Expression motifs that confer tissue and development-specific expression in plants

The present invention relates to the use of DNA sequence motifs to regulate gene expression in a tissue- or developmental-specific manner in transgenic plants. The invention generally relates to the engineering and use of G-box related sequence motifs, specifically Iwt and PA motifs, which function as cis-elements of promoters, to regulate the expression of heterologous genes in transgenic plants. PA enhances high level expression in roots, low level expression in leaves and little or no expression in seeds. By contrast, Iwt confers preferential expression in seeds, but in a developmentally-regulated manner.

TABLE OF CONTENTS 
1. Introduction 
2. Background Of The Invention 
2.1 Manipulation Of Plant Promoters 
2.2 G-Box Elements 
2.3 TAF-1 Transactivating Factor 
3. Summary Of Invention 
3.1 Definitions 
4. Description Of The Figures 
5. Detailed Description Of The Invention 
5.1 PA and Iwt Motifs 
5.2 PA or Iwt Motif Modified Promoters 
5.3 Engineering A Gene Of Interest Controlled By The Modified Promoters 
5.4 Production Of Transgenic Plants And Plant Cells 
5.5 Expression Of Target Gene Products In Transgenic Plants 
6. Example: Tissue-Specific And Developmentally Regulated Expression Of 
Target Gene Products In Transgenic Tobacco 
6.1 Materials And Methods 
6.1.1 G-Box-Related Constructs 
6.1.2 Transgenic Plants 
6.1.3 Histochemical Staining 
6.1.4 Gus Enzyme Assays 
6.1.5 Northern Analysis 
6.2 Results 
6.2.1 Engineered Transgenic Plants 
6.2.2 Expression Of Target Gene Product In Mature Plants 
6.2.3 Expression Of Target Gene Produced During Seed Development 
6.2.4 Expression Of Target Gene Product In Young Seedlings 
6.2.5 Expression Pattern Of TAF-1 mRNA 
1. INTRODUCTION 
The present invention relates to the use of two G-box related nucleotide 
sequences to direct tissue-specific and developmentally-regulated gene 
expression of target gene products in transgenic plants. 
2. BACKGROUND OF THE INVENTION 
One of the important challenges in genetic engineering of plants is 
constructing novel promoters that have precisely tailored tissue-specific 
or developmentally-regulated activities. The availability of tailored 
promoters would enable the expression of engineered traits in only the 
desired tissue or organ locations, or in particular developmental stages. 
Such exact control of transgene expression is highly desirable to enhance 
the biological efficiency and performance predictability of trangenic 
plants. However, attempts to engineer such systems have been met with 
limited success. 
2.1 Manipulation of Plant Promoters 
Plant RNA polymerase II promoters, like those of other higher eukaryotes, 
have complex structures and are comprised of several distinct elements. 
One essential element is the TATA box. It determines the transcription 
initiation site and is typically located -35 to -25 basepairs (bp) 
upstream of the initiation site, which is defined as position +1 
(Breathnack and Chambon, 1981, Ann Rev. Biochem. 50: 349-383; Messing et 
al., 1983, In: Genetic Engineering of Plants, Kosuge, Meredith and 
Hollaender, (eds.), pp. 211-227). Another common element is located 
between -70 and -100 bp upstream and has the consensus sequence CCAAT. In 
plants, the CCAAT box may have a different consensus sequence and has been 
termed the AGGA box. (Messing et al. ibid). Besides TATA and CCAAT boxes, 
virtually all eukaryotic promoters studied to date contain additional 
upstream DNA sequences that regulate promoter activity (Benoist and 
Chambon, 1981, Nature 290: 304-310; Gruss et al., 1981, Proc. Nat. Acad. 
Sci USA 78: 943-947; Khoury and Gruss, 1983, Cell 27: 313-314.) These 
upstream regulatory sequences are variously known as enhancers or upstream 
activating sequences. Such sequences are variable in length and may extend 
from around -100 bp to 1,000 bp or more 5' upstream of the transcription 
initiation site. 
Early attempts in developing novel plant promoters involved recombining 
regulatory sequences of one promoter with parts of another promoter (Fluhr 
et al., 1986, Science 232: 1106-1112; Ellis et al., 1987, EMBO J. 6: 
11-16). Such constructs involved adding a heterologous regulatory sequence 
to an active promoter with its own partial or complete regulatory 
sequences (Ellis et al., id.; Strittmatter and Chua, 1987, Proc. Nat. 
Acad. Sci. USA 84: 8986-8990; Poulsen and Chua, 1988, Mol. Gen. Genet. 
214: 16-23; Comai et al., 1991, Plant Molec. Biol. 15: 373-381). 
Alternatively, modified promoters have also been developed by adding a 
heterologous regulatory sequence to the 5' upstream region of an inactive, 
truncated promoter, i.e. a promoter that spans only the core TATA and, 
sometimes, the CCAAT elements, (Fluhr et al., 1986, ibid; Strittmatter and 
Chua, 1987, Proc. Nat. Acad. Sci. USA 84: 8986-8990; Aryan et al., 1991, 
Mol. Gen. Genet. 225: 65-71). While such recombinations can produce 
promoters with novel activities, they generally have not been particularly 
useful in producing tailored promoters with precisely controlled or 
limited activities. That is because most the known regulatory sequences 
conferred manifold complex tissue-, developmental- or environmental 
activities. 
Dissection of plant regulatory sequences have shown that they are typically 
comprised multiple distinct "cis-elements" each appearing to confer a 
different segment of the original spectrum of activity (See Strittmatter 
and Chua, ibid; Ellis et al., ibid; Benfey et al., 1990, EMBO J 9: 
1677-1684). The molecular basis of such activity seemed to reside in a 
cis-element's ability to bind trans-acting protein factors that regulate 
transcription initiation. Some cis-elements may bind with more than one 
factor, and these factors, in turn, may interact with different affinities 
with more than one cis-element (Johnson, P. F. and McKnight, S. L., 1989, 
Ann. Rev. Biochem. 58: 799-839). Despite the appreciation of cis-element's 
role in regulating transcription, no general understanding has emerged on 
what exactly comprises a cis-element or how cis-elements can be used, in 
isolated forms, to modify active promoters or construct novel promoters. 
2.2 G-Box Elements 
In plants, the G-box element or sequence motif exemplifies the promiscuity 
of certain DNA sequences in their interaction with multiple binding 
proteins. This element was first identified as an 11 bp sequence (5' 
C/A-ACACGTGGCA 3') (SEQ. ID NO:1) located upstream of many genes encoding 
the small subunit of ribulose biphosphate carboxylase (Giuliano et al., 
1988, Proc. Natl. Acad. Sci. USA 85: 7089-7093). Since then, cis-elements 
comprised of G-box or related sequence motifs with the CACGTG 
hexanucleotide core have been identified in the promoters of a diverse set 
of unrelated genes, including those controlled by visible and ultraviolet 
light (Schulze-Lefert et al., 1989, EMBO J. 8: 651-656), abscisic acid 
(ABA) (Guiltinan et al., 1990, Science 250: 267-271), wounding (Rosahl et 
al., 1986, Mol. Gen. Genet. 203: 214-220) or, anaerobiosis (DeLisle & 
Ferl, 1990, Plant Cell 2: 547-557. Recent data indicate that plant nuclear 
extracts contain a number of binding activities with specificity for G-box 
and related sequences (Staiger et al., 1991, Eur. J. Biochem. 199: 
519-527; Williams et al., 1992, Plant Cell 4: 485-496). Indeed, several 
cDNA clones encoding proteins that specifically interact with cis-elements 
containing the CACGTG core have been isolated: wheat EmBP-1 (Guiltinan et 
al., 1990, Science 250: 267-271), wheat HBP-la (Tabata et al., 1991, EMBO 
J. 10: 1459-1467), tobacco TAF-1 (Oeda et al., 1991, EMBO J. 10: 
1793-1802), parsley CPRF-1, 2, 3 (Weisshaar et al., 1991, EMBO J. 10: 
1777-1786, and Arabidopsis GBF-1, 2, 3 (Schindler et al., 1992, EMBO J. 
11: 1261-1273). Interestingly, all these proteins belong to the bZIP class 
of transcription factors, i.e., they possess a basic domain abutting a 
leucine repeat. It has been shown that bZIP proteins bind as dimers to 
their target sites (cf. Johnson & McKnight, 1989, Annu. Rev. Biochem. 58: 
799-839). 
2.3 TAF-1 Transactivating Factor 
The TAF-1 cDNA clone was isolated from a tobacco cDNA expression library by 
screening for proteins that show affinity for motif I (Iwt, 5' GTACGTGGCG 
3') (SEQ. ID NO:2), a conserved sequence found in promoters of different 
ABA-responsive genes (see Skriver & Mundy, 1990, Plant Cell 2: 503-512 for 
a review). Because TAF-1 is a bZIP protein and because Iwt contains a 
G-box related core sequence TACGTG, the question arose whether the 
preferred binding site of TAF-1 might be the perfect palindrome PA, 5' 
GCCACGTGGC 3' (SEQ. ID NO:3) which contains the G-box hexameric core 
sequence CACGTG. Experiments showed that, indeed, TAF-1 binds to PA with a 
higher affinity (about 70 times) than to Iwt (Oeda et al., 1991, EMBO J. 
10: 1793-1802). 
The finding that TAF-1 can bind in vitro to two related sequence motifs, PA 
and Iwt, although with different affinities, poses interesting questions 
regarding binding and transcription activation in vivo. Transient 
expression of the TAF-1 gene in tobacco leaves showed that the factor is 
capable of activating the expression of Iwt motif containing promoters. 
Further, Northern blot analysis demonstrated that TAF-1 was highly 
expressed in roots but poorly expressed in leaves and stems (Oeda et al., 
1991, EMBO J. 10: 1793-1802). Thus, in the simplest case, if both motifs 
interact with TAF-1 in vivo, then they should confer an expression pattern 
similar to that exhibited by TAF-1 expression; however, their activities 
in the expressing tissues may differ quantitatively, reflecting in some 
way their different binding affinity for the factor. Alternatively, if the 
motifs interact with different factors in vivo, then different expression 
profiles would be expected. Other more complicated scenarios are also 
possible. 
The potential roles of Iwt and PA motifs in regulating gene expression 
remain to be determined. The presence of Iwt sequences as G-box related 
motifs in the 5' upstream regions of many plant promoters suggests that 
the Iwt motif might function as a cis-element in regulating transcription. 
A possible cis-element role for the PA motif is less apparent, since PA is 
an artificial derivative of the Iwt motif and is not known to be present 
in the regulatory regions of plant genes. Further, although TAF-1 
activates Iwt motif containing promoters, it is not known what other 
transcriptional factors activate promoters that contain either the Iwt or 
PA motif. 
3. SUMMARY OF INVENTION 
The present invention relates to the use of DNA sequence motifs to regulate 
gene expression in a tissue- or developmental-specific manner in 
transgenic plants. The invention generally relates to the engineering and 
use of G-box related sequence motifs, specifically Iwt and PA motifs, 
which function as cis-elements of promoters, to regulate the expression of 
heterologous genes in transgenic plants. PA enhances high level expression 
in roots, low level expression in leaves and little or no expression in 
seeds. By contrast, Iwt confers preferential expression in seeds, but in a 
developmentally-regulated manner. 
In accordance with the present invention, the Iwt or PA motif may be used 
in monomeric or various multimeric forms to modify promoters. Further, 
each motif may also be used singly or multiply, and in combinations with 
other motifs, to produce novel cis-elements with a specifically tailored 
spectrum of tissue- or developmental-specificities. 
Promoters modified with Iwt or PA motif have a variety of uses, including, 
but not limited to, expressing or overexpressing proteins, anti-sense 
RNAs, and ribozymes in plant cell expression systems, in plant cultures, 
or in stably transformed plants. The gene expression under control of such 
modified promoters may be induced by hormone treatments, developmental 
processes, or differentiation processes. 
The use of the PA- or Iwt-motif modified promoters may have particular 
value in engineering agronomically important plants. Such recombinant 
promoters would enable precise control of phenotypic traits in engineered 
plants and may be beneficially used to produce transgenic plants that have 
enhanced nutritional value; that have modified differentiation and 
development programs; or are resistant to a variety of factors including 
pests, pathogens, adverse environmental conditions or chemicals. 
The invention is based, in part, on the surprising finding that the closely 
related Iwt and PA motifs, which differ in only two out of ten base-pairs, 
confer dramatically different tissue- and developmental-specificities to 
the promoters they modify. Iwt tetramers confer embryo-specific 
expression, whereas PA tetramers confer high level root expression, low 
level leaf expression, and no seed expression. The invention is 
illustrated herein by way of working examples in which the GUS reporter 
gene driven by Iwt- or PA-modified promoters were engineered into 
transgenic tobacco plants. GUS activity in different tissues and at 
different stages of plant development was analyzed quantitatively and 
qualitatively. The expression pattern of TAF-1 mRNA in different tissues 
of tobacco plants was also analyzed. These results demonstate that Iwt 
promotes expression in developing and mature seeds whereas PA confers 
preferential expression in roots. The results also demonstrate that TAF-1 
mRNA is not expressed in seeds, although it is highly expressed in roots. 
On the basis of the foregoing results, interactions of TAF-1 with PA and 
Iwt sequences for regulating gene expression are proposed. 
3.1 Definitions 
The terms listed below, as used herein, will have the meaning indicated. 
CaMV=Cauliflower Mosaic Virus 
cDNA=complementary DNA 
DAF=days after fertilization 
DNA=deoxyribonucleic acid 
GUS=1,3 .beta.-Glucuronidase 
MU=5' TGACTGTTCT 3' (SEQ. ID NO:4) 
PA=5' GCCACGTGGC 3' (SEQ. ID NO:3) 
PCR=polymerase chain reaction 
PEG=polyethylene glycol 
polyA=polyadenylated 
rbcS=ribulose bisphosphate carboxylase small subunit 
RNA=ribonucleic acid 
Iwt=5' GTACGTGGCG 3' (SEQ. ID NO:2)

5. DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the use of two closely related DNA 
sequence motifs that confer very different developmental- and 
tissue-specific expression patterns on plant promoters. The invention is 
illustrated by working examples involving the design of transgenic tobacco 
plants containing recombinant DNA sequences comprised of tetramers of Iwt 
or PA sequence motifs regulating the expression of a truncated promoter 
(-90 35S promoter). The perfect palindromic PA motif conferred on the 
chimeric gene construct high level expression in roots and low level 
expression in leaves and no expression in seeds. In contrast, the wild 
type Iwt motif conferred on the chimeric gene construct 
developmental-specific expression in seed tissues. 
5.1 PA and Iwt Motifs 
PA (SEQ. ID NO:3) and Iwt (SEQ. ID NO:2) motifs and multimers of these 
motifs, described in FIG. 1, may be obtained by chemical synthesis or by 
cloning from recombinant DNA constructs or appropriate cellular source 
containing such motifs or multimers. For example, chemical synthesis 
methods well known to those skilled in the art can be used to synthesize 
the motifs and their multimers reported in FIG. 1. See, for example, 
Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7: 215-233; Chow and 
Kempe, 1981, Nuc. Acids Res. 9: 2807-2817. These sequences may be 
synthesized with additional flanking sequences that contain appropriate 
and useful restriction enzyme sites for facilitating subsequent 
manipulations of these motifs containing sequences. 
Alternatively, synthetic DNA sequences that are partially or wholly 
homologous to the desired motif or multimer may be used as hybridization 
probes in cloning DNA fragments containing such motifs or multimers from 
appropriate genomic sources. The cloned fragments are then mapped and 
sequenced to pin-point the locations of the motif-containing sequences. 
Smaller fragments containing such sequences are then subcloned and further 
modified as desired. The recombinant DNA methods for doing all of the 
aforementioned steps of isolation, characterization, and manipulation are 
well known to those skilled in the art and may be found in reference 
sources such as Sambrook et al., 1989, Molecular Cloning, A Laboratory 
Manual, Cold Spring Harbor Laboratory Press, Plainview, N.Y. 
Alternatively, synthetic DNA sequences that are partially or wholly 
homologous to the desired motif or multimer may also be used as primers in 
polymerase chain reactions (PCR) to amplify and isolate motif-containing 
sequences from any appropriate DNA source. This approach may also be used 
to modify such motif-containing sequences before amplification and 
isolation, or to construct the desired motif-containing sequence de novo, 
based entirely on the use of overlapping primers. Protocols for executing 
all of the aforementioned PCR procedures are well known to those skill in 
the art, and may be found in reference sources such as Gelfand, 1989, PCR 
Technology, Principles and Applications for DNA Amplification, (Ed.), H. 
A. Erlich, Stockton Press, N.Y., and Current Protocols In Molecular 
Biology, Vol. 2, Ch. 15, Eds., Ausubel et al., John Wiley & Sones, 1988. 
Sequences containing motifs and multimers isolated using any of the 
aforementioned approaches may be cloned into convenient bacterial or viral 
vectors including, but not limited to, plasmids, cosmids, or phages to 
facilitate any further desired manipulations. Such manipulations of the 
motif-containing or multimer-containing fragment may include mutagenesis, 
addition or removal of sequences. For example, useful alterations may 
include the insertion of other cis-element sequences, sequence motifs, or 
restriction enzyme sites; conversely the alterations may include the 
removal of such sequences and sites. 
5.2 PA or Iwt Motif Modified Promoters 
The PA (SEQ. ID NO:3) and Iwt (SEQ. ID NO:2) motifs of the invention may be 
used in either monomeric or multimeric form to modify any promoter. A 
motif or a multimet of the motif, isolated or constructed as described 
above, can be attached to the 5'-end of a promoter fragment using 
recombinant DNA methods well know to those skilled in the art. The 
modified promoter may be a truncated promoter that has been deleted for 
parts or the whole of sequences 50 basepairs or more 5'upstream of the 
transcription initiation site; such truncated promoters may or may not 
retain the AGGA box. Alternatively, the promoter to be modified may be an 
intact promoter retaining most, if not all, of its upstream enhancer or 
regulatory sequences. 
The recombinant DNA methods for making the PA- or Iwt-modified promoter 
constructs are well known to those skill in the art (e.g., see Sambrook et 
al., 1989, Molecular Cloning A Laboratory Manual. Cold Spring Harbor 
Press). For example, the transcription initiation site of the cloned 
promoter to be modified can be mapped by methods such as primer extension, 
RNA/S1 nuclease protection or other equivalent methods. Thereafter, 
restriction enzyme sites can be constructed at the desired 5' upstream 
location of the promoter fragment using methods such as in vitro 
mutagenesis or PCR amplification. Alternatively, such sites may be 
introduced by resectioning the promoter fragment with Bal31 exonuclease or 
a 5'-3' exonuclease/mung bean nuclease mix, followed by ligating a 
oligonucleotide linker containing the desired restriction enzyme sites. 
Finally, the desired motif or motif multimer can be cloned into the new 5' 
upstream restriction enzyme sites. 
In a preferred embodiment of the present invention, a tetramer of the PA or 
Iwt motif is attached to the 5'-end of a truncated plant promoter. Such 
truncated promoters typically comprise of sequences starting at or about 
the transcription initiation site and extending to no more than 150 bp 5' 
upstream. These truncated promoters generally are inactive or are only 
minimally active. Examples of such truncated promoters may include, among 
others, a "minimal" CaMV 35S promoter whose 5' end terminates at position 
-46 bp with respect to the transcription initiation site (Skriver et al., 
Proc. Nat. Acad. Sci. USA 88: 7266-7270); the truncated "-90 35S" promoter 
in the X-GUS-90 vector (Benfy and Chua, 1989, Science 244: 174-181); a 
truncated "-101 nos" promoter derived from the nopaline synthase promoter 
(Aryan et al., 1991, Mol. Gen. Genet. 225: 65-71); and the truncated maize 
Adh-1 promoter in pADcat 2 (Ellis et al., 1987, EMBO J. 6: 11-16). 
In another preferred embodiment of the present invention, the Iwt or PA 
motif tetramer is attached to the promoter region that is between 50 to 
100 basepairs 5' upstream of the transcription initiation site. This 
location allows the tetramer to strongly exert its regulatory effect on 
transcription. 
In other embodiments of the present invention, it may be desirable to 
change the number of PA or Iwt motifs attached to a promoter or to vary 
the distance between a motif-containing sequence and the promoter core. 
These variations may be used to modulate the regulatory activity of the 
Iwt or PA motifs. Additional embodiments of the present invention also 
include promoter constructs containing other types of sequence motifs, in 
addition to the Iwt or PA motifs. Such additions may be used to expand the 
expression spectra of the modified promoter. 
5.3 Engineering a Gene of Interest Controlled by the Modified Promoters 
The PA or Iwt motif-modified promoters of the present invention may be used 
to direct the expression of any desired RNA product or protein gene 
product. Useful RNA products include, but are not limited to, "antisense" 
RNA or ribozymes. Such recombinant construct generally comprise a PA or 
Iwt motif-modified promoter, as described herein, ligated to the nucleic 
acid sequence encoding the desired gene product. 
Where the desired gene product is a protein, the DNA construct is designed 
so that the protein coding sequence is ligated in phase with the 
translational initiation codon downstream of the promoter. Where the 
promoter fragment is missing a 5' leader sequences, a DNA fragment 
encoding both the protein and its 5' RNA leader sequence is ligated 
immediately downstream of the transcription initiation site. 
Alternatively, an unrelated 5' RNA leader sequence may be used to bridge 
the promoter and the protein coding sequence. In such instances, the 
design should be such that protein coding sequence is ligated in phase 
with the initiation codon present in the leader sequence, or ligated such 
that no initiation codon interposed between the transcription initiation 
site and the first methionine codon of the protein. 
Further, it may be desirable to include additional DNA sequences in the 
protein expression constructs. Examples of additional DNA sequences 
include, but are not limited to, those encoding: a 3' untranslated region; 
a transcription termination and polyadenylation signal; an intron; a 
signal peptide (which facilitate the secretion of the protein); or a 
transit peptide (which targets the protein to a particular cellular 
compartment such as the nucleus, chloroplast, mitochondria, or vacuole). 
The recombinant construct of the present invention may include a selectable 
marker for propagation of the construct. For example, a construct to be 
propagated in bacteria preferably contains an antibiotic resistance gene, 
such as one that confers resistance to kanamycin, tetracycline, 
streptomycin, or chloramphenicol. Suitable vectors for propagating the 
construct include plasmids, cosmids, bacteriophages or viruses, to name 
but a few. 
In addition, the recombinant constructs may include plant-expressible 
selectable or screenable marker genes for isolating, identifying or 
tracking plant cells transformed by these constructs. Selectable markers 
include, but are not limited to, genes that confer antibiotic resistances, 
(e.g., resistance to kanamycin or hygromycin) or herbicide resistance 
(e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate). 
Screenable markers include, but are not be limited to, genes encoding 
.beta.-glucuronidase (Jefferson, 1987, Plant Molec Biol. Rep 5: 387-405), 
luciferase (Ow et al., 1986, Science 234: 856-859), B protein that 
regulate anthocyanin pigment production (Goff et al., 1990, EMBO J 9: 
2517-2522). 
In embodiments of the present invention which utilize the Agrobacterium 
tumefacien system for transforming plants (see below), the recombinant 
constructs may additionally comprise the left and right T-DNA border 
sequences flanking the DNA sequences to be transformed into the plant 
cell. The proper design and construction of such T-DNA based 
transformation vectors are well known to those skill in the art. 
5.4 Production of Transgenic Plants and Plant Cells 
A recombinant construct containing a gene of interest placed under the 
control of the Iwt or PA motif-modified promoter, as described herein, is 
used to transform a plant cell or to genetic engineer plants. The gene of 
interest may be a heterologous gene or a gene endogenous to the plant. In 
a preferred embodiement, the Agrobacterium tumefaciens is employed to 
introduce a PA or Iwt-containing recombinant construct into a plant. Such 
transformation preferably use a binary Agrobacterium T-DNA vector (Bevan, 
1984, Nucl. Acid Res. 12: 8711-8721), and the co-cultivation procedure 
(Horsch et al., 1985, Science 227: 1229-1231). Generally, Agrobacterium is 
used to transform dicotyledonous plants (Bevan et al. 1982 Ann. Rev. Genet 
16: 357-384; Rogers et al., 1986, Methods Enzymol. 118: 627-641). 
Agrobacterium also may be used to transfer DNA to a wide range of 
monocotyledonous plants as well (Hernalsteen et al., 1984, EMBO J 3: 
3039-3041; Hooykass-Van Slogteren et al., 1984, Nature 311: 763-764; 
Grimsley et al., 1987, Nature 325: 1677-179; Boulton et al., 1989, Plant 
Mol. Biol. 12: 31-40.; Gould et al., 1991, Plant Physiol. 95: 426-434). 
Alternative methods for introducing PA and Iwt containing constructs into 
plants or plant cells may also be utilized, particularly if the desired 
target is a monocotyledonous plant or plant cell. These methods include, 
but are not limited to, protoplast transformation through calcium-, PEG- 
or electroporation-mediated uptake of naked DNA (Paszkowski et al., 1984, 
EMBO J 3: 2717-2722, Potrykus et al. 1985, Molec. Gen. Genet. 199: 
169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82: 5824-5828; 
Shimamoto, 1989, Nature 338: 274-276). Other methods for transforming 
monocotyledonous plants include microinjection, silicon carbide-mediated 
DNA uptake (Kaeppler et al., 1990, Plant Cell Reporter 9: 415-418), and 
microprojectile bombardment (Klein et al., 1988, Proc. Nat. Acad. Sci. USA 
85: 4305-4309; Gordon-Kamm et al., 1990, Plant Cell 2: 603-618). 
The Iwt- or PA-modified gene construct may be introduced into a wide 
variety of plants and plant cell systems using the transformation methods 
described above. Preferred transformed plants or plant cells are those of 
maize, wheat, rice, soybean, tomato, tobacco, carrots, potato, sugar 
beets, yam, Arabidopsis, or petunia. 
Transformed cell or plant may be identified and isolated by selecting or 
screening for the marker genes present on the transformating DNA. 
Selection and screening methods in the various plant systems are well 
known to those skilled in the art. A number of physical and biochemical 
methods may be used, either independently or together with phenotypic 
selection or screening, to identify transformants containing the PA and 
Iwt recombinant constructs. These methods include, but are not limited to: 
1) Southern analysis or PCR amplification for detecting and determining 
the structure of the recombinant DNA insert; 2) Northern blot, S-1 RNase 
protection, primer-extension or reverse transcriptase-PCR amplification 
for detecting and examining the RNA transcript; 3) enzymatic assays for 
detecting the products of the PA or Iwt driven genes, where the gene 
product is an enzyme or ribozyme; 4) protein gel electrophoresis, western 
blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where 
the gene product is a protein. Additional techniques such as in situ 
hybridization, enzyme staining, and immunostaining, may be used to detect 
the presence or expression of the recombinant construct in a particular 
plant organ or tissue. These assays are well known to those skilled in the 
art. 
5.5. Expression of Target Gene Products in Transgenic Plants 
The present invention may be advantageously used to direct the expression 
of a variety of gene products. These gene products include, but are not 
limited to, proteins, anti-sense RNA and ribozymes. 
In particular embodiments of the present invention, by way of illustration, 
and not by limitation, the Iwt motif-modified promoters which confer 
developmental-specific expression in seed may be used to express a storage 
protein such as a phaseolin, a gliadin, a zein, etc. In addition, Iwt 
motif-modified promoters may be used to express non-plant proteins of 
exceptional nutritional quality (e.g., casein, ovalbumin) as well as plant 
proteins engineered to have enhanced nutritional quality (e.g., increased 
methionine and/or lysine content). The Iwt motif enables the expression of 
the storage protein to occur during embryogenesis and, thereby, can 
produce seeds with such enhanced nutritional qualities. The Iwt-modified 
promoter may also advantageously be used to express insect inhibitory 
proteins such as the cowpea Bowman-Birk protease inhibitors which have 
protective effects against herbivorous insects (Hilder et al., 1989, Plant 
Molec. Biol. 13: 701-710). The embryo-specific expression of such proteins 
would deter or suppress insect predation of seeds and, thereby, increase 
seed yields in engineered crop plants. 
Other applications of Iwt-modified promoters include controlling the 
expression of anti-sense, ribozyme or protein genes whose products 
interfere with basic metabolic or particular embryo-specific functions, 
and expressing antisense or ribozyme molecules designed to remove 
undesirable traits. The expression of such genes using the Iwt-modified 
promoters would synchronize and focus the expression of the gene product 
to the intended target function in the embryo and, thereby, enable 
specific and efficient alteration of embryo development, structure or 
metabolism. 
PA-modified promoters, which confer root-specific expression, may be 
advantageously used to express various pest-resistance and 
disease-resistance genes. Pest resistance genes include the protease 
inhibitor proteins discussed above, as well as various Bacillus 
thuringiensis endotoxin proteins. PA-modified promoters may also be used 
to express disease resistance genes, such as, for example, virus coat 
protein, anti-sense RNA or ribozyme genes for anti-virus protection 
(Gadani et al., 1990, Arch. Virol 115: 1-21); lysozymes and maganins for 
anti-bacterial protection; or glucanases, osmotins or chitinases for 
anti-fungal protection. The use of a PA-motif modified promoter would 
localize and enhance the expression of such a pest-resistance or 
disease-resistance gene in the roots and, to a lesser extent, in the 
leaves, thereby, effecting resistance to soil and foliar diseases and 
pests, such as nematodes and insects. The engineering of such sequences 
under the control of PA-modified promoters is particularly advantageously 
applied to root vegetables, including, but not limited to, sugar beets, 
carrots, turnips, parsnips, etc. 
Other applications of PA modified promoters include controlling the 
expression of anti-sense, ribozyme or protein genes whose products 
interfere with basic metabolic or particular root or leaf functions. The 
expression of such genes under the control of a PA-modified promoter would 
synchronize and focus the expression of these gene products to the 
intended target functions in the root or leaf and, thereby, enable 
specific and efficient alterations of development, structure or metabolism 
of these two organs. 
Genes encoding transactivators that bind a PA or Iwt motif and activate 
transcription may also be genetically engineered into the host plant cell 
containing the PA or Iwt construct. A preferred transactivator is the 
TAF-1 protein. Transforming plants with a transactivator genes such as 
TAF-1 expands the tissue and developmental range of a PA-or 
Iwt-motif-containing promoter. For example, to achieve light-regulated, 
leaf-specific expression from a Iwt or PA construct, a rbcS promoter, 
which has that expression pattern, is used to drive the TAF-1 gene. Since 
TAF-1 activates PA- or Iwt-motif-containing constructs, TAF-1 factor would 
act to couple the expression patterns of PA or Iwt motif containing 
constructs to that of the rbcS promoter. In other embodiments, the same 
approach is used to expand the plant range of a PA- of Iwt-containing 
promoter. There, a PA- or Iwt-containing construct may be employed in 
plants which do not naturally contain transactivators that recognize these 
motifs by engineering in a cognate transactivator, such as TAF-1, under 
the control of a promoter that drives expression in the particular plant. 
In the working examples described infra, the tobacco transcriptional 
activator, TAF-1, binds in vitro to two G-box like sequences, PA and Iwt, 
and its binding affinity for PA is about 70 times higher than that for Iwt 
(Oeda et al., 1991, EMBO J. 10: 1793-1802). These examples also compare 
the in vivo expression patterns conferred by these two short sequences 
with that of a negative control, Imu, a motif I mutant which is unable to 
bind to the activator in vitro (Oeda et al., 1991, EMBO J. 10: 1793-1802). 
The results show that PA and Iwt tetramers, which differ only in 2 bp per 
10 bp repeat (FIG. 1B) confer strikingly different expression patterns in 
transgenic tobacco plants. The Imu tetramer, on the other hand, appears to 
be inactive as a cis-regulatory element because it has no noticeable 
effect on the expression of the X-GUS-90 promoter. FIG. 6 summarizes in a 
schematic form the expression patterns obtained with constructs 1 to 4 at 
different stages of plant development. 
PA confers high level expression in roots, low level in leaves, and little 
or no expression in seeds (FIGS. 3A-3L and 4A-4F) Iwt, on the other hand, 
confers preferential expression in seeds but in a 
developmentally-regulated manner. GUS activity appears only at 15 DAF and 
increases steadily thereafter (FIGS. 3A-3L), and in mature seeds the 
activity is detected in the entire embryo as well as the endosperm. 
Although some GUS activity is found in cotyledons of germinating seedlings 
(FIGS. 4A-4F), this could be due to residual GUS enzyme synthesized during 
seed development. 
The contrasting and almost non-overlapping expression profiles conferred by 
PA and Iwt strongly suggest that these two cis-regulatory elements 
interact with different transactivating factors in vivo. These results 
favor the hypothesis that the transcription activator TAF-1 is the cognate 
factor for PA for the following two reasons: (1) PA is a high affinity 
binding site for the activator (Oeda et al., 1991, EMBO J. 10: 1793-1802), 
and (2) the expression pattern conferred by PA parallels that of TAF-1. 
Bombardment experiments using a 35S-TAF-1 chimeric construct demonstrated 
that a transient increase in the expression level of TAF-1 can activate an 
Iwt-linked transgene in tobacco leaves (Oeda et al., 1991, EMBO J. 10: 
1793-1802). Notwithstanding this observation, the example detailed herein 
demonstrate that Iwt is inactive in root even though this organ expresses 
TAF-1 at a high level. This result suggests that the high concentration of 
TAF-1 in roots is still unable to compensate for the low binding affinity 
of Iwt for this factor. 
In comparison to other known seed-specific promoters, the expression 
pattern conferred by Iwt is of particular interest because it appears to 
direct expression in both embryos and endosperm. The cis-acting elements 
studied thus far from seed-specific genes promote expression in specific 
regions of the seed (cf. Goldberg et al., 1989, Cell 56: 149-160 for a 
review; Bustos et al., 1991, EMBO J. 10: 1469-1479). Only hex-3 (5' 
GGACGCGTGGC 3') (SEQ. ID NO:5), a mutant derivative of the hex motif 
located in the wheat histone H3 promoter (Tabata et al., 1989, EMBO J. 10: 
1459-1467), has been shown to confer activity in the embryo, as well as 
the endosperm of tobacco seeds (Lam & Chua, 1991, J. Biol. Chem. 250: 
17131-17135). Whether Iwt and hex-3 interact with the same or different 
factors remains to be determined. Iwt (GTACGTGGCG) (SEQ. ID NO:2) shares 
striking sequence homology (7 out of 9 bp are identical) to the opaque 2 
target site (TCTACGTGGA) (SEQ. ID NO:6) in the 5' region of the 22-kD zein 
gene which is expressed predominantly in maize endosperm (Schmidt et al., 
1992, Plant Cell 4: 689-700). 
An important consideration for the interpretation of the expression studies 
described infra is that the constructs were made in the context of the -90 
35S promoter (FIG. 1A). As described previously, this promoter confers 
expression in the radicle of the embryo, the radicle pole of the endosperm 
and the tips of young and mature roots (Benfey et al., 1989, Science 244: 
174-181). By contrast, construct 2 which contains the PA tetramer confers 
strong expression in the entire root including root hairs (FIGS. 4A and 
4B). In addition, the GUS activity in roots containing construct 2 is 
about 10 times higher than those containing the -90 35S promoter alone 
(FIG. 2). A 21-bp element, designated as as-1, is responsible for the root 
expression of the -90 35S promoter (Lam et al., 1989, Proc. Natl. Acad. 
Sci. USA 86: 7890-7894). It is possible that the strong expression in 
root, as well as the weak expression in leaf, obtained with construct 2 
are generated by a synergistic interaction between PA tetramer and the 
as-1 element. Similar consideration may also apply to the preferential 
seed expression seen with construct 3. 
To the best of applicant's knowledge, this is the first report that two 
closely-related plant regulatory sequences such as PA and Iwt can confer 
completely different expression patterns in transgenic plants. Clearly, 
these two sequences can be used to direct tissue-specific and 
developmentally-regulated expression of target genes for basic research as 
well as biotechnological applications. 
6. EXAMPLE: TISSUE-SPECIFIC AND DEVELOPMENTALLY REGULATED EXPRESSION OF 
TARGET GENE PRODUCTS IN TRANSGENIC TOBACCO 
The expression patterns conferred by two G-box related motifs, a perfect 
palindromic sequence (PA, 5' GCCACGTGGC 3') (SEQ. ID NO:3) and motif I 
(Iwt, 5' GTACGTGGCG 3') (SEQ. ID NO:2), were analyzed in transgenic 
tobacco plants. A mutant version of motif I, Imu, was used as a negative 
control. PA is not known to be present in the promoters of plant genes, 
whereas Iwt is a conserved sequence found in ABA-inducible promoters. 
Previous studies demonstrated that PA and Iwt, but not Imu, can bind to 
the tobacco transcription activator TAF-1 in vitro, with the PA sequence 
showing a 70-fold higher affinity as compared to Iwt (Oeda et al., 1991, 
EMBO J. 10: 1793-1802). Tetramers of PA and Iwt, which differ by only 2 
base pairs per 10 base-pair repeat, confer very different tissue-specific 
and expression patterns in transgenic tobacco plants. PA confers 
preferential expression in root tissues with a low level of activity in 
leaves, whereas Iwt directs developmentally regulated expression in seeds 
beginning from 15 DAF until seed maturation. Imu appears to be inactive as 
it gives the same expression pattern as the -90 35S promoter control. 
Norther blot analysis showed that the expression pattern of TAF-1 mRNA is 
similar to that directed by PA, suggesting that TAF-1 may be involved in 
the transcriptional regulation of PA. 
6.1 Materials and Methods 
6.1.1 G-Box-Related Constructs 
Construct 1 contains the -90 35S promoter fragment from -90 to +8 CaMV 35S 
(A domain) fused to the GUS coding sequence (Jefferson et al., 1987, EMBO 
J. 6: 3901-3907). The 3' end from the pea rbcS-3C gene was placed 
downstream of the GUS sequence to provide for a polyadenylation signal. 
This construct was inserted into pMON505 to give the X-GUS-90 vector, 
which was described in detail elsewhere (Benfey & Chua, 1989, Science 244: 
174-181). For construct 2, a head-to-tail tetramer of the perfect 
palindromic sequence (PA) 5'-GCCACGTGGC-3' (SEQ. ID NO:3) was synthesized 
with a HindIII site at the 5' end and a XhoI site at the 3' end. The 
tetramer was then cloned between the HindIII (5') and XhoI (3') sites into 
a pEMBL12 derivative, sequenced and, then inserted at -90 of X-GUS-90. The 
same strategy was used to generate constructs 3 and 4 except that the 
tetramers synthesized corresponded to the wild type motif I (Iwt) 
5'-GTACGTGGCG-3' (SEQ. ID NO:2) and the mutant motif I derivative (Imu) 
5'-TGACTGTTCT-3' (SEQ. ID NO:4), respectively. 
6.1.2 Transgenic Plants 
Constructs were mobilized into Agrobacterium tumefaciens GV3111SE, and 
tobacco plants (Nicotiana tabacum c.v. SR1) were used for transformation. 
Shoots were regenerated on medium containing 200 .mu.g/ml kanamycin 
(Rogers et al., 1986, Methods Enzymol. 118: 627-640). After rooting, 
transgenic plantelets were transferred to soil and grown in a growth 
chamber, or maintained, by cuttings, in Plantcons (Tm) containing MS media 
supplemented with 3% sucrose, 0.7% agar, 100 .mu.g/ml kanamycin, and 500 
.mu.g/ml carbenicillin. Primary transformants were allowed to 
self-fertilize and seeds were collected, sterilized, and germinated on 
supplemented MS media (see above). Seedlings were maintained at 26.degree. 
C. under 16 hours of light and 8 hours of dark. Seven to ten independent 
transgenic plants were analyzed for each construct. 
6.1.3 Histochemical Staining 
GUS histochemical staining was carried out essentially as described by 
Jefferson et al., 1987, EMBO J. 6: 3901-3907. The development stages of 
the seeds used for the assays were determined by tagging flowers of 
primary transformants when petals had fully expanded (0 DAF). At various 
intervals hereafter (10 DAF, 15 DAF, and 20 DAF), capsules were removed 
and 200 .mu.m sections were prepared with a cryotome. Sections were 
stained by placing them directly into a histochemical substrate solution 
containing 1 mM 5-bromo-4-chloro-3-indolyl glucuronidase (X-Gluc) and 50 
mM sodium phosphate buffer (pH 7.0) on a microscope slide for 12 hours in 
a humidified chamber at 37.degree. C. Mature seeds were stained as 
described by Benfey & Chua, 1989, Science 244: 174-181. In order to rule 
out possible diffusion of the GUS enzyme or dye from one tissue to another 
during incubation, embryos were removed from endosperms prior to 
incubation with the substrate. Seven- and 15-days old seedlings were 
removed from the Petri dishes, and placed directly into the X-Gluc 
solution and incubated as described for capsule sections (see above). 
After the incubation, chlorophyll was cleared as described previously 
(Benfey & Chua, 1989, Science 244: 174-181. 
6.1.4 GUS Enzyme Assays 
GUS enzyme assays were performed mainly as described by Jefferson et al., 
1987, EMBO J. 6: 3901-3907. Extracts were made from leaves and roots of 
seven week-old plants grown on supplemented MS media (see above), from 
leaves of seven to ten week-old plants frown in a greenhouse, and from 
mature seeds. Ten micrograms of protein were incubated with 4-methyl 
umbelliferyl glucuronide (MUG) solution for 60 minutes at 37.degree. C. 
The reactions were stoped by adding 2.5 ml of 0.2M sodium carbonate, and 
fluorescence was measured with a Perkin-Elmer LS5 fluorimeter by using a 
solution of 100 nM 4-methylumbelliferone (MU) in 0.2M sodium carbonate for 
calibration. 
6.1.5 Northern Analysis 
PolyA-plus RNA was extracted from seeds at 20 DAF, and from leaves and 
roots of seven week-old transgenic tobacco plants grown on supplemented 
media (see above). RNA was electrophoresed in glyoxal gels and blotted 
according to standard protocols. Filters were hybridized to the 1.2 Kb 
labeled EcoRI fragment of TAF-1 cDNA (Oeda et al., 1991, EMBO J. 10: 
1793-1802), or to the .beta.-ATPase cDNA (Boutry and Chua, 1985) in a 
solution of 50% formamide, 5.times. SSC, 100 .mu.g/ml sonicated salmon 
sperm DNA, 0.5% SDS, 5.times. Denhardt's at 42.degree. C. for 20 hours. 
Filters were washed in 1.times. SSC, 0.5 SDS at 65.degree. C. for one hour 
and autoradiographed. 
6.2 Results 
6.2.1 Engineered Transgenic Plants 
To investigate the functional properties of PA and motif I (Iwt), the 
head-to-tail tetramers comprising tandem copies of these sequences were 
inserted upstream of the truncated -90 35S promoter in the X-GUS-90 vector 
(construct 1) to generate constructs 2 and 3, respectively. The X-GUS-90 
vector contains the -90 to +8 (A domain) region of the CaMV 35S promoter 
fused to the .beta.-glucuronidase (GUS) coding sequence, with a 3' 
fragment of the pea rbcS-3C gene (Benfey & Chua, 1989, Science 244: 
174-181). As a negative control, a tetramer of a motif I mutant, 
TGACTGTTCT (SEQ. ID NO:4), designated as Imu, previously shown to have no 
binding activity for TAF-1 (Oeda et al., 1991, EMBO J. 10: 1793-1802) was 
synthesized. The Imu tetramer was also inserted upstream of the truncated 
35S promoter in the X-GUS-90 vector to give construct 4. FIG. 1A shows the 
structures of these chimetic constructs and compares the sequences of the 
different tetramers used (FIG. 3). The chimeric constructs were 
transferred to Agrobacterium tumefaciens, and tobacco leaf discs were 
transformed as described in Material and Methods. The expression of the 
GUS reporter gene was analyzed in primary transformants as well as in 
their progeny at different developmental stages. 
6.2.2 Expression of Target Gene Product in Mature Plants 
GUS activity was measured quantitatively in leaves and roots from at least 
7 independent transgenic plants for each construct (FIG. 1A). FIG. 2A 
shows that leaves from plants carrying constructs 3 and 4 expressed a 
similar level of GUS activity as those from plants containing the X-GUS 90 
vector alone (construct 2). By contrast, GUS activity in leaves harboring 
construct 2 was three to five times higher. The value of GUS activity in 
the leaves was not affected by the growth conditions of the plants (soil 
or tissue culture grown plants). In the case of roots, constructs 1 and 4 
gave similar GUS expression level whereas constructs 2 and 3 were about 
ten and two times higher, respectively (FIG. 2B). These results indicate 
that, in mature plants, PA can confer a high level of expression in roots 
and, to a lesser degree, in leaves. On the contrary, Iwt and Imu appear to 
be relatively inactive in these tissues. The 5- to 10-fold higher level of 
GUS activity conferred by construct 1 (X-GUS-90 vector) in roots as 
compared to leaves confirms our previous observation that domain A (-90 to 
+8) of the CaMV 35S promoter shows preferential expression in roots 
(Benfey et al., 1989, EMBO J. 8: 2195-2202). Because constructs 2, 3, and 
4 were all made in the context of the -90 35S promoter, it is possible 
that the expression patterns obtained with PA reflect the interaction of 
this sequence with domain A. 
6.2.3 Expression of Target Gene Produced During Seed Development 
R1 seeds from transgenic plants containing constructs 1 through 4 were 
analyzed at several stages of development. For this purpose, seeds were 
harvested at 10, 15, and 20 days after petals had fully expanded (DAF), 
and also at maturity. Seeds were sectioned, stained for GUS activity, and 
the stained sections examined by light microscopy as described in 
Materials and Methods. In the case of mature seeds, the GUS activity in 
soluble extracts were also determined quantitatively (FIG. 2C). 
Seeds that contained PA or Imu did not show any specific GUS staining when 
compared to control seeds containing the X-GUS-90 vector alone (FIGS. 
3A-3C, and FIGS. 3D-3F). In all these three cases, GUS activity was 
localized, beginning at 15 DAF (not shown), in the radicle of the embryo 
and in the endosperm cells at the radicle pole (FIGS. 3A-3F). By contrast, 
Iwt conferred regulated expression in developing seeds. GUS expression was 
first detected in embryos from seeds at 15 DAF (FIGS. 3G-3I). The 
expression progressively increased until seed maturity when GUS activity 
was present in the entire embryo as well as the endosperm (FIGS. 3J-3L). 
The different seed expression patterns conferred by PA or Imu on one hand, 
and Iwt on the other hand, were also reflected in quantitative GUS assay 
of extracts prepared from mature seeds (FIG. 2C). Seeds with Iwt showed 
about 10 times more GUS activity than seeds with PA or Imu, which gave the 
same level of activity as seeds from control plants (construct 1). 
These results indicate that whereas PA and Imu are inactive in seeds, Iwt 
can confer specific expression in the embryo and endosperm. However, this 
expression appears to be developmentally regulated during seed development 
because GUS activity appears at 15 DAF and subsequently increases until 
seed maturity. GUS expression in the radicle of the embryos, and in the 
endosperm cells at the radicle pole, was likely due to the -90 to +8 35S 
CaMV promoter fragment (A domain) from the X-GUS-90 vector, as already 
described (Benfey et al., 1989, EMBO J. 8: 2195-2202). 
6.2.4 Expression of Target Gene Product in Young Seedlings 
R1 seeds from transgenic plants containing constructs 1 through 4 were 
sterilized and germinated on media as described in Materials and Methods. 
Seedlings were removed at 7 days (stage of 2 cotyledons) and 15 days 
(stage of 2 cotyledons and first 2 leaves), and then processed for 
detection of GUS activity at the cellular level. FIG. 4A shows that 7 
day-old seedlings containing PA showed strong GUS activity in roots and 
weak GUS activity in cotyledons. In seedlings containing Iwt, strong GUS 
activity was evident in the cotyledons, as well as at the root tips (FIG. 
4E). As a negative control, seedlings containing Imu demonstrated 
identical expression pattern to that of X-GUS-90 control seedlings, i.e. 
GUS activity was only detected at the root tips (FIG. 4C). At 15 days, 
seedlings containing PA showed very strong staining in roots, and 
intermediate staining in the cotyledons and in the first two leaves (FIG. 
4B). Expression from Iwt was detected in the cotyledons and at the root 
tips; however, no GUS activity was visible in the first two leaves (FIG. 
4F). In seedlings containing Imu or the X-GUS-90 vector alone (control 
seedlings), staining was only obtained at the root tips (FIG. 4D). 
These results demonstrate that PA and Iwt confer tissue-specific expression 
in seedlings and, moreover, this expression is developmentally regulated 
during seed germination. In the case of PA, expression is very strong in 
roots and, to a lesser degree, in cotyledons and leaves. It should be 
stressed that this pattern of expression appears only after seed 
germination. On the other hand, in young seedlings containing the Iwt 
sequences, expression is detected in cotyledons but, interestingly, not in 
leaves. In all of the four constructs, the expression detected in the root 
tip can be attributed to the CaMV 35S A domain in the X-GUS-90 vector 
(Benfey et al., 1989, EMBO J. 8: 2195-2202). 
6.2.5 Expression Pattern of TAF-1 mRNA 
A previously characterized cDNA clone encodes a tobacco transcription 
activator designated as TAF-1 (Oeda et al., 1991, EMBO J. 10: 1793-1802). 
TAF-1 is a bZIP protein that binds to both Iwt and PA, albeit with 
striking different affinity, but not to Imu. Because the expression 
patterns conferred by cis-regulatory elements may reflect the abundance of 
the transcription factors that interact with them, the expression pattern 
of TAF-1 mRNA was compared to those conferred by PA and Iwt. In a previous 
report, TAF-1 mRNA was shown to be 10 to 20 times more abundant in roots 
than in leaves and stems (Oeda et al., 1991, EMBO J. 10: 1793-1802). The 
transcriptional activity of Iwt in tobacco seeds (FIGS. 2A-2C and FIGS. 
3A-3F) prompted the analysis of the expression of TAF-1 mRNA in this 
organ. The TAF-1 mRNA level in developing seeds at 20 DAF was inrestiguted 
because at this stage of seed development, Iwt already conferred a high 
GUS expression level (see above). FIG. 5A, shows that TAF-1 mRNA was 
neither detected in developing seeds (20 DAF), nor in leaves. A longer 
exposure of the autoradiogram only revealed a faint band corresponding in 
size to that of the TAF-1 mRNA in leaves. As an internal control for the 
Northern blot analysis, the mRNA coding for the constitutively expressed 
mitochondrial .beta.-ATPase gene (Boutry & Chua, 1985, EMBO J. 4: 
2159-2165) was present at a higher level in seeds as opposed to leaves and 
roots (FIG. 5B). These results suggested that the expression pattern of 
TAF-1 mRNA is similar to that conferred by PA (see above). Among the three 
G-box-binding factors (GBFs) recently cloned from Arabidopsis, GBF3 which 
shares sequence homology with TAF1 is also highly expressed in roots with 
little expression in leaves (Schindler et al., 1992, EMBO J. 11: 
1261-1273). It would be interesting to see whether GBF3 is also poorly 
expressed in seeds, as is the case with TAF-1 reported here. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: G-box element sequence 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MACACGTGGCA11 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: TAF-1 binding motif 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GTACGTGGCG10 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: TAF-1 binding motif 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GCCACGTGGC10 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: TAF-1 binding motif related sequence 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TGACTGTTCT10 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: Hex-motif related sequence 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GGACGCGTGGC11 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: Opaque 2 binding site 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TCTACGTGGA10 
__________________________________________________________________________