Download Multiple Domains Exist within the Upstream Activator

The Plant Cell, Vol. 1, 925-936, September 1989 O 1989 American Society of Plant Physiologists
Multiple Domains Exist within the Upstream Activator
Sequence of the Octopine Synthase Gene
Scott M. Leisner and Stanton 8. Gelvin'
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
It is known that a 16-base pair palindrome (ACGTAAGCGCTTACGT)located upstream of the ocs gene can activate
a maize adhl promoter in a transient expression system [Ellis et al. (1987). EMBO J. 6, 11-16; Ellis et al. (1987).
EMBO J. 6, 3203-32081. We have determined that this palindrome is also essential for ocs promoter activity in
tobacco calli. In addition, sequences immediately adjacent to this palindrome, both 5' and 3', modulate its activity.
The palindrome is sensitive to the differentiated state of the plant cells in which it resides; it is active in calli and
the leaves of small shoots but is inactive in the leaves of rooted plants. We have tested upstream sequences from
two other T-DNA genes that have homology to this palindrome for their ability to activate the octopine synthase
promoter in tobacco calli. The upstream region from the mannopine synthase gene can activate the octopine
synthase promoter, but an upstream region from the gene implicated in octopine and nopaline secretion cannot
activate the promoter.
INTRODUCTION
The Gram-negative soil bacterium Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous
and some monocotyledonous plants. Virulent strains of
this bacterium harbor a large tumor-inducing (Ti) plasmid
and transfer a portion of this plasmid, the transferred DNA
(T-DNA), to a susceptible plant cell. The T-DNA, shown
schematically in Figure 1, integrates into the plant nuclear
genome where its genes are expressed. Some T-DNA
genes encode enzymes that synthesize plant hormones,
resulting in the tumor phenotype. Other T-DNA genes
direct the synthesis and secretion of unusual amino acid
and sugar derivatives called opines. The inciting strain of
Agrobacterium can utilize these opines as a carbon and
sometimes a nitrogen source. Agrobacterium strains can
be classified based upon their ability to catabolize particular opines. (For recent reviews of the crown gall tumorigenesis process, see Nester et al., 1984; Binns and
Thomashow, 1988.)
The regulationof T-DNA gene expression is an interesting problem because the genes are normally harbored on
a prokaryotic plasmid but are subsequently expressed in
eukaryotes. T-DNA genes possess all of the sequence
elements required for transcription in plants. These elements have been studied for several T-DNA genes, including those involved in cytokinin biosynthesis, tmr (de Pater
et al., 1987); agropine biosynthesis, ags (Bruce, Bandyopadhyay, and Gurley, 1988); octopine biosynthesis, ocs
(Ellis et al., 1987a, 1987b; Leisner and Gelvin, 1988);
' To whom correspondence should be addressed
mannopine biosynthesis, mas (DiRita and Gelvin, 1987);
nopaline biosynthesis, nos (An, 1987; Mitra and An, 1989);
and a gene encoding a 780-nucleotide mRNA from TR
(Bruce, Bandyopadhyay, and Gurley, 1988). These T-DNA
genes contain TATA boxes that set the site of transcription
initiation, and many contain upstream elements, located
more than 1O0 bp from the transcription initiation site, that
modulate the levels of transcription. The nos gene, the ocs
gene, and the 780 gene all contain upstreamtranscriptional
activating sequences that can activate promoters when
present in either orientation (Ellis et al., 1987a, 1987b;
Leisner and Gelvin, 1988; Bruce, Bandyopadhyay, and
Gurley, 1988; Mitra and An, 1989). The ags element cannot
activate the promoter for the 780 gene in either orientation
(Bruce, Bandyopadhyay, and Gurley, 1988). The tmr element has not been tested for its ability to activate promoters independent of its orientation. The 780 gene element activates its promoter when separated by 613 bp
(Bruce, Bandyopadhyay, and Gurley, 1988). Each of the
other elements, however, cannot function when placed
more than several hundred base pairs from a promoter (de
Pater et al., 1987; Leisner and Gelvin, 1988; Bruce, Bandyopadhyay, and Gurley, 1988; Mitra and An, 1989). As
with many plant upstream activating sequences (Timko et
al. 1985), these T-DNA upstream elements more closely
resemble yeast upstream activation sites than transcriptional enhancers.
We are interested in the upstream element of the octopine synthase (ocs) gene. The product of this gene condenses arginine and pyruvate to form the opine, octopine
926
The Plant Cell
TL
b
5
7
ims
-------D--+
1
.
2
I m r onstml ocs
780
- 4
4' 3'
mas
ogs
375
2
Figure 1. Schematic Map of the Bipartite T-DNA of pTiA6.
Arrowheads denote the T-DNA borders and arrows indicate the
direction of transcription of various open reading frames. (For
reviews of the various T-DNA genes, see Garfinkel et al., 1981;
Nester et al., 1984; Binns and Thomashow, 1988.) Genes 5 and
7 encode mRNAs of unknown function. The tmsl and tms2 genes
encode enzymes of a two-step pathway for the biosynthesis of
auxins, and the tmr gene encodes an enzyme for the biosynthesis
of cytokinins. The ons gene encodes a protein involved in octopine
and nopaline secretion (Messens et al., 1985), and the tml gene
regulates tumor size in some plant species. The ocs gene encodes
an enzyme that synthesizes octopine (Hack and Kemp, 1980),
and the 780 gene (4') encodes a 780-nucleotide-longmRNA of
unknown function (Karcher, DiRita, and Gelvin, 1984). The 3'
gene also encodes an mRNA of unknown function. The mas gene
(2') and the 1' gene encode the enzymes of a two-step pathway
for the synthesis of mannopine (Ellis, Ryder, and Tate, 1984;
Komro et al., 1985), and the ags gene encodes an enzyme that
cyclizes mannopineto yield agropine (Ellis, Ryder, and Tate, 1984;
Komro et al., 1985). Dark boxes at the 5' ends of the ocs and
mas genes indicate the presence of orientation-independent upstream elements that activate the ocs promoter. The open box
for the ons gene indicates that the upstream element cannot
activate the ocs promoter. In this study, we investigated sequences from -333 to -1 16 from the transcription start site for
the ocs gene, -318 to -213 from the transcription start for the
mas gene, and 363 bp to 170 bp upstream from the translation
start site for the ons gene.
(Hack and Kemp, 1980). Previously, Ellis and co-workers
(Ellis et al., 1987b) determined that a 16-bp palindrome
(ACGTAAGCGCTTACGT) located upstream of the ocs
gene could activate a heterologous promoter in transient
expression assays. We found that a fragment (from -333
to -1 16) containing this palindrome is also important in
activating the ocs promoter in tobacco calli when stably
incorporated into the genome (Leisner and Gelvin, 1988).
This fragment activated the ocs promoter when present in
either orientation, but was position-dependent. In this paper, we present a more detailed analysis of the octopine
synthase upstream activator sequence. The 16-bp palindrome is sufficient to activate the ocs promoter in tobacco
calli. However, sequences adjacent to the palindrome influente its activity. We have additionally extended our analysis of the ocs upstream activator sequence to transgenic
tobacco plants. Whereas the palindrome is sufficient to
activate the ocs promoter in tobacco calli and the leaves
of small shoots, it is insufficient to do so in the leaves of
rooted plants. A portion of the 16-bp palindrome is found
in the upstream regions of two other T-DNA genes, the
mannopine synthase (mas) gene (Ellis, Ryder, and Tate,
1984; Komro et al., 1985; DiRita and Gelvin, 1987) and
the gene implicated in octopine and nopaline secretion
(ons)from transformed plant cells (Messens et al., 1985).
Restrictionendonuclease fragments from the mas and ons
genes, containing sequences similar to the palindrome,
were tested for their ability to activate the ocs promoter.
RESULTS
Analysis of the Octopine Synthase Upstream Activator
Sequence
An upstream activator sequence (UAS) regulates transcription of the octopine synthase gene (Ellis et al., 1987a,
1987b; Leisner and Gelvin, 1988). To determine which
sequences present within the UAS are required for activity
in tobacco calli, we cloned various restriction endonuclease
fragments of the ocs activator element into pOCSA2.
These fragments are described in Figures 2 and 3. After
mobilizationof plasmids harboring these fragments cloned
upstream of the ocs gene into A. tumefaciens strain
LBA4404, we infected tobacco leaf discs and selected
kanamycin-resistant calli. We pooled 20 or more calli,
assayed them for octopine synthase activity (Otten and
Schilperoort, 1978), and determined the amount of octopine synthesized per microgram of protein. Figure 4A
shows the results of a typical octopine synthase assay.
Figure 48 shows that octopine synthase activity correlates
with the steady-state leve1 of ocs mRNA in tobacco calli.
These data corroborate our previous findings (Leisner and
Gelvin, 1988).
5' and 3' Deletion Analysis of the Octopine Synthase
UpstreamActivator Sequence
Calli containing the construction pOCSA2 lacking the ocs
upstream element had little detectable octopine synthase
activity (Figure 3 and Figure 4A). The activity of calli
harboring the 5' deletion plasmids pSaul33, pAlulO6, and
pRsa61 (Figure 3) indicated that a region important for
activator function is located between the Alul (-222) and
Rsal (-177) sites. (The transcription initiation site of the
ocs gene is designated as +1.) The 3' deletion construction pRsal31 indicated that the 3' boundary of an active
region is located upstream of the Rsal site at -177.
Therefore, we cloned the 45-bp fragment between the Alul
(-222) and Rsal (-177) sites (pAIRa45, Figure 3) onto the
ocs promoter. This construction resulted in full octopine
synthase activity when transferred to tobacco calli (Figure
3 and Figure 4A).
To localize further the active sequences present within
the pAIRa45 fragment, we cloned the 38-bp fragment from
-222 to -1 89 (pHin38) onto the ocs promoter (Figure 3).
ocs Upstream Activator Sequence Domains
A
pSLl
Ac
I
927
Calli incited by bacteria harboring this plasmid had little
detectable octopine synthase activity (Figure 3). The
pAIRa45 fragment contains a 16-bp palindrome
(ACGTAAGCGCTTACGT from -1 92 to -1 77),and this
palindrome is truncated in the plasmid pHin38. Because
lack of octopine synthase activity correlated with lack of a
complete palindrome, we hypothesized that the palindrome could be responsible for the activity directed by the
fragment contained in pAIRa45. To test this hypothesis,
C
Figure 2. lntermediate Plasmids Used in the Construction of
Octopine Synthase Upstream Activator Deletions.
(A) The plasmid pSLl (Leisner and Gelvin, 1988). pSLl contains
the entire ocs upstream activator. Relevant restriction endonuclease sites for subcloning different fragments of the activator are
shown.
(B) The intermediate plasmids pG1, pAl, and pX1. All three of
these plasmids are derived from pSLl as described in Methods.
The fragments derived from pG1, pAl , and pX1 are shown below
each plasmid map. The fragments from the plasmid pG1 are
labeled PD for promoter-dista1and PP for promoter-proximal.
(C) The plasmids pUX13 and pOCSA2. pUX13 is pUC13 with the
Smal site converted to Xhol by linker addition. We have described
the constructionof pOCSA2 previously (Leisner and Gelvin, 1988).
The arrow indicates the direction of transcription of the ocs gene.
The BamHl site is at -1 16 bp relativeto the transcriptioninitiation
site.
Restriction endonuclease sites are abbreviated as follows: Ac,
Accl; Ac-B, Accl converted to BamHI; A, Alul; A-B, Alul converted to BamHI; B, BamHI; D, Dral; E, EcoRI; H, Hindlll; Hc,
Hincll; M, Mspl; P, HinPI; Ps, Pstl; R, Rsal; S, Sall; Sa, Sau3a;
Sa/B, Sau3a-BamHI fusion not cleavable by BamHI; Sm, Smal;
Ss, Sstl; T, Taql; X, Xhol; Xb, Xbal. The area containing diagonal
lines indicates the 16-bp palindrome.
PSRj
P S I
-
t w - . . . -
M
+ms-
NXSAZ+m$-
34
2
1653
99
34
2
Figure 3. Deletion Analysis of the Octopine Synthase Upstream
Activator Sequence.
The arrow with ocs in the middle indicates the orientation of the
octopine synthase gene. The promoter fragment used begins at
-1 16 bp relative to the transcription start site. Sequences to the
left of -116 in the construction pENl contain the upstream
activator. The upstream activator sequence extends from -333
to -1 16, and the relevant restriction enzyme sites are marked.
The locations of the SV40 enhancer core sequence, a potential
2-DNA-forming sequence, and the 16-bp palindrome are marked
as black boxes. The location of the palindrome is also marked as
an open box on each construction. The arrowheads for each
construction indicate the orientation of the construction relative
to the promoter; an arrow pointing to the right indicates the wildtype orientation. The amount of octopine (pmol) made per microgram of protein during a 2-hr incubation is indicated, as is the
relative leve1 of octopine synthase activity (normalized to pEN1 as
100%) for each construction. One unit of octopine synthase
activity is equivalent to 0.8 pmol of octopine synthesized per hour
per microgram of protein. pENl synthesized about 2500 pmol of
octopine.
928
The Plant Cell
OJ
Incubation
time
(Hrs)
o~~2
Incubation
time
<
5E
I
0.
8
3 2
C~2
6~2
C/1
0.
(Hrs)
ARG —
ARG-
OCT—
OCT —
2.0 -
Figure 4. Octopine Synthase Assay of Various ocs Activator Deletions.
(A) Electrophoretogram of octopine synthase assays performed on 20 pooled calli incited with the constructions indicated above. Positions
of octopine (OCT) and arginine (ARG) are marked. Numbers above indicate the time of incubation in hours. Origin (+) is at the bottom.
The amount of protein in each extract was 9.3 ^g for pEN1, 15.8 ^g for pENR1, 7.5 ,ug f°r pAIRa45, 24 ng for pPAL16, 10.3 /jg for
pOCSA2, 7.4 jig for pAITaR54 and 11.8 ^g for pAluR106. The amount of octopine made after 2 hr was 2498 pmol of octopine for pEN1,
1070 pmol for pENR1, 1636 for pAIRa45, 2059 pmol for pPAL16, 53.9 pmol for pOCSA2, 3817 pmol for pAITaR54 and 5980 pmol for
pAluR106.
(B) Correlation of octopine synthase activity with the steady-state level of octopine synthase mRNA. Electrophoretogram of octopine
synthase assays performed on 20 pooled calli incited with the constructions indicated above. Positions of octopine (OCT) and arginine
(ARG) are marked. Numbers above indicate the time of incubation in hours. Origin (+) is at the bottom. The amount of protein in each
extract was 9.3 ng for pEN1, 15.8 ^g for pENR1, and 10.3 ^g for pOCSA2. The amount of octopine made after 2 hr was 2498 pmol for
pEN1, 1070 pmol for pENR1, and 53.9 pmol for pOCSA2. The graph below is a densitometer tracing of an RNA gel blot of total RNA
isolated from pooled calli incited with pEN1, pENR1, and pOCSA2. We subjected 15 ^g of total RNA to electrophoresis through a 1.5%
agarose gel containing 6% formaldehyde, blotted the RNA onto nitrocellulose (Karcher, DiRita, and Gelvin, 1984), and hybridized the filter
with a Oral fragment [12221 to 13685 (Barker et al., 1983)] encompassing the ocs gene. We scanned an autoradiograph of the blot with
a densitometer. The peaks shown below the electrophoretogram indicate the amount of ocs mRNA present in the calli harboring each
construction. The ordinate for this figure is relative OD.
we synthesized an oligonucleotide containing the sequence of the palindrome and cloned it onto the 5' end of
the ocs gene. When mobilized into tobacco calli, this
construction, pPAL16, was also active (Figure 3 and Figure
4A). Therefore, the palindrome is necessary and sufficient
to stimulate the ocs promoter in tobacco calli. These data
correlate with the findings of Ellis et al. (1987b). The
constructions pHin70, pHinR70, pHin38, pHinR38, and
pSaHi69 all contain half of the palindrome and are inactive
in tobacco calli. The palindrome must therefore be complete in order for the UAS to function. Oligonucleotides
corresponding to either half of the palindrome, pHPPD and
ocs Upstream Activator Sequence Domains
pHPPP, are also nonfunctional when cloned onto the ocs
gene (Figure 3).
Although the palindrome alone can activate the ocs
promoter in tobacco calli, it is not as active as are the
larger elements pEN1 or pAIRa45 (Figure 3 and Figure
4A). These data indicate that sequences present between
-222 and -193, within pAIRa45, augment the palindrome
to provide full activity. A subset of these additional sequences 5' to the palindrome contained within pHin38 [the
5' palindrome augmenting sequence (5' PAS)] cannot
augment the activity of a truncated palindrome, suggesting
that the 5' PAS cannot function by itself.
Sequences downstreamof the palindrome also augment
the activity of the palindrome. The plasmids pAluR106
(-222 to -1 16) and pAITaR54 (-222 to -1 70) are both
equally active in tobacco calli, and the activity of both is
greater than that of the larger element pENl (Figure 3 and
Figure 4A). In both of these constructions, the palindrome
is 24 bp closer to the promoter than it is in pENl , and both
contain potential Z-DNA stretches. The palindrome is 120
bp from the TATA box in pAITaR54, pAluRl06, and
pAIRaR45. However, the activity of pAIRaR45 is equivalent
to that of pEN1. These results suggest that the enhancement of activity for pAITaR54 and pAluRlO6 over that of
pEN1 is most likely due to the presence of additional
sequences 3' to the palindrome (termed the 3' palindrome
augmenting sequence, or 3' PAS), rather than the closer
proximity of the palindrome to the promoter. pENl also
has the 3' PAS but is less active than pAlulO6, pAluRlO6,
and pAlTaR54 because it may contain inhibitorysequences
(see below). The 3' PAS cannot augment the activity of a
truncated palindrome (pHin70), suggesting that the 3' PAS
cannot function on its own (Figure 3).
Sequences between the Sau3a (-249) and Alul(-222)
sites also modulate the activity of the octopine synthase
UAS. The activity of the pSau133 fragment is equivalent
to that of the larger element (pENl), whereas the activity
of the pAlulO6 fragment is greater (Figure 3). One possible
interpretation of these data is that inhibitory sequences
are located between the Alul and Sau3a sites. Interestingly, pAIRa45 lacks these putative inhibitory sequences
but is only as active as is pENl . These data suggest that
not only must the putative inhibitory sequences be removed, but also that the potential 2-DNA sequence must
be present to elicit additional activity. These data also
explain why pEN1 is less active than are pAlul06, pAluR106, and pAITaR54.
When we cloned the restriction endonuclease fragments
pSau84, pMsp42, and pMpSa42 upstream of the ocs
gene, we observed no octopine synthase activity in tobacco calli. These data indicate that sequences upstream
of the Sau3a site (-249), including a SV40 enhancer core
sequence, are not sufficient for activity. Neither do these
sequences augment the activity of either pRsa61 (pSR1)
or pAIRa45 (pSA1). The plasmid pSR1 is inactive, as are
pRsa61 and pSau84. These data indicate that the Sau84
929
fragment cannot activate an already inactive Rsa61 fragment. In addition, the sequences between the Sau3a
(-249) and Rsal (-177) sites are absolutely required for
activity. The construction pSA1 is only as active as is
AIRa45. These data indicatethat the Sau84 fragment does
not augment the activity of the AIRa45 fragment and has
no stimulatory effect even on positively acting sequences.
We had previously shown that a dimer of the Sau84 (-333
to -249) fragment in the reverse orientation could also
activate the ocs promoter (Leisner and Gelvin, 1988). We
have subsequently been unable to reproduce this result.
Table 1 shows the number of base pairs between the
palindrome and the TATA box for a number of the constructions. In general, when the orientation of the various
UAS fragments is such that the palindrome is closer to the
ocs promoter, these constructions are more active than
are those in which the palindrome is further away. For
example, pENR1 is about 40% as active as is pENl . The
palindrome is 231 bp and 146 bp from the TATA box for
pENRl and pENl , respectively. pRsal31 has the same
activity as does pENl , whereas pRsaR131 has the same
activity as does pENR1. The palindrome is 98 bp and 212
bp from the TATA box for pRsal31 and pRsaR131,
respectively.
The activity of both pAIRa45 and pAIRaR45 is approximately the same as is the activity of pENl . The palindrome
is 91 bp and 120 bp from the TATA box for pAIRa45 and
pAIRaR45, respectively. Likewise, pAlulO6 and pAluRlO6
are both approximately equally active, and the activity of
both is greater than that of pENl . In these constructions,
the palindrome is 146 bp and 120 bp from the TATA box
for pAlulO6 and pAluRl06, respectively (Table 1). These
data indicate that UAS fragments can activate the ocs
promoter to wild-type levels when the palindrome is 91 bp
Table 1. Distance of Palindrome from TATA Box
Construction
Distance from
TATA Box
Relative Activity
in Calli"
bP
pENl
pENRl
pSaul33
pAlulO6
pAluRlO6
pRsal31
pRsaRl31
pAIRa45
pSAl
pAIRaR45
pAITaR54
pPALl6
146
231
146
146
120
98
212
91
91
120
120
88
1O0
41
99
160
21o
90
36
97
99
105
215
36
numbers are relative to pENl . pENl synthesized 167 pmol
of octopine per microgram of protein during a 2-hr incubation.
a The
930
The Plant Cell
to 146 bp from the TATA box, but when the palindrome is
moved greater than 200 bp from the TATA box, activity is
reduced. These data corroborate our previous findings
that octopine synthase activity is abolished when the activator is separated from the promoter by 608 bp (the
palindrome is 754 bp from the TATA box; Leisner and
Gelvin, 1988).
Various UAS SubfragmentsFunction Differently in
Shoots and in Calli
We obtained transgenic shoots from tobacco calli containing pENl, pENRl , pAlulO6, pAIRa45, and pPAL16. For
each construction, we pooled leaves from 10 plants and
tested them for octopine synthase activity. Leaves were
tested at two separate times during development: When
they were 1 cm to 2 cm high, and when they were 8 cm
to 12 cm high and had formed roots. Table 2 shows the
activity of the ocs element in calli, the leaves of small
shoots, and the leaves of rooted plants. If the construction
was designated as ocs-positive, the pooled leaves contained 20% or more of the octopine synthase activity of
the construction pEN1 in that tissue. ocs-Negative plants
contained 2% or less of the octopine synthase activity of
the construction pEN1 in that tissue. All of the constructs
that are active in calli are also active in the leaves of small
shoots and the leaves of rooted plants except for pPAL16.
This construction is active in calli and the leaves of small
shoots but is inactive in the leaves of rooted plants.
Table 2. Activity of UAS Constructions in Calli, Small Shoots,
and Rooted Plants
~
pENl
pENRl
pAlulO6
pAIRa45
pPALl6
pOCSA2
Activity in
Calli"
Activity in
Leaves
of Small
Shoots"
+
+
+
+
+
+
+
+
4-
-
+
-
Activity in
Leaves
of
Rooted
Plants*
+
+
+
+
-
+ indicates that the pooled plant tissue contained 20% or more
of the octopine synthase activity of the construction pENl in that
tissue. - indicates that the pooled plant tissue contained 2% or
less of the octopine synthase activity of the construction pENl in
that tissue. The activity of the construction pENl in calli, the
leaves of small shoots, and the leaves of rooted plants was 0.83
pmol, 0.17 pmol, and 0.23 pmol of octopine made per hour per
microgram of protein, respectively.
Do Upstream Regions of Other T-DNA Genes That
Have Homology to the Palindrome also Contain
Upstream Activator Sequences That Can Activate the
ocs Promoter?
Because the 16-bp palindrome is an essential motif in the
octopine synthase UAS (Ellis et al., 1987b), we scanned
the 5'-flanking sequences of other T-DNA genes for homology to the palindrome. The 5'-flanking sequences of
both a gene involved in mannopine biosynthesis(mas) and
the gene implicated in octopine and nopaline secretion
(ons) contain sequences homologous to the ocs palindrome. Figure 5A shows schematically the regions containing these sequences homologous to the palindrome
used in this study. Figure 58 shows the DNA sequences
of these regions as determined by Barker et al., 1983 and
DeGreve et al., 1983.
In a previous study of the 5'-flanking sequence of the
mannopine synthase gene, we hypothesizedthe existence
of a transcriptional activating element located between
-31 8 bp and -1 30 bp from the transcription start site
(DiRita and Gelvin, 1987). This region also contains 7
contiguous base pairs homologous to the promoter-dista1
portion of the palindrome (ACGTAAG). To determine
whether this element had the properties of an upstream
activator, we cloned a subfragment of this region from
-318 to -213, containing sequences homologous to the
palindrome, onto the ocs promoter in both orientations.
We subsequently mobilized these constructions into tobacco cells and assayed octopine synthase activity. We
observed octopine synthase activity for both of these
constructions. The mas element activates the ocs gene to
a greater extent in the correct orientation than in the
reverse orientation (Figure 5A). Neither orientation of the
mas element functions as well as does the ocs activator
to stimulate the ocs gene. These results indicate that an
upstream region containing homology to the palindrome
from another T-DNA gene functions as an upstream activator of a heterologous (ocs) promoter. It does not, however, prove that the 7 bp containing homology to the ocs
palindrome are responsible for the activity of the mas
activator.
The 5' end of the ons gene also has some homology (6
bp) to the promoter-dista1 portion of the ocs palindrome
(CGTAAG). We cloned a fragment (-363 bp to -170 bp
upstream from the ATG) from the ons 5'-flanking sequence
onto the ocs promoter. This ons sequence did not activate
the ocs promoter in tobacco calli (Figure 5A).
a
DlSCUSSlON
We have examined the octopine synthase upstream activator sequence in detail. Deletion analysis indicated that
an essential sequence was located between 222 bp and
ocs Upstream Activator Sequence Domains
A
Onwne Relalive
Synlhase Octoptne
AcawIy
Synlhase
(unW
Acln~ly
-
-__
6-- 3 4
m'
2
ACAGGCCMA ITCGCTCTTA G C C G T A C M T ATTACTCACC GGTCCGATGC
EB1:
enr
CCCCCATCGT AGGTGMGGT G G M A T T M T GATCCATCTT GAGACCACAG G C C C A C M C A
............................................................
.......... - - - T T A G T T l CCACGAMGT A T T A T G T T M A T T I T A M K T TTTCGATGTA
CCTACCAGTT T C C T C M G G G TCCACCAAM ACCThAGCGC TTACCIACAT GGTCGATMG
- A C A l T l l T C MATCAGTGC GCMGACGTG W
T
A
T M T G T G G C T A T M T T G T M M A T M A C T A T-TGT
T CTGACCTAGT TTTTATTTTT
CCGTCTTATG TATMTTTGT
A A M C G C M T TTGTAGATCI T M C A T C C M CCTCCCTTTC A G G G A T C - - -
..........
C T A C T M T T T CCTCCTTTAT TTCGGCCTGT ACGACATCCC M C C C G G - - -
..........
C T M A T G T T T M T A T A T A T C ATAGMCGCA A T M A T A T T A MTATAGCGC T T T T A T C M A
.....................................
TATMATACA TCATTACMG TTGTTTATAT TTCGGGT
Figure 5. Activity of Putative Upstream Activator Sequences from
Severa1T-DNA Genes.
(A) Schematic diagram of the regions from several T-DNA genes
containing sequences homologous to the palindrome and their
orientation with respect to the ocs gene. Box with x in it represents the ocs promoter beginning at -116 bp relative to the
transcriptionstart site; arrow with ocs in it represents the octopine
synthase structural gene, and the arrow indicates the direction of
transcription. The rectangle with the thick arrow represents the
different putative upstream activator sequences; the direction of
the arrow indicates the orientation of the activator. The ocs
element extends from -333 bp to -1 16 bp relative to the transcription start site, the mas element extends from -318 bp to
-21 3 bp relativeto the transcriptionstart site, and the ons element
extends from 363 bp to 170 bp upstream from the translation
start site. The picomoles of octopine made during a 2-hr incubation
per microgram of protein and the relative leve1 of octopine synthase activity (normalized to pEN1 as 100%) are indicated as in
Figure 3.
( 8 ) The DNA sequences of upstream regions containing sequences homologous to the palindrome. The ocs element extends
from -333 bp to -1 16 bp upstream from the transcription initiation site. The mas element extends from -318 bp to -213 bp
upstream from the transcription initiation site. The ons element
extends from -363 bp to -1 70 bp upstream from the translation
initiation site. The palindrome homologies, including the palindrome itself for ocs, are underlined. The three sequences are
arranged so that the palindrome homologies are aligned.
931
177 bp upstream from the transcription initiation site. This
region contains a 16-bp palindrome (ACGTAAGCGCTTACGT), between -1 92 and -1 77, that can autonomously
activate the ocs promoter in tobacco calli. This palindrome
can also activate the maize Adhl promoter in transient
expression assays (Ellis et al., 1987b). Many other eukaryotic genes, such as the c-fos gene (Greenberg, Siegfried,
and Ziff, 1987), are also regulated by palindromic sequences. Sequences exhibiting dyad symmetry are believed to interact with dimeric proteins and, in the case of
c-fos, proteins are known to bind to the palindrome. The
ocs palindrome also interacts with a nuclear protein (Singh
et al., 1989).
The ocs palindrome must be intact to function. The
element functions well when present in several locations
from 91 bp to 146 bp from the TATA box (Table 1).
Therefore, it most likely does not need to be on a particular
face of the DNA helix with respect to the TATA box. When
the palindrome is moved greater than 200 bp from the
TATA box, its activity diminishes.
Although the palindrome is the only motif in the ocs
activator that can function autonomously, other sequences
also play a subtle role in modulating the activity of the
palindrome. In tobacco calli, the palindrome (pPAL16) is
about one-thirdas active as is the larger element contained
in pEN1. Because sequences between -222 and -1 77
(pAIRa45) activate the ocs promoter to wild-type levels,
these data indicate that sequences between -222 and
-193 augment the activity of the palindrome. Within this
5' PAS is an 8-bp sequence (CCTCAAGG) that is also
found between the TATA box of the ocs gene and the
transcription initiation site. Whether or not this sequence
plays a role in the regulation of transcription of the ocs
gene remains to be determined.
If the sequences between -222 and -168 (pAITaR54)
are cloned onto the ocs promoter, activity greater than
that of the entire element is observed. In addition to the
palindrome and the 5' PAS, this fragment contains an
intact, potential Z-DNA-forming sequence. A potential ZDNA-forming sequence is an essential part of the nos
upstream activator sequence (An, 1987; Mitra and An,
1989). It is possible that the potential Z-DNA-forming
sequence may mark the DNA backbone in such a way as
to allow proteins to bind to the palindrome more efficiently
(An, 1987). It is also possible that the alternating purine
and pyrimidine tract may not form Z-DNA but the 3' PAS
may also be a protein binding site.
The pSaul33 fragment (-249 to -1 16) activates the
ocs promoter to wild-type levels but pAlulO6 (-222 to
-1 16) directs greater than wild-type activity. These data
suggest that sequences between -249 and -222 may be
inhibitory.To observe greater than wild-type activity, these
putative inhibitory sequences must be removed, but the
3' PAS must also be present in addition to the palindrome.
These data indicate a complex interaction between the
putative 5' inhibitory sequences and the 3' PAS. There-
932
The Plant Cell
fore, the octopine synthase UAS may be regulated by
interactions between both positive and negative elements.
The pea rbcS3A gene and a soybean leghemoglobin gene
are also regulatedby positive and negative elements (Kuhlemeier et al., 1987; Stougaard et al., 1987).
The 16-bp palindrome activates the ocs promoter in
both calli and the leaves of small shoots, but is inactive in
the leaves of rooted plants. These results suggest that the
activity of the palindrome is sensitive to the differentiated
state of the tissue in which it resides. In addition, the larger
the fragment harboring the 16-bp palindrome, the more
active are the constructions in rooted plants. The ST-LS7
gene from potato also requires more 5’-flanking sequence
for expression in differentiated tissues than in calli (Stockhaus et al., 1987). These observations may indicate that
the regulation of gene expression in differentiated tissues
is more complex than in calli.
We have shown that either half of the palindrome by
itself is not able to stimulate the ocs promoter. This result
is interesting because the promoter-dista1 7 bp of the
palindrome (ACGTAAG) is found within the 5’-flanking
sequences of severa1 other genes, such as the cauliflower
mosaic virus 35s promoter, the potato protease inhibitor
II gene, the soybean lectin Lel gene, and the mas and ons
genes (Barker et al., 1983; Vodkin, Rhodes, and Goldberg,
1983; Odell, Nagy, and Chua, 1985; Thornburg et al.,
1987),that are expressed in plants. This sequence is found
within a portion of the mannopine synthase (mas) upstream activating sequence essential for activity of the
mas promoter (Figure 5). A 100-bp fragment of the mas
upstream region harboring the ACGTAAG sequence can,
in either orientation, stimulate an ocs promoter. The situation with the ocs UAS may be similar to that of the serum
response element of the c-fos gene. The essential components of both the ocs UAS and the c-fos serum regulatory elements are palindromes. Neither of the two activates
its own promoters if only half of the palindrome is present,
whereas half of the palindrome sequence can be found in
essential upstream regions of other genes (Elsholtz et al.,
1986; Greenberg, Siegfried, and Ziff, 1987). It is also
possible, however, that the presence of sequences homologous to a portion of the ocs palindrome is fortuitous
and that these sequences play no role in the activity of the
other genes.
We tested an upstream portion from the ons gene for
its ability to stimulate the ocs promoter. This fragment also
has homology to a portion of the palindrome (CGTAAG).
The ons fragment does not activate the ocs gene in either
orientation. These data suggest that the ACGTAAG sequence, if important in activating the mas and ons genes,
may need to be within the proper sequence context in
order to augment the ocs promoter. It is also possible that
the ons upstream fragment does not interact synergistically with the ocs promoter. Although the ocs element can
activate the maize Adh 7 promoter in transient expression
assays, it cannot activate the 780 gene promoter in sunflower tumors (Ellis et al., 1987a, 1987b; Bruce, Bandyopadhyay, and Gurley, 1988). These data indicate that not
all promoter-activator pairs function in a productive manner. This has also been shown for the SV40 enhancer;
although it stimulates the ,8-globin promoter by at least
100-fold, it cannot stimulate the a-globin promoter (Khoury
and Gruss, 1983).
In a previous study of the ocs element, Ellis et al. (1987b)
indicated that the 16-bp palindrome was sufficient for
activity in transient expression assays. These authors
tested the effects of ocs activator fragments on the maize
Adh7 promoter using chloramphenicol acetyltransferase
as a reporter of gene activity. We studied the effects of
ocs activator fragments on the ocs promoter in stable
expression experiments using the ocs gene as a reporter
of gene activity. Ellis et al. (1987b) did not test the role of
sequences flanking the palindrome, nor the ability of the
palindrome to function in rooted plants. We have found
that sequences flanking the palindrome play a subtle role
in the stable expression of the ocs gene in tobacco calli.
The palindrome is also not sufficient for expression in the
leaves of rooted plants. We are currently analyzing the
various ocs activator fragments for tissue-specificexpression and determining at which stage of development they
function or are inactive.
METHODS
Materials
Restriction endonucleases were purchased from Bethesda Research Laboratories and used according to the manufacturer’s
specifications. DNA polymerase I Klenow fragment and T4 DNA
ligase were obtained from Pharmacia LKB Biotechnology, Inc.
and used according to the manufacturer’s specifications. o~-~*PdCTP was purchased from Amersham. DNA fragments were
labeled with an Amersham nick translation kit. Reagents for the
octopine synthase assay and antibiotics were purchased from
Sigma. Reagents for determining protein concentrations were
purchased from Bio-Rad and used according to the manufacturer’s specifications.
Strains and Culture Conditions
fscherichia coli strains were grown in LB medium (Maniatis,
Fritsch, and Sambrook, 1982). Agrobacterium strain LBA4404
(Hoekema et al., 1983) was grown in AB minimal medium (Lichtenstein and Draper, 1986). Antibiotic concentrations,when used,
were for E. coli: kanamycin, 50 pg/mL; ampicillin, 100 gg/mL; for
Agrobacterium tumefaciens: kanamycin, 100 gg/mL; rifampicin,
1 O pg/mL. These experimentswere conducted under P1 containment conditions as specified by the National lnstitutes of Health
Recombinant DNA Guidelines.
ocs Upstream Activator Sequence Domains
Construction of Plasmids Harboring Deletions of the
Octopine Synthase Activator
All plasmids were constructed using basic recombinant DNA
techniques (Maniatis, Fritsch, and Sambrook, 1982). We have
previously described the construction of the plasmids pEN1,
pENRl , pSaul33, pAlulO6, pRsa61, pSau84, and pOCSA2 (Leisner and Gelvin, 1988). pAluRlO6 was constructed by cleaving
pAlulO6 with BamHI, religating the fragments, and screening
colonies for the insert in the reverse orientation. All of the other
ocs activator subfragments except the synthetic oligonucleotides
were derived from either pSLl , pG1, pAl , or pX1, as described
in Figure 2. pUX13 was constructed by converting the Smal site
of pUCl3 to Xhol by linker addition(Figure 2C). We have described
the construction of pSLl previously (Leisner and Gelvin, 1988).
The BamHl to Sau3a fragment from pSL1 (Figure 2A) was cloned
into the BamHl site of pUX13 (Figure 2C), resulting in the plasmid
pG1 (Figure 28). pAl (Figure 2B) was constructed by converting
the Alul site of the Alul to BamHl fragment from pSLl (Figure 2A)
to BamHl by linker addition. This fragment was subsequently
cloned into the BamHl site of pUC13. The BamHl insert of pSLl
(Figure 2A) was cleaved with HinPl , the fragment ends were filled
in with Klenow fragment and converted to Xhol sites by linker
addition. This fragment was then cloned into the Xhol site of
pUX13 (Figure 2C), resulting in pX1 (Figure 28).
The BamHl insert of pAl (Figure 28) was cleaved with HinPl ,
the fragment ends were filled in with Klenow fragment, and the
HinPl sites of both fragments convertedto Bglll by linker addition.
Both halves of the pAl fragment were subsequently cloned into
the BamHl site of pOCSA2 (Figure 2C), to yield pHinR70 and
pHinR38 (Figure 3). The BamHl insert of pAl (Figure 28) was
cleaved with HinPl , the ends were filled in with Klenow fragment,
and the HinPl site converted to Xhol by linker addition. This 70bp fragment was cloned into the BamHl and Xhol sites of pUX13
(Figure 2C). The resulting plasmid was cleaved with BamHl and
Xhol. and the 70-bp fragment was cloned into the BamHl and Sal1
sites of pOCSA2 (Figure 2C), yielding pHin70 (Figure 3). The Xhol
insert of pX1 (Figure 2B) was cleaved with Alul, and the 38-bp
fragment was cloned into the Hincll and Xhol sites of pUX13
(Figure 2C). The resulting plasmid was cleaved with Hindlll and
Xhol, and the 38-bp fragment was cloned into the Hindlll and Sal1
sites of pOCSA2 (Figure 2C), yielding pHin38 (Figure 3). The Xhol
insert of pX1 (Figure 28) was cleaved with Sau3a, and the 69-bp
fragment was cloned into the Xhol and BamHl sites of pUX13
(Figure 2C). The resulting plasmid was cleaved with Hindlll and
Xhol, and the 69-bp fragment was cloned into the Hindlll and Sal1
sites of pOCSA2 (Figure 2C), resulting in pSaHi69 (Figure 3).
The BamHl insert of pSLl (Figure 2A) was cleaved with Rsal,
and the 133-bp fragment was cloned into the Hincll site of pUXl3
(Figure 2C). The resulting plasmids were screened for the Rsal
fragment in both orientations. A plasmid harboring the Rsal fragment in the correct orientation and a plasmid harboring the Rsal
fragment in the reverse orientation were cleaved with Hindlll and
BamHI, and the 131-bp fragments were cloned separately into
the Hindlll and BamHl sites of pOCSA2 (Figure 2C), resulting in
pRsal31 and pRsaR131 (Figure 3). respectively. The BamHl
insert of pAl (Figure 28) was cleaved with Taql, and the 54-bp
fragment was cloned into the BamHl and Accl sites of pUX13
(Figure 2C). The resulting plasmid was cleaved with Hindlll and
BamHI, and the 54-bp fragment was cloned into the Hindlll and
933
BamHl sites of pOCSA2 (Figure 2C), resultingin pAITaR54 (Figure
3). The BamHl insert of pAl (Figure 28) was cleaved with Rsal,
Xhol linkers were attached to the Rsal site, and the 45-bp fragment was cloned into the BamHl and Xhol sites of pUX13 (Figure
2C). The resulting plasmid was cleaved with either Hindlll and
Xhol, or BamHl and Xhol. These fragments were cloned separately into the Hindlll and Sall, or the BamHl and Sal1 sites of
pOCSA2 (Figure 2C), generating the plasmids pAIRa45 and
pAIRaR45 (Figure 3), respectively.
The plasmid pSauR84 (Figure 3) was constructed by cutting
the plasmid pG1 (Figure 28) with BamHl and Xhol, and cloning
the 84-bp fragment into the BamHl and Sal1 sites of pOCSA2
(Figure 2C). The plasmid pG1 was cleaved with Hindlll and Xhol,
and the 84-bp fragment was cloned into the Hindlll and Sal1 sites
of pAIRa45 and pRsa61 (Figure 3), yielding pSAl and pSRl
(Figure 3), respectively. The BamHl to Xhol activator fragment of
pG1 (Figure 2B) was cleaved with Mspl, and both halves were
purified by preparative gel electrophoresis (Maniatis, Fritsch, and
Sambrook, 1982). The Mspl fragment end was filled in with
Klenow fragment, converted into an Xhol site by linker addition
for the promoter-dista1half of the pG1 (Figure 28) fragment, or to
BamHl for the promoter-proximalhalf, and the resulting BamHl to
Xhol fragments were cloned individually into the BamHl and Xhol
sites of pUX13 (Figure 2C). The resulting plasmids harboring the
promoter-dista1 and promoter-proximal halves of pG1 were
cleaved with Hindlll and Xhol, and the excised fragments were
subsequently cloned separately into the Hindlll and Sal1 sites of
pOCSA2 (Figure 2C), resulting in pMsp42 and pMpSa42 (Figure
3), respectively.
Construction of Plasmids Harboring Oligonucleotides
Five different oligonucleotides were synthesized and cloned into
pOCSA2 (Figure 2C).
The oligonucleotides were as follows:
(1) 5’-TCGAACGTAAG-3 ’
3 ’ - TGCATTCCTAG- 5 ’
(2)
(3) 5’-TCGACTTACGT- 3 ‘
3 ’ - GAATGCACTAG- 5 ’
(4)
(5) 5’-GATCACGTAAGCGCTTACGT- 3 ‘
(5)
3 ‘ - TGCATTCGCGAATGCACTAG-5’
Oligonucleotide pair 1 and 2 was cloned into the Sal1 and BamHl
sites of pOCSA2 (Figure 2C). This resulted in the loss of the Sal1
and Xbal sites but retentionof the BamHl site. These sites allowed
us to demonstrate the presence of the oligonucleotides in the
new plasmid pHPPD (Figure 3). The oligonucleotide pair 3 and 4
was also cloned into the Sal1 and BamHl sites of pOCSA2 (Figure
2C), resulting in the loss of the BamHl and Xbal sites but the
retention of the Sal1 site. These sites allowed us to demonstrate
the presence of the oligonucleotides in the resulting plasmid
pHPPP (Figure 3). Oligonucleotide number 5 was annealed to
itself and cloned into the BamHl site of pOCSA2 (Figure 2C), thus
destroying the BamHl site. pOCSA2 derivatives that did not cut
with BamHl (and possibly contained the oligonucleotide) were
digested with Xbal, the fragment ends filled in with Klenow fragment and d2P-dCTP, and the fragments cleaved with Dral.
pOCSA2 was treated in the same manner. The resulting labeled
DNA was subjected to electrophoresis through a 4% polyacryl-
934
The Plant Cell
amide gel, and the gel was autoradiographed. pOCSA2 (Figure
2C) yielded a 100-bp labeled Xbal to Dral fragment, but pOCSA2
containing the oligonucleotide yielded a 121-bp fragment. A plasmid containing the 121-bp Xbal to Dral fragment was designated
pPALl6 (Figure 3).
Constructionof Plasmids Harboring Upstream Fragments
from the mas and ons Genes
The upstream region of the mas gene, -31 8 to -213 relative to
the transcription initiation site [base pairs 20513 to 20407 (Barker
et al., 1983)] was isolated from the plasmid pKan2-318 (DiRita
and Gelvin, 1987) by digestion with Xhol and Haelll, the ends
were filled in with Klenow fragment, and Xhol linkers were a!
tached. The resulting fragment was cloned into the Sal1 site of
pOCSA2 in both orientations, yielding pMASl and pMASRl
shown in Figure 5A. BamHl fragment 17 [base pairs 13774 to
9062 (Barker et al., 1983)] was cleaved with Ddel and Rsal [base
pairs 9634 to 9836 (Barker et al., 1983)l. This fragment contains
the upstream region of the ons gene from 363 bp to 170 bp
upstream of the translation start site. (The transcription start site
of this gene is not known.) The ends of this 203-bp fragment were
filled in with Klenow fragment, and this fragment was cloned into
the Hincll site of pUX13, resulting in the plasmid p03. pONSRl
(Figure 5A) was constructed by cleaving p03 with Hindlll and
BamHI, and cloning the 203-bp fragment into the Hindlll and
BamHl sites of pOCSA2. p03 was also cleaved with Hindlll, the
fragment ends were filled in with Klenow fragment, and the Hindlll
site was converted to Bglll by linker addition. The resulting plasmid, called p03', was cleaved with Bglll and Xhol, and the 203bp fragment was cloned into the BamHl and Sal1 sites of pOCSA2,
resulting in pONS1 (Figure 5A).
Conjugation of Plasmids into A. tumefaciens
Plasmids were mobilized into A. tumefaciens strain LBA4404 by
triparental mating (Bevan, 1984) using the E. coli strain harboring
the recombinant plasmid, E. coli strain MM294/pRK2013 (Ditta et
al., 1980), and A. tumefaciens strain LBA4404. Agrobacterium
transconjugants were selected on AB minimal plates containing
0.5% glucose, 1O pg/mL rifampicin, and 1O0 pg/mL kanamycin.
lnfectionof Plants, Octopine Synthase Assays, and RNA Gel
Blot Analysis
Tobacco leaf discs were infected with the Agrobacterium strains
harboring recombinant plasmids (Horsch et al., 1985), and kanamycin-resistant calli were assayed for octopine synthase activity
(Otten and Schilperoort, 1978) with the following modifications.
Twenty different calli of equal weight were pooled for each construction and were ground in a small mortar and pestle with
grinding buffer (Otten and Schilperoort, 1978). The tissue extract
was centrifuged at 1800 x g (4000 rpm in a SA600 rotor) for 5
min. The supernatant solution was transferred to a 1.5-mL microcentrifuge tube and centrifuged in a microcentrifuge for 10 sec.
The supernatant solution was then divided into two separate
microcentrifuge tubes. One tube was used to determine the
protein concentration (Bradford, 1976). Ten microliters of the
supernatant solution from the other tube was mixed with 10 pL
of reaction mix containing arginine, pyruvate, and NADH (Otten
and Schilperoort, 1978). Eight microliters of this mixture was
immediately spotted onto a sheet of Whatman 3MM paper, and a
second 8-pL sample was spotted after 2 hr of incubationat 25OC.
The papers were dried, subjected to electrophoresis, and stained
as previously described (Leisner and Gelvin, 1988). The papers
were photographed using Polaroid type 55 film at an exposure
time of 4 min. The negative was prepared according to instructions
from Polaroid. The negative was scanned with the densitometer
attachment of a Beckman mcdel DU8 spectrophotometer, the
peaks cut out of the chart recorder paper, and weighed. The
amount of octopine in each spot was determined by comparison
with known octopine standards, and this value was normalized to
the protein concentrationof the original tissue extract. The octopine synthase assay is linear in the protein concentration range
utilized, and the intensity of the image on the photograph is within
the linear response range (data not shown). One unit of octopine
synthase activity is equivalent to 0.83 pmol of octopine made per
hour per microgram protein. Assay samples contained approximately 15 pg of protein. Seven different pools of calli were tested
for the construction pENl and the variation was minimal: the
average was 89.9 f 13.2 units. Only numbers greater than 2 SD
from the mean (i.e., <64 or >116) were considered as significantly
different from the activity of pENl . Calli containing pENl synthesized approximately 2500,pmol of octopine per assay. The value
for pENl was arbitrarily set at 100%. For transgenic plants and
shoots, the leaves from 1O plants harboring the same construction
were pooled and tested for octopine synthase activity as described above. The second leaf from the top of the 2-cm shoots
was pooled, and leaf discs were cut out by a No. 6 size cork
borer from the rooted (8 cm to 12 cm tall) plants. Octopine
synthase-positive plants contained 20% or more of the octopine
synthase activity of the construction pENl in that tissue. Octopine
synthase-negative plants contained 2% or less of the octopine
synthase activity of the construction pENl in that tissue. RNA
was isolated from 20 pooled calli for each construction, and RNA
gel blot analysis was done as described by Karcher, DiRita, and
Gelvin (1984). The autoradiogram was scanned with a
densitometer.
ACKNOWLEDGMENTS
We thank Marlaya Wyncott, Lillian Habeck, and Yu-Mei Wang for
their technical assistance; Dr. Avtar Handa and Denise Tieman
for assistance with the densitometer; Brad Goodner for critical
reading of the manuscript; and Wilma Foust for help in the
preparationof the manuscript.This work was supported by grants
from the U.S. Department of Agriculture (87-CRCR-1-2443) and
a National Science Foundation Presidential Young lnvestigator
Award (DMB8351152) to S.B.G., with matching funds from Agrigenetics Research Associates. S.M.L. held a Proctor and Gamble
Research Fellowship during part of the course of this work.
Received May 19, 1989; revised July 17, 1989.
ocs Upstream Activator Sequence Domains
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Plant Cell 1989;1;925-936
DOI 10.1105/tpc.1.9.925
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