Proc. Natl. Acad. Sci. USA Vol. 85, pp. 4310-4314, June 1988 Cell Biology An enhancer-like element present in the promoter of a T-DNA gene from the Ti plasmid of Agrobacterium tumefaciens (transcription/Ti plasmid/780 gene /Agrobacterinum tumefaciens) WESLEY B. BRUCE*, RAM BANDYOPADHYAYt, AND WILLIAM B. GURLEY Department of Microbiology and Cell Science, 1059 McCarty Hall, University of Florida, Gainesville, FL 32611 Communicated by Mary-Dell Chilton, January 19, 1988 (receivedfor review August 19, 1987)

ABSTRACT The promoter of the 780 gene of T-right analysis by 5' and internal deletions identified a distal [Thomashow, M., Nutter, R., Montoya, A., Gordon, M. & promoter domain referred to as the "activator" element (ca. Nester, E. (1980) Cel 19, 729-739] from Agrobacterium tume- - 440 to -229 bp) that was shown to be essential for 780 faciens Ti plasmid (pTil5955) was shown to contain an up- promoter function and to possess some spacial flexibility in stream cis-acting element (activator) having enhancer-like its ability to function at various distances from the start of properties. To characterize the properties of this promoter transcription. Here we demonstrate that this element can element, it was placed in both polarities, upstream and activate a - 37-bp 5' deletion (A- 37) of the 780 promoter downstream of a A- 37 deletion mutant of the 780 gene. The independently of orientation and spacing when located 5' to A- 37 deletion contains the entire 780 gene with the 5' flank- TATA. Upstream promoter domains of the ocs and the ags ing sequences deleted upstream of TATA to - 37. The effect of gene that are presumably analogous to the 780 activator were the activator on in vivo transcriptional activity was assessed by also examined in both polarities to determine if sequences S1 nuclease mapping utilizing a homologous reference gene as from heterologous transferred DNA (T-DNA) promoters an internal standard. Transcript levels from sunflower crown could also activate the 780 gene A- 37 promoter. For each gall tumors were between 127% and 90% of the wild-type 780 promoter construction, transcriptional analyses were con- gene depending on the polarity of the activator element when ducted by using pooled RNA isolated from 200-300 trans- placed directly upstream to the 780 gene A- 37 promoter. formed sunflower tumors utilizing a reference gene vector Repositioning the activator element 613 base pairs further system that incorporated a reference copy of the 780 gene as upstream increased transcription by 2-fold regardless of po- an internal standard (11). larity. However, the activator element did not promote tran- scription when placed in either polarity -=200 base pairs downstream of the poly(A) addition site. Upstream promoter MATERIALS AND METHODS fragments (TATA-distal) from the octopine synthase (ocs) and Plasmid Construction: 780 Activator Element/780 A- 37 agropine synthase (ags) genes were also tested for activation of Deletion Clone. Test and reference copies of the 780 gene the A- 37 construction. The ocs sequences activated transcrip- were incorporated into the T-DNA of A. tumefaciens tion of the A- 37 deletion to 14% of wild-type levels when (Ag5260, derived from strain 15955) by means of a pAC- placed upstream in the B (reverse) orientation. All other YC184-based shuttle vector designated pW9-TD:A&-37 (11, constructions with the ocs and ags sequences showed no 12). DNA fragments containing the 780 activator were intro- enhancement of promoter activity. duced 5' (BamHI) or 3' (HindIII) relative to the test gene (Fig. 1). The test and reference genes contain 37 and 290 bp of 5' Genetic analysis of many viral and animal genes has identi- flanking sequences, respectively. The 5' untranslated leader fied regulatory elements known as enhancers (1, 2). Gener- of the reference gene contains an internal 8-bp deletion to ally, enhancers are cis-acting elements functioning indepen- facilitate the discrimination of test and reference gene tran- dently oforientation at various distances from TATA and the scripts by S1 nuclease analysis. Unique restriction sites are start oftranscription, 5' or 3' to the gene (3-5). Enhancer-like present in pW9-TD:A- 37 that allow insertion of DNA elements in plant promoters have been found in two genes of fragments upstream of position - 37 bp (BamHI) and =200 pea: the small subunit gene of ribulose-bisphosphate carbox- bp downstream (HindIII) of the poly(A) addition site of the ylase (RbcS) (6-8) and the light-harvesting chlorophyll a/b- test gene. To allow upstream insertion of the 780 gene binding protein (Cab) gene (9). A 240- to 280-base-pair (bp) activator fragments in both polarities, the Sal I sites of the region (ca. - 330 to - 50 bp from the start of transcription) BamHI-Sal I fragments (- 476 to - 112 bp or - 476 to - 200 of the RbcS gene imparts light inducibility and tissue speci- bp) from the appropriate 780 3' deletion clones (11) were ficity on the nopaline synthase (nos) and the cauliflower converted to BamHI by linker addition (18). mosaic virus 35S promoters when placed 5' to these promot- The 780 activator was also moved upstream by the inser- ers in either orientation (7). A portion of the ocs promoter tion of a 613-bp 4X174 Hae III fragment [1173-1779 bp (19)] from - 292 to - 116 bp was recently demonstrated to func- into the Sal I site located between the 780 - 476/ - 112-bp tion in the 5' position independently of orientation in the activator fragment and the - 37-bp deletion end point of the stimulation of transcription from the maize alcohol dehy- test gene. This insertion/substitution resulted in the 780 drogenase (Adh-1) gene in a transgenic system (10). activator being positioned 538 bp upstream to its original We have previously described a cis-acting element present in the wild-type promoter. The effect of the 46X174 in the promoter of the 780 gene from T-right (34) of the location pTi15955 of Agrobacterium tumefaciens that seems to pos- sess attributes of a eukaryotic enhancer (11). Transcriptional Abbreviations: RTL, relative transcript level; T-DNA, transferred DNA; OCS, octopine synthase; AGS, agropine synthase. *Present address: Plant Gene Expression Center, 800 Buchanan The publication costs ofthis article were defrayed in part by page charge Street, Albany, CA 94170. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Biochemistry and Pharmacology, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Tufts University, 136 Harrison Avenue, Boston, MA 02111.

Downloaded by guest on October 1, 2021 4310 Cell Biology: Bruce et al. Proc. Nati. Acad. Sci. USA 85 (1988) 4311 A. BamHI or HindIII site of 37 and screened for pTi15955 pW9-TD:A- T-L T-R polarity by restriction analysis. Triparental Conjugation and Tumor Formation. Reference 1 4 6ab OCS 4 ' 3 2 ' 1 ' 0 gene shuttle vectors were transferred from Escherichia coli to -a -* 4- 0. -**.. -_r - A. tumefaciens Ag5260 (11) by a triparental conjugation Bam Hi 17a 2 I I .. procedure as described by Fraley et al. (20). Tumors were Hindlil B ||| E T C T D incited on the stems of sunflower (Helianthus annuus cv. Eco Rl Large Grey) seedlings, and the plants were grown as de- scribed (21). Following -15 days of tumor growth, an kb average of 200-300 tumors for each promoter construction AGS was harvested and immediately frozen in liquid nitrogen. A By S1 Nuclease Analysis. Transcript levels of the A- 37 test 78 0 -476/-112 gene and 780 reference gene were assayed by S1 nuclease hybrid protection that was performed with z15 ug of -476/-200 S poly(A)-enriched RNA isolated from pooled sunflower tu- B S mors as described (11). The hybridization probe used in the S1 nuclease analysis was a 135-bp (+ 60 to -74 bp) 5' B. end-labeled DNA fragment isolated from the 780 gene 5' B. (~~~~~~~~ deletion clone A-74 (11). Relative transcript levels (RTLs) were determined from cpm values of the S1 nuclease hybrid-protected bands that were cut out of polyacrylamide gels and subjected to deter- I mination of radioactivity by Cerenkov counting. The RTL 7 80 pW9-1 values were calculated as a percentage from the ratio of cpm reference 43 (' i gene 1 values of the test gene to the 780 reference gene divided by RSP the ratio of cpm values of the wild-type 780 gene (-476 bp of 5' flanking sequences) to the reference gene. All RTLs represent an average derived from three to five independent S1 nuclease hybrid-protection experiments. 6b 6a RESULTS FIG. 1. Construction of reference gene vectors containing T-DNA5'-flanking regions upstream or downstream of A- 37780 test Stimulation of Transcription by Upstream Placement of the gene. (A) Restriction map of the T-DNA of pTi15955 (13). Small 780 Activator. The 780 activator enhanced mRNA levels from arrows denote position and polarities of transcripts present in T-left 0' 90%o to 210% when located at various positions upstream of (14) and T-right (15-17). Transcripts 4' and correspond to the 780 the 780 A -37 deletion (test gene). Fig. 2A depicts an and ags genes, respectively. The large arrows represent the 5' an flanking regions of the three T-DNA genes used for the fragment autoradiograph of S1 nuclease mapping gel showing polarity constructions. OCS, octopine synthase; AGS, agropine protected DNA fragments corresponding to the major tran- synthase. (B) Introduction of5'-flanking regions into the reference scripts derived from the test (upper bands) and reference gene shuttle vector. The 5' flanking region fragments modified with (lower bands) genes. A summary diagram of the construc- BamHI or HindIII linkers were inserted in the 5' or 3' locations, tions tested and the RTLs obtained is shown in Fig.. 3. respectively, of the 780 A -37 deletion (test gene) in both polarities. Although the A -37 deletion mutation by itself showed only Construction of the test gene and the reference gene shuttle vector 2%, or less, activity compared to the wild-type promoter, was as described (11). The hatched area is the Sph I-BamHI fragment placement of the 780 activator upstream of position -37 of pACYC184 (12). The solid black areas are the 780 reference and resulted in. a near restoration of wild-type activity in some test genes. The stippled area is the Sph I-BamHI fragment from the BamHI 17a segment of T-left, used for homologous recombination cases and in others exceeded wild-type levels. The activator - - with the Ti plasmid. The black triangle denotes the Taq I deletion in construction 476/ 112 5' A, in which the 780 wild-type the 780 reference gene leader and "Cam"' denotes the chloramphen- promoter between positions - 112 and -37 was replaced icol-resistance gene. The 25-bp Sal I-BamHI fragment immediately with 25 bp of the polylinker from pUC19, was transcribed at upstream of the test gene is part of the polylinker from pUC19. A, 93% of the wild-type level. Promoter activity was not AccI; B, BamHI; H, HindIII;S, SalI; Sp, SphI. significantly affected by the orientation of the activator at this location since the - 476/-1125' B construction had a RTL fragment alone on transcription of the A- 37 clone was tested value of90%o. The - 476- to - 112-bp DNA fragment contains by placing it in both polarities directly upstream. not only the activator element[-476 to ca. -221 bp (11)l but To introduce the 780 activator element downstream of the also additional sequences from -220 to - 112 that lie test gene, the BamHI-Sal I fragment ( - 476/ -112 bp) from between the activator and TATA. Removal of 163 bp of this the - 112-bp 3' deletion clone was modified by addition of DNA between -200 and -37 (-476/-200 5' A) and HindIII linkers (18) and then inserted into the HindIII site of replacement with 25 bp of pUC19 polylinker resulted in pW9-TD:A- 37 in both orientations. The polarities of the transcriptional activity of 127%. Reversing the orientation of inserted 780 activator elements and the 4X174 fragments the - 476/ - 200-bp fragment ( - 476/ -2005' B) reduced the were determined by restriction analysis. RTL to 92%. These results indicated that the 3' border of the Plasmid Construction: ocs and ags 5' Upstream Fragments. activator element is located 5' to position -220, which is Sequences between the putative CCAAT boxes and the consistent with the previous 5' deletion analysis delimiting respective 5' T-DNA borders from the ocs and ags genes the activator from -476 to -221 (11). These results also were placed in both polarities, 5' and 3' to the 780 gene A- 37 show that the activator is able tofunction with the 780 TATA deletion clone (test gene). The BamHI-Acc I fragment of the in the absence ofotherupstream,or TATA-proximal, ele- ocs gene (216 bp) and the BamHI-Sal I fragment of a 3' ments. deletion mutant of the ags gene (182 bp) were modified by The 780 activator (-476 to - 112) was also able to activate either BamHI or HindIII linker addition (18). The "linkered" the test gene promoter at long range. Placement of the ocs and ags promoter fragments were then ligated into the - 476/-112 fragment 650 bp upstream of the start of tran- Downloaded by guest on October 1, 2021 4312 Cell Biology: Bruce et al. Proc. Natl. Acad. Sci. USA 85 (1988)

11 *1 A. WT 1 2 3 4 5 6 7 8 9 l0 r *47 -11 2-72s RTL WT _{_1i3-0 00 Mj

4761-I 12 5' A 93 -471 -4112 ___~ _ _ _ WORM . a- 476cs412 s' B 90 _-@._ _ -1 12 -476

476/-200 5' A 127 -476 -200 476/-200 5' 92 -200 -476 -37 B. WT 12 13 14 15 16 17 18 19 20 780 AOX 210 -476 -.112 (613 bp) I 780 S/OX 4 183 -112 -476

OX A *X174 412.0

"..N-+-- VVVV""- Ab IImA^ z09_ ox a - ,>vs A, _ _ _ ,,,v, ., ^ _,

analysis of 780 test gene -37 Do +926 FIG. 2. Autoradiograph of S1 nuclease 0 activity. Approximately 15 ug of poly(A) RNA from sunflower * 780 3' A GENE ~ 2.1 tumors was used for all lanes. Lane WT, RNA from tumors -476 :I1 are containing the wild-type and reference 780 genes. Lanes 11 and 20 - a "M" refers to the 780 from different hybridization experiment. 780 3' B 2.0 major promoter transcripts and "R" refers to the reference gene -1 12- -4761 transcripts. (A) Lanes: 1, -476/-112 5' A; 2, -476/-112 5' B; 3, 2.0 -476/- 200 5' B; 4, 780 (-476/- 112) A/+X; 5, 780 B/4X; 6,4AX A; A- 3 7 7, XX B; 8, 780 3' A; 9, 780 3' B; 10, 780 A-37; and 11, -476/-200 5'A. (B) Lanes: 12, OCS 5'A; 13, OCS 5'B; 14, AGS5'A; 15, AGS 5' B; 16, OCS 3' A; 17, OCS 3' B; 18, AGS 3' A; 19, AGS 3' B; and FIG. 3. Schematic representation of the 780 activator in various 20, 780 i\- 37 gene. positions relative to the 780 test gene. The 780 constructions correspond to the S1 nuclease analysis in Fig. 2A. The vertical-lined scription (780 A/+X) by the insertion of 613 bp of Sal arrows represent either the - 476/- 112-bp or - 476/- 200-bp frag- I-linkered 4X174 sequences (Fig. 3) resulted in a 2-fold ments from the 780 promoter as designated. The open and black increase in transcription relative to the wild-type level. The boxes denote the regions of the 780 promoter from - 112 to - 38 bp - to + 1 respectively. The stippled region represents the reverse polarity ofthe activator element in the same location and 37 bp, 780 transcribed and 3' flanking sequences from + 1 bp to -150 bp (780 B/OX) also stimulated activity to greater than wild-type downstream of the poly(A) addition site (+ 926). The hatched box is levels (183%). The 4X174 fragment alone in either orientation the #X174 Hae III 603-bp fragment with the 10-bp Sal I linkers had a RTL <2%, similar to the test gene alone. added. The panel at the right shows the RTLs representing percent- The activity level of construct - 476/ - 112 5' A was ages relative to the 780 wild-type gene activity (WT). Usually RTLs significantly lower than the activity of a similar 780 promoter varied ± 1o, or less, whereas the RTLs s2.1 varied only ± 0.9%o of wild-type activity between experiments. construction, ID - 112/ - 37, which was reported to be 140% of the wild-type level (11). The internal deletion mutant represented 1-2% of the major promoter activity (data not - -112 and -37 ID 112/-37 has sequences between bp shown) and were not investigated further. replaced with a 6-bp Sal I linker. Addition of 19 bp of pUC19 Evaluation of TATA-Distal Regions of the ocs and ags polylinker between the - 112-bp and - 37-bp deletion sites in Promoters. The promoter regions immediately upstream of in constructing the -476/-112 5' A promoter resulted the putative CCAAT boxes of the ocs and ags genes were one-third less transcription relative to the ID - 112/-37 examined for their ability to promote activity of the 780 test deletion mutant. It is unclear whether altered spacing of gene. A TATA-distal portion of the ocs promoter from

between - cis-acting elements, changed helical orientation cis positions - 330 to 115 bp and the ags promoter region from elements, or the actual sequence of the additional 19 bp positions - 314 to - 155 bp were placed separately in both caused the reduction in activity. polarities, upstream and downstream of the 780 test gene. The 780 activator element was not able to cause any The autoradiographs of S1 nuclease mapping gels assaying detectable stimulation of transcription when placed down- the ocs/780 and ags/780 constructions are shown in Fig. 2B stream of the test gene. Positioning the - 476/ - 112-bp with the calculated RTL values for each construction given fragment -200 bp downstream from the poly(A) addition site in Fig. 4. Placing these heterologous promoter fragments resulted in no enhancement ofactivity in either polarity. This either immediately upstream of the 780 TATA or =200 bp result is similar to the studies ofthe light-inducible enhancers downstream of the poly(A) addition site failed to stimulate from RbcS and Cab genes that do not activate transcription transcription (<2% activity) with one exception; the ocs of the nos or cauliflower mosaic virus 35S promoter while in fragment in the B orientation (OCS 5' B, Fig. 4) resulted in the downstream location (6, 9). a 7-fold increase in transcription (14% of wild-type activity). The predominant transcripts of the 780 gene initiated from 25 to 30 bp downstream ofTATA in all constructions (Fig. 2). DISCUSSION However, some low-level transcripts were seen initiating upstream of - 37 bp in constructions containing the activator The 780 promoter has been shown to consist of three major in the 5' position. These spurious upstream start sites functional domains: the TATA, an upstream element (TATA- Downloaded by guest on October 1, 2021 Cell Biology: Bruce et al. Proc. Natl. Acad. Sci. USA 85 (1988) 4313 downstream (9). The enhancer region of the cauliflower -476 -12-1 1 2 * v mosaic virus 35S promoter is bidirectional and stimulates the 0 0 promoters ofT-DNA genes S and 7 (22). It is noteworthy that WT _ stimulation of gene 5 transcription occurs when the 35S enhancer is positioned -2000 bp downstream of the pro- 2.0 moter. Certain T-DNA promoters seem to be more readily -330 f-115 stimulated by downstream enhancers than others, since no stimulation is observed when the 35S enhancer is positioned OCS SI < 1 4 downstream of the nos promoter (22). In the case of the 780 -115 -330--- activator, it is unclear whether the lack of 3' function is a or AGS V A 2.0 general property of this enhancer-like element simply -314 characteristic of the particular 780 gene fusions examined. -133 The insertion of 19 bp (unique out of 25 total) of linker AGS S5 B 42.0 sequences between the activator fragment (- 476 to - 112) -133 -314 and - 37 resulted in a reduction in activity from a RTL of 143 -37 +926 obtained previously (11) to a value of 93 (Fig. 3). Not all oCsV A _ 1-2.o insertions, however, cause a decrease in activity since -330 -115 insertion ofthe 613-bp Hae III fragment of4X174 at the same position resulted in a >2-fold stimulation in activity. OCQ B _II 2.0 The presence of an enhancer-like element in the promoter -115 -330 of the 780 gene raises the possibility that other T-DNA genes AGS 3' A 2.0 may have this type of promoter structure. For example, the -314 -133 nos gene contains a series of repeats that correspond with the 5' boundary of the promoter that may have enhancer-like AGS 3' B 42.0 activity (23). We tested TATA-distal domains from two other -133 -314 T-DNA genes to evaluate their ability to potentiate transcrip- -37 tion from the deleted 780 promoter. When TATA-distal 4 .. A-3 7 4f *4e*4. . 2.0 regions of the ocs and ags promoters were placed in both orientations upstream of the 780 TATA, transcriptional activity was not stimulated in all but one case (Fig. 4). The FIG. 4. Schematic representaion of the ocs and ags fragments exception was the B orientation ofthe ocs fragment (- 330 to in various positions relative to the 780 test gene. The ocs and ags - 115) that resulted in a RTL of 14 representing a 7-fold constructions correspond to the S1 nuclease analysis in Fig. 2B. The enhancement. Neither the ocs not the acti- light hatched arrows represent the ocs fragment (- 330 to -115 bp ags fragments relative to the original ocs gene start site), whereas the dark hatched vated transcription when positioned 3' to the 780 gene. arrows represent ags promoter fragment (- 314 to - 133 bp relative The lack of strong 5' activity with the ocs fragment is to the original the ags gene start site). "WT" refers to the wild-type surprising since these sequences enable inducible expression 780 gene (+ 1 to + 926). The panel at the right shows the RTLs ofthe maize Adh-1 promoter to occur in tobacco when placed representing percentages of 780 wild-type gene activity. The RTLs either upstream ofposition - 100 or 10 bp upstream ofTATA varied to within ± 0.9%o of wild-type activity between experiments. (10, 24), suggesting that this region of the ocs gene has enhancer-like properties. The ocs fragment contains a 16-bp proximal element), and a TATA-distal activator element (11). inverted repeat (5' ACGTAAGCGCTTACGT 3') that alone is The TATA-distal location (-440 to -229) in the wild-type sufficient to activate the maize Adh-1 promoter when placed gene and flexibility in spacing requirements of the activator upstream of TATA in either orientation (24). The reason(s) suggested that this element may possess other enhancer-like that the B orientation of the ocs fragment, and not the A properties. Additional characteristics of enhancers include orientation, activated transcription is unclear. One possibil- the ability to stimulate transcription independently of orien- ity that invokes a DNA-looping mechanism (25-27) involves tation and distance in heterologous and homologous promot- orientation-specific differences in the spacing of cis elements ers. In the present study we further establish that the 780 within the ocs fragment and the 780 TATA. For example, the activator imparts two of these characteristics by demonstrat- ocs 16-bp inverted repeat is positioned 56 bp further upstream ing its ability to stimulate transcription in a bidirectional in the B orientation and therefore may be less constrained in manner over relatively large distances from TATA in the bending toward the TATA region. absence of other upstream elements. The activator element The lack of activity by the ags and ocs upstream promoter increased transcription up to 105-fold when placed 5' to the fragments may reflect a requirement for the presence ofother TATA of the 780 gene. The 780 activator can also enhance upstream elements in addition to the TATA for optimal transcription of heterologous genes since, when inserted at function in sunflower tumors. In the case of the ags fragment position - 140, unpublished results from our laboratory (John this seems likely since deletion of sequences in the TATA- Ingersoll and W.B.G.) indicate that the 780 activator stimu- proximal domain (-165 to -40) of ags abolishes transcrip- lates transcriptional activity of the maize Adh-) promoter in tional activity in sunflower tumors (unpublished). It is not sunflower tumors. However, in contrast to many animal viral known if a similar requirement exists for the ocs enhancer- and cellular enhancers, the 780 activator did not stimulate like region when expressed in sunflower. The 780 activator transcription when positioned 3' to the gene. seems to differ from the ags TATA-distal domain by not The lack of 3' activity has also been seen with enhancer- having a strong functional requirement for additional up- like elements from other plant genes. The upstream region of stream elements. In this respect, the 780 activator is similar the RbcS gene that imparts light inducibility and tissue to the activator element present in the T-cyt gene (cytokinin specificity does not promote specific transcription of the nos production, gene 4 of T-left) promoter that also requires only promoter fusion gene when placed downstream (6). Simi- a TATA for wild-type activity (28). The T-cyt activator, larly, a 247-bp region (- 347 to -100 bp) from the Cab gene however, does not appear to be a typical enhancer-like also confers light induction and tissue specificity on the element since it does not function when the distance between constitutive nos promoter when placed 5' in either polarity it and TATA is increased over that ofthe wild-type gene (28). but does not influence the nos promoter when positioned Alternatively, the lack of stimulation of the 780 TATA by Downloaded by guest on October 1, 2021 4314 Cell Biology: Bruce et al. Proc. Natl. Acad. Sci. USA 85 (1988) heterologous promoter elements from the ocs and ags genes 11. Bruce, W. B. & Gurley, W. B. (1987) Mol. Cell. Biol. 7, 59-67. may be due to incompatibility between promoter components 12. Chang, A. C. Y. & Cohen, S. N. (1978) J. Bacteriol. 134, for mosaic promoters comprised of 1141-1156. similar to that reported 13. Barker, R. F., Idler, K. B., Thompson, D. V. & Kemp, J. D. the TATA from the herpes simplex thymidine kinase gene (1983) Plant Mol. Biol. 2, 335-350. and TATA-proximal modules from the rabbit B-globin gene 14. Willmitzer, L., Dhaese, P., Schreier, P. H., Schmalenbach, (29). W., Van Montagu, M. & Schell, J. (1983) Cell 19, 729-739. Enhancers are a class of promoter elements that act over 15. Salomon, F., Deblaere, R., Leemans, J., Hernalsteens, J.-P., large distances to stimulate transcription and seem to be Van Montagu, M. & Schell, J. (1984) EMBO J. 3, 141-146. uniquely associated with eukaryotic genes. Prokaryotic 16. Karcher, S. J., DiRita, V. J. & Gelvin, S. B. (1984) Mol. Gen. components that Genet. 194, 159-165. genes such as gal, ara, and deo contain 17. Winter, J. A., Wright, R. L. & Gurley, W. B. (1984) Nucleic show long-range cooperativity (30-32), but these regulatory Acids Res. 12, 2391-2406. sequences act to repress promoter activity. DNase I protec- 18. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular tion ("footprinting") studies indicate that the 780 activator Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold interacts specifically with proteins from crude nuclear ex- Spring Harbor, NY). tracts of soybean (33). Hence, it seems likely that the 19. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, activators of T-DNA genes have evolved to utilize trans- A. R., Fiddes, J. C., Hutchinson, C. A., III, Slocombe, P. M. acting factors that are conserved in a wide range of dicoty- & Smith, M. (1977) Nature (London) 265, 687-695. ledonous plants. Although T-DNA is of bacterial origin, the 20. Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P., ocs Flick, J., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, presence of enhancer-like activators in the 780 and genes C. L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffmann, suggests that their adaptation to the transcriptional apparatus N. L. & Woo, S. C. (1983) Proc. Natl. Acad. Sci. USA 80, of higher plants has resulted in promoters that are truly 4803-4807. eukaryotic in function and organization. 21. Gurley, W. B., Czarnecka, E., Nagao, R. T. & Key, J. L. Czarnecka for (1986) Mol. Cell. Biol. 6, 559-576. We thank Richard Amasino, Dulce Barros, and Eva 22. Kay, R., Chan, A., Daly, M. & McPherson, J. (1987) Science critical review of this manuscript. This work was supported by a 236, 1299-1302. contract from Lubrizol Genetics, Inc. This paper is Florida Agricul- 23. An, G., Ebert, P. R., Yi, B.-Y. & Choi, C.-H. (1986) Mol. Gen. tural Experiment Station Journal Series no. 8963. Genet. 203, 245-250. 1. Dynan, W. S. & Tjian, R. (1985) Nature (London) 316, 774- 24. Ellis, J. G., Llewellyn, D. J., Walker, J. C., Dennis, E. S. & 778. Peacock, W. J. (1987) EMBO J. 6, 3203-3208. 2. Khoury, G. & Gruss, P. (1983) Cell 33, 313-314. 25. Hochschild, A. & Ptashne, M. (1986) Cell 44, 681-687. 3. Banedji, J., Rusconi, S. & Schaffner, W. (1981) Cell 27, 26. Mossing, M. C. & Record, M. T., Jr. (1986) Science 233, 299-308. 889-892. 4. de Villiers, J. & Schaffner, W. (1981) Nucleic Acids Res. 9, 27. Shore, D. & Baldwin, R. L. (1983) J. Mol. Biol. 170, 957-981. 6251-6254. 28. DePater, B. S., Klinkhamer, M. P., Amesz, P. A., de Kam, 5. Moreau, P., Hen, R., Wasylyk, B., Everrett, R., Gaub, M. P. R. J., Memelink, J., Hoge, J. H. C. & Schilperoort, R. A. & Chambon, P. (1981) Nucleic Acids Res. 9, 6047-6069. (1987) Nucleic Acids Res. 15, 8267-8281. 6. Timko, M., Kausch, A., Castresana, C., Fassler, J., Herrera- 29. Cochran, M. D. & Weissman, C. (1984) EMBO J. 3, 2453-2459. Estrella, L., Van den Broeck, G., Van Montagu, M., Schell, J. 30. Majumdar, A. & Adhya, S. (1984) Proc. Natl. Acad. Sci. USA & Cashmore, A. (1985) Nature (London) 318, 579-582. 81, 6100-6104. 7. Fluhr, R., Kuhlemeier, C., Nagy, F. & Chua, N.-H. (1986) 31. Dunn, T. M., Hahn, S., Ogden, S. & Schleif, R. F. (1984) Proc. Science 232, 1106-1112. Natl. Acad. Sci. USA 81, 5017-5020. 8. Morelli, G., Nagy, F., Fraley, R. T., Rogers, S. G. & Chua, 32. Dandanell, G., Valentin-Hansen, P., Larsen, J. E. L. & Ham- N.-H. (1985) Nature (London) 315, 200-204. mer, K. (1987) Nature (London) 325, 823-826. 9. Simpson, J., Schell, J., Van Montagu, M. & Herrera-Estrella, 33. Bruce, W. B. (1987) Dissertation (UniversityofFlorida, Gaines- L. (1986) Nature (London) 323, 551-554. ville, FL). 10. Ellis, J. G., Llewellyn, D. J., Dennis, E. S. & Peacock, W. J. 34. Thomashow, M., Nutter, R., Montoya, A., Gordon, M. & (1987) EMBO J. 6, 11-16. Nester, E. (1980) Cell 19, 729-739. Downloaded by guest on October 1, 2021