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An Enhancer-Like Element Present in the Promoter of a T-DNA Gene

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An Enhancer-Like Element Present in the Promoter of a T-DNA Gene 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.
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