DNA sequences of PCR products obtained by PAL-C1
primer overlapped perfectly with the 5¢-end sequence of
a cDNA (data not shown). The sizes of PCR products
ranged from 400 to 1300 bp (when PAL-C1 was used for
walking).
Aligned DNA sequences of three PCR products
(generated by the arbitrary 10 mers AP140, AP122 and
AP103) are given in Fig. 4. DNA sequences were de-
posited in DDBJ, EMBL and Genbank under Accession
Nos. AB016713±AB016715. The longest walk into the
5¢-¯anking region was obtained with AP140 (1014 bp
upstream of the start codon), followed by AP122
(510 bp) and AP103 (352 bp). Although the region close
to the coding sequences is highly conserved, there are
extensive dierences between the products obtained with
Fig. 4 Aligned DNA sequences
of three TAIL-PCR products
(ampli®ed with the arbitrary
primers AP140, AP122 and
AP103) obtained from the
5¢-¯anking regions of Pal genes
of D. bulbifera. The putative
TATA box (sequence 5¢-TAT-
TTAA-3¢) and MYB recogni-
tion elements (MRE consensus
sequence: 5¢-ACCAACC-3¢) are
boxed
558

the APs in regions further upstream (a 7-bp insertion in
the AP103 sequence at position ± 168 from the start
codon; no sequence homology between AP140 and
AP122 sequences upstream of position ± 355). These
observations indicate that we have isolated the 5¢-
¯anking regions of dierent Pal loci. Indeed Pal genes
are known to form a multigene family (Logeman et al.
1995; Wanner et al. 1995). In the regions isolated, a
putative TATA box and several MREs [MYB reco gni-
tion elements; 5¢-G(G/T)T(A/T)G(G/T)T-3¢; type II
MYB consensus sequence; Yang and Klessig 1996] could
be identi®ed. MREs were previously identi®ed in boxes
P and L of parsley Pal genes, and obviously are address
sites for the MYB-like protein BPF-1 (de Costa e Silva
et al. 1993; Feldbru
È
gge et al. 1997).
Isolation of the 5¢-¯anking region
of the Pgi gene from D. tokoro
After testing 16 arbit rary 10 mers , ®ve primers were
found to result in the desired products. Two examples
are shown in Fig. 3. The sizes of the PCR products
obtained ranged from 200 to 1500 bp (when PGI-C1 was
used for walking). The DNA sequence of the longest
PCR product (obtained with arbitrary primer AP125)
has been deposited in DDBJ, EMBL and Genbank un-
der the Accession No. AB016716 .
Transient expression of the GUS gene driven
by the isolated 5¢-¯anking regions
Isolated 5¢-¯anking regions of Pal and Pgi genes were
fused to the gus gene, and their activity was tested by
transient transformation after delivery into tobacco BY2
cells by particle bombardment (Fig. 5). Three plasmids
bearing Pal 5¢-¯anking regions (pRG108 for
PAL-AP140, pRG109 for PAL-AP122 and pRG110 for
PAL-AP103) and a plasmid carrying the Pgi 5¢-¯anking
region (pRG103 for PGI-AP125) were found to drive
expression of the gus gene (Fig. 5). This indicates that
the isolated 5¢ ¯anking regions contain the minimal
promoter elements necessary for the transcription of the
genes located downstream.
Responses of the promoters to external stimuli were
tested by culturing the transformed tobacco BY2 cells on
media containing various substances. As shown in
Fig. 5, the presence of SA in the medium signi®cantly
increased the level of expression of gus driven by all
three Pal 5¢-¯anking region constructs (pRG108,
pRG109 and pRG110), relative to the expression level of
the control (water), whereas culture ®ltra te of Botrytis
and N-acetylchitohexamer had no eect.
Yang and Klessig (1996) have shown that the myb
oncogene homolog from tobacco (myb1) is rapidly in-
duced (within 15 min) by externall y applied SA, and
that Myb1 protein speci®cally binds to a Myb-binding
consensus sequence, 5¢-G(G/T)T(A/T)G(G/T)T-3¢,
found in the promoter region of the tobacco PR-1a gene,
the transcription of which is induced several hours after
myb1 induction. They suggest that the tobacco myb1
gene encodes a signaling component that acts down-
stream of SA, probably participating in transcriptional
activation of PR genes that contribute to plant disease
resistance. In view of the presence of multiple MREs in
the 5¢-¯anking regions of D. bulbifera Pal genes, we as-
sume that upon the treatment of transformed tobacco
BY2 cells with SA, myb1 was induced and Myb1 sub-
sequently bound to MRE of the Pal promoter regions of
the reporter plasmids, thereby activating the expression
of gus.
Versatility of the TAIL-PCR method
for the isolation of promoters
Using the TAIL -PCR technique with two modi®cations,
we have successfully and rapidly isolated the 5¢-¯anking
Fig. 5 A Schematic outline of the strategy for transient expression
analysis using tobacco BY2 cells B Structure of the transient
expression vectors carrying Pal (pRG108, pRG109 and pRG110)
and Pgi (pRG103) 5¢-¯anking regions. C Transient expression of gus
genes after introduction of plasmids into tobacco BY2 cells by particle
bombardment. Some 24 h after plasmid introduction, cells were
placed on medium supplemented either with water (WT), Botrytis
cinerea culture ®ltrate (BT), N-acetochitohexamer (CH) or salicylic
acid (SA). Experiments were performed in triplicate. The ordinate
represents the ratio between the GUS and LUC measurements
559

regions of three Pal genes from D. bulbifera and the Pgi
gene from D. tokoro. Transient expression studies
showed that all the isolated sequences are transcription-
ally functional. In the case of the Pal gene, 5¢-¯anking
regions of multiple loci could be recovered. This dem-
onstrates that we can isolate the promoter regions of
most of the members of a particular gene family by
systematically testing a large number of 10 mers of
arbitrary sequence, in combination with consensus
degenerate gene-speci®c primers. This versatile method
recommends itself for the isolation of regulatory ele-
ments of genes from any organism.
Acknowledgements We thank Dr. C. Kirchner (University of
Frankfurt) for provision of pRT101-LUC, and Dr. M. Nishihara
and Ms. K. Ohmiya (Iwate Biotechnology Research Center) for
instructions on the use of tobacco BY2 cells. R. T. appreciates an
Alexander-von-Humboldt Fellowship (No. IV-1-7121-1028559).
Research of the authors was supported by GTZ (95.2072.7-001.00).
Thanks are due to Dr. K. H. Wolpers (GTZ) for promoting our
yam research.
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