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The Plant Cell, Vol. 1, 925-936, September 1989 O 1989 American Society of Plant Physiologists Multiple Domains Exist within the Upstream Activator Sequence of the Octopine Synthase Gene Scott M. Leisner and Stanton 8. Gelvin' Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 It is known that a 16-base pair palindrome (ACGTAAGCGCTTACGT)located upstream of the ocs gene can activate a maize adhl promoter in a transient expression system [Ellis et al. (1987). EMBO J. 6, 11-16; Ellis et al. (1987). EMBO J. 6, 3203-32081. We have determined that this palindrome is also essential for ocs promoter activity in tobacco calli. In addition, sequences immediately adjacent to this palindrome, both 5' and 3', modulate its activity. The palindrome is sensitive to the differentiated state of the plant cells in which it resides; it is active in calli and the leaves of small shoots but is inactive in the leaves of rooted plants. We have tested upstream sequences from two other T-DNA genes that have homology to this palindrome for their ability to activate the octopine synthase promoter in tobacco calli. The upstream region from the mannopine synthase gene can activate the octopine synthase promoter, but an upstream region from the gene implicated in octopine and nopaline secretion cannot activate the promoter. INTRODUCTION The Gram-negative soil bacterium Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous and some monocotyledonous plants. Virulent strains of this bacterium harbor a large tumor-inducing (Ti) plasmid and transfer a portion of this plasmid, the transferred DNA (T-DNA), to a susceptible plant cell. The T-DNA, shown schematically in Figure 1, integrates into the plant nuclear genome where its genes are expressed. Some T-DNA genes encode enzymes that synthesize plant hormones, resulting in the tumor phenotype. Other T-DNA genes direct the synthesis and secretion of unusual amino acid and sugar derivatives called opines. The inciting strain of Agrobacterium can utilize these opines as a carbon and sometimes a nitrogen source. Agrobacterium strains can be classified based upon their ability to catabolize particular opines. (For recent reviews of the crown gall tumorigenesis process, see Nester et al., 1984; Binns and Thomashow, 1988.) The regulationof T-DNA gene expression is an interesting problem because the genes are normally harbored on a prokaryotic plasmid but are subsequently expressed in eukaryotes. T-DNA genes possess all of the sequence elements required for transcription in plants. These elements have been studied for several T-DNA genes, including those involved in cytokinin biosynthesis, tmr (de Pater et al., 1987); agropine biosynthesis, ags (Bruce, Bandyopadhyay, and Gurley, 1988); octopine biosynthesis, ocs (Ellis et al., 1987a, 1987b; Leisner and Gelvin, 1988); ' To whom correspondence should be addressed mannopine biosynthesis, mas (DiRita and Gelvin, 1987); nopaline biosynthesis, nos (An, 1987; Mitra and An, 1989); and a gene encoding a 780-nucleotide mRNA from TR (Bruce, Bandyopadhyay, and Gurley, 1988). These T-DNA genes contain TATA boxes that set the site of transcription initiation, and many contain upstream elements, located more than 1O0 bp from the transcription initiation site, that modulate the levels of transcription. The nos gene, the ocs gene, and the 780 gene all contain upstreamtranscriptional activating sequences that can activate promoters when present in either orientation (Ellis et al., 1987a, 1987b; Leisner and Gelvin, 1988; Bruce, Bandyopadhyay, and Gurley, 1988; Mitra and An, 1989). The ags element cannot activate the promoter for the 780 gene in either orientation (Bruce, Bandyopadhyay, and Gurley, 1988). The tmr element has not been tested for its ability to activate promoters independent of its orientation. The 780 gene element activates its promoter when separated by 613 bp (Bruce, Bandyopadhyay, and Gurley, 1988). Each of the other elements, however, cannot function when placed more than several hundred base pairs from a promoter (de Pater et al., 1987; Leisner and Gelvin, 1988; Bruce, Bandyopadhyay, and Gurley, 1988; Mitra and An, 1989). As with many plant upstream activating sequences (Timko et al. 1985), these T-DNA upstream elements more closely resemble yeast upstream activation sites than transcriptional enhancers. We are interested in the upstream element of the octopine synthase (ocs) gene. The product of this gene condenses arginine and pyruvate to form the opine, octopine 926 The Plant Cell TL b 5 7 ims -------D--+ 1 . 2 I m r onstml ocs 780 - 4 4' 3' mas ogs 375 2 Figure 1. Schematic Map of the Bipartite T-DNA of pTiA6. Arrowheads denote the T-DNA borders and arrows indicate the direction of transcription of various open reading frames. (For reviews of the various T-DNA genes, see Garfinkel et al., 1981; Nester et al., 1984; Binns and Thomashow, 1988.) Genes 5 and 7 encode mRNAs of unknown function. The tmsl and tms2 genes encode enzymes of a two-step pathway for the biosynthesis of auxins, and the tmr gene encodes an enzyme for the biosynthesis of cytokinins. The ons gene encodes a protein involved in octopine and nopaline secretion (Messens et al., 1985), and the tml gene regulates tumor size in some plant species. The ocs gene encodes an enzyme that synthesizes octopine (Hack and Kemp, 1980), and the 780 gene (4') encodes a 780-nucleotide-longmRNA of unknown function (Karcher, DiRita, and Gelvin, 1984). The 3' gene also encodes an mRNA of unknown function. The mas gene (2') and the 1' gene encode the enzymes of a two-step pathway for the synthesis of mannopine (Ellis, Ryder, and Tate, 1984; Komro et al., 1985), and the ags gene encodes an enzyme that cyclizes mannopineto yield agropine (Ellis, Ryder, and Tate, 1984; Komro et al., 1985). Dark boxes at the 5' ends of the ocs and mas genes indicate the presence of orientation-independent upstream elements that activate the ocs promoter. The open box for the ons gene indicates that the upstream element cannot activate the ocs promoter. In this study, we investigated sequences from -333 to -1 16 from the transcription start site for the ocs gene, -318 to -213 from the transcription start for the mas gene, and 363 bp to 170 bp upstream from the translation start site for the ons gene. (Hack and Kemp, 1980). Previously, Ellis and co-workers (Ellis et al., 1987b) determined that a 16-bp palindrome (ACGTAAGCGCTTACGT) located upstream of the ocs gene could activate a heterologous promoter in transient expression assays. We found that a fragment (from -333 to -1 16) containing this palindrome is also important in activating the ocs promoter in tobacco calli when stably incorporated into the genome (Leisner and Gelvin, 1988). This fragment activated the ocs promoter when present in either orientation, but was position-dependent. In this paper, we present a more detailed analysis of the octopine synthase upstream activator sequence. The 16-bp palindrome is sufficient to activate the ocs promoter in tobacco calli. However, sequences adjacent to the palindrome influente its activity. We have additionally extended our analysis of the ocs upstream activator sequence to transgenic tobacco plants. Whereas the palindrome is sufficient to activate the ocs promoter in tobacco calli and the leaves of small shoots, it is insufficient to do so in the leaves of rooted plants. A portion of the 16-bp palindrome is found in the upstream regions of two other T-DNA genes, the mannopine synthase (mas) gene (Ellis, Ryder, and Tate, 1984; Komro et al., 1985; DiRita and Gelvin, 1987) and the gene implicated in octopine and nopaline secretion (ons)from transformed plant cells (Messens et al., 1985). Restrictionendonuclease fragments from the mas and ons genes, containing sequences similar to the palindrome, were tested for their ability to activate the ocs promoter. RESULTS Analysis of the Octopine Synthase Upstream Activator Sequence An upstream activator sequence (UAS) regulates transcription of the octopine synthase gene (Ellis et al., 1987a, 1987b; Leisner and Gelvin, 1988). To determine which sequences present within the UAS are required for activity in tobacco calli, we cloned various restriction endonuclease fragments of the ocs activator element into pOCSA2. These fragments are described in Figures 2 and 3. After mobilizationof plasmids harboring these fragments cloned upstream of the ocs gene into A. tumefaciens strain LBA4404, we infected tobacco leaf discs and selected kanamycin-resistant calli. We pooled 20 or more calli, assayed them for octopine synthase activity (Otten and Schilperoort, 1978), and determined the amount of octopine synthesized per microgram of protein. Figure 4A shows the results of a typical octopine synthase assay. Figure 48 shows that octopine synthase activity correlates with the steady-state leve1 of ocs mRNA in tobacco calli. These data corroborate our previous findings (Leisner and Gelvin, 1988). 5' and 3' Deletion Analysis of the Octopine Synthase UpstreamActivator Sequence Calli containing the construction pOCSA2 lacking the ocs upstream element had little detectable octopine synthase activity (Figure 3 and Figure 4A). The activity of calli harboring the 5' deletion plasmids pSaul33, pAlulO6, and pRsa61 (Figure 3) indicated that a region important for activator function is located between the Alul (-222) and Rsal (-177) sites. (The transcription initiation site of the ocs gene is designated as +1.) The 3' deletion construction pRsal31 indicated that the 3' boundary of an active region is located upstream of the Rsal site at -177. Therefore, we cloned the 45-bp fragment between the Alul (-222) and Rsal (-177) sites (pAIRa45, Figure 3) onto the ocs promoter. This construction resulted in full octopine synthase activity when transferred to tobacco calli (Figure 3 and Figure 4A). To localize further the active sequences present within the pAIRa45 fragment, we cloned the 38-bp fragment from -222 to -1 89 (pHin38) onto the ocs promoter (Figure 3). ocs Upstream Activator Sequence Domains A pSLl Ac I 927 Calli incited by bacteria harboring this plasmid had little detectable octopine synthase activity (Figure 3). The pAIRa45 fragment contains a 16-bp palindrome (ACGTAAGCGCTTACGT from -1 92 to -1 77),and this palindrome is truncated in the plasmid pHin38. Because lack of octopine synthase activity correlated with lack of a complete palindrome, we hypothesized that the palindrome could be responsible for the activity directed by the fragment contained in pAIRa45. To test this hypothesis, C Figure 2. lntermediate Plasmids Used in the Construction of Octopine Synthase Upstream Activator Deletions. (A) The plasmid pSLl (Leisner and Gelvin, 1988). pSLl contains the entire ocs upstream activator. Relevant restriction endonuclease sites for subcloning different fragments of the activator are shown. (B) The intermediate plasmids pG1, pAl, and pX1. All three of these plasmids are derived from pSLl as described in Methods. The fragments derived from pG1, pAl , and pX1 are shown below each plasmid map. The fragments from the plasmid pG1 are labeled PD for promoter-dista1and PP for promoter-proximal. (C) The plasmids pUX13 and pOCSA2. pUX13 is pUC13 with the Smal site converted to Xhol by linker addition. We have described the constructionof pOCSA2 previously (Leisner and Gelvin, 1988). The arrow indicates the direction of transcription of the ocs gene. The BamHl site is at -1 16 bp relativeto the transcriptioninitiation site. Restriction endonuclease sites are abbreviated as follows: Ac, Accl; Ac-B, Accl converted to BamHI; A, Alul; A-B, Alul converted to BamHI; B, BamHI; D, Dral; E, EcoRI; H, Hindlll; Hc, Hincll; M, Mspl; P, HinPI; Ps, Pstl; R, Rsal; S, Sall; Sa, Sau3a; Sa/B, Sau3a-BamHI fusion not cleavable by BamHI; Sm, Smal; Ss, Sstl; T, Taql; X, Xhol; Xb, Xbal. The area containing diagonal lines indicates the 16-bp palindrome. PSRj P S I - t w - . . . - M +ms- NXSAZ+m$- 34 2 1653 99 34 2 Figure 3. Deletion Analysis of the Octopine Synthase Upstream Activator Sequence. The arrow with ocs in the middle indicates the orientation of the octopine synthase gene. The promoter fragment used begins at -1 16 bp relative to the transcription start site. Sequences to the left of -116 in the construction pENl contain the upstream activator. The upstream activator sequence extends from -333 to -1 16, and the relevant restriction enzyme sites are marked. The locations of the SV40 enhancer core sequence, a potential 2-DNA-forming sequence, and the 16-bp palindrome are marked as black boxes. The location of the palindrome is also marked as an open box on each construction. The arrowheads for each construction indicate the orientation of the construction relative to the promoter; an arrow pointing to the right indicates the wildtype orientation. The amount of octopine (pmol) made per microgram of protein during a 2-hr incubation is indicated, as is the relative leve1 of octopine synthase activity (normalized to pEN1 as 100%) for each construction. One unit of octopine synthase activity is equivalent to 0.8 pmol of octopine synthesized per hour per microgram of protein. pENl synthesized about 2500 pmol of octopine. 928 The Plant Cell OJ Incubation time (Hrs) o~~2 Incubation time < 5E I 0. 8 3 2 C~2 6~2 C/1 0. (Hrs) ARG — ARG- OCT— OCT — 2.0 - Figure 4. Octopine Synthase Assay of Various ocs Activator Deletions. (A) Electrophoretogram of octopine synthase assays performed on 20 pooled calli incited with the constructions indicated above. Positions of octopine (OCT) and arginine (ARG) are marked. Numbers above indicate the time of incubation in hours. Origin (+) is at the bottom. The amount of protein in each extract was 9.3 ^g for pEN1, 15.8 ^g for pENR1, 7.5 ,ug f°r pAIRa45, 24 ng for pPAL16, 10.3 /jg for pOCSA2, 7.4 jig for pAITaR54 and 11.8 ^g for pAluR106. The amount of octopine made after 2 hr was 2498 pmol of octopine for pEN1, 1070 pmol for pENR1, 1636 for pAIRa45, 2059 pmol for pPAL16, 53.9 pmol for pOCSA2, 3817 pmol for pAITaR54 and 5980 pmol for pAluR106. (B) Correlation of octopine synthase activity with the steady-state level of octopine synthase mRNA. Electrophoretogram of octopine synthase assays performed on 20 pooled calli incited with the constructions indicated above. Positions of octopine (OCT) and arginine (ARG) are marked. Numbers above indicate the time of incubation in hours. Origin (+) is at the bottom. The amount of protein in each extract was 9.3 ng for pEN1, 15.8 ^g for pENR1, and 10.3 ^g for pOCSA2. The amount of octopine made after 2 hr was 2498 pmol for pEN1, 1070 pmol for pENR1, and 53.9 pmol for pOCSA2. The graph below is a densitometer tracing of an RNA gel blot of total RNA isolated from pooled calli incited with pEN1, pENR1, and pOCSA2. We subjected 15 ^g of total RNA to electrophoresis through a 1.5% agarose gel containing 6% formaldehyde, blotted the RNA onto nitrocellulose (Karcher, DiRita, and Gelvin, 1984), and hybridized the filter with a Oral fragment [12221 to 13685 (Barker et al., 1983)] encompassing the ocs gene. We scanned an autoradiograph of the blot with a densitometer. The peaks shown below the electrophoretogram indicate the amount of ocs mRNA present in the calli harboring each construction. The ordinate for this figure is relative OD. we synthesized an oligonucleotide containing the sequence of the palindrome and cloned it onto the 5' end of the ocs gene. When mobilized into tobacco calli, this construction, pPAL16, was also active (Figure 3 and Figure 4A). Therefore, the palindrome is necessary and sufficient to stimulate the ocs promoter in tobacco calli. These data correlate with the findings of Ellis et al. (1987b). The constructions pHin70, pHinR70, pHin38, pHinR38, and pSaHi69 all contain half of the palindrome and are inactive in tobacco calli. The palindrome must therefore be complete in order for the UAS to function. Oligonucleotides corresponding to either half of the palindrome, pHPPD and ocs Upstream Activator Sequence Domains pHPPP, are also nonfunctional when cloned onto the ocs gene (Figure 3). Although the palindrome alone can activate the ocs promoter in tobacco calli, it is not as active as are the larger elements pEN1 or pAIRa45 (Figure 3 and Figure 4A). These data indicate that sequences present between -222 and -193, within pAIRa45, augment the palindrome to provide full activity. A subset of these additional sequences 5' to the palindrome contained within pHin38 [the 5' palindrome augmenting sequence (5' PAS)] cannot augment the activity of a truncated palindrome, suggesting that the 5' PAS cannot function by itself. Sequences downstreamof the palindrome also augment the activity of the palindrome. The plasmids pAluR106 (-222 to -1 16) and pAITaR54 (-222 to -1 70) are both equally active in tobacco calli, and the activity of both is greater than that of the larger element pENl (Figure 3 and Figure 4A). In both of these constructions, the palindrome is 24 bp closer to the promoter than it is in pENl , and both contain potential Z-DNA stretches. The palindrome is 120 bp from the TATA box in pAITaR54, pAluRl06, and pAIRaR45. However, the activity of pAIRaR45 is equivalent to that of pEN1. These results suggest that the enhancement of activity for pAITaR54 and pAluRlO6 over that of pEN1 is most likely due to the presence of additional sequences 3' to the palindrome (termed the 3' palindrome augmenting sequence, or 3' PAS), rather than the closer proximity of the palindrome to the promoter. pENl also has the 3' PAS but is less active than pAlulO6, pAluRlO6, and pAlTaR54 because it may contain inhibitorysequences (see below). The 3' PAS cannot augment the activity of a truncated palindrome (pHin70), suggesting that the 3' PAS cannot function on its own (Figure 3). Sequences between the Sau3a (-249) and Alul(-222) sites also modulate the activity of the octopine synthase UAS. The activity of the pSau133 fragment is equivalent to that of the larger element (pENl), whereas the activity of the pAlulO6 fragment is greater (Figure 3). One possible interpretation of these data is that inhibitory sequences are located between the Alul and Sau3a sites. Interestingly, pAIRa45 lacks these putative inhibitory sequences but is only as active as is pENl . These data suggest that not only must the putative inhibitory sequences be removed, but also that the potential 2-DNA sequence must be present to elicit additional activity. These data also explain why pEN1 is less active than are pAlul06, pAluR106, and pAITaR54. When we cloned the restriction endonuclease fragments pSau84, pMsp42, and pMpSa42 upstream of the ocs gene, we observed no octopine synthase activity in tobacco calli. These data indicate that sequences upstream of the Sau3a site (-249), including a SV40 enhancer core sequence, are not sufficient for activity. Neither do these sequences augment the activity of either pRsa61 (pSR1) or pAIRa45 (pSA1). The plasmid pSR1 is inactive, as are pRsa61 and pSau84. These data indicate that the Sau84 929 fragment cannot activate an already inactive Rsa61 fragment. In addition, the sequences between the Sau3a (-249) and Rsal (-177) sites are absolutely required for activity. The construction pSA1 is only as active as is AIRa45. These data indicatethat the Sau84 fragment does not augment the activity of the AIRa45 fragment and has no stimulatory effect even on positively acting sequences. We had previously shown that a dimer of the Sau84 (-333 to -249) fragment in the reverse orientation could also activate the ocs promoter (Leisner and Gelvin, 1988). We have subsequently been unable to reproduce this result. Table 1 shows the number of base pairs between the palindrome and the TATA box for a number of the constructions. In general, when the orientation of the various UAS fragments is such that the palindrome is closer to the ocs promoter, these constructions are more active than are those in which the palindrome is further away. For example, pENR1 is about 40% as active as is pENl . The palindrome is 231 bp and 146 bp from the TATA box for pENRl and pENl , respectively. pRsal31 has the same activity as does pENl , whereas pRsaR131 has the same activity as does pENR1. The palindrome is 98 bp and 212 bp from the TATA box for pRsal31 and pRsaR131, respectively. The activity of both pAIRa45 and pAIRaR45 is approximately the same as is the activity of pENl . The palindrome is 91 bp and 120 bp from the TATA box for pAIRa45 and pAIRaR45, respectively. Likewise, pAlulO6 and pAluRlO6 are both approximately equally active, and the activity of both is greater than that of pENl . In these constructions, the palindrome is 146 bp and 120 bp from the TATA box for pAlulO6 and pAluRl06, respectively (Table 1). These data indicate that UAS fragments can activate the ocs promoter to wild-type levels when the palindrome is 91 bp Table 1. Distance of Palindrome from TATA Box Construction Distance from TATA Box Relative Activity in Calli" bP pENl pENRl pSaul33 pAlulO6 pAluRlO6 pRsal31 pRsaRl31 pAIRa45 pSAl pAIRaR45 pAITaR54 pPALl6 146 231 146 146 120 98 212 91 91 120 120 88 1O0 41 99 160 21o 90 36 97 99 105 215 36 numbers are relative to pENl . pENl synthesized 167 pmol of octopine per microgram of protein during a 2-hr incubation. a The 930 The Plant Cell to 146 bp from the TATA box, but when the palindrome is moved greater than 200 bp from the TATA box, activity is reduced. These data corroborate our previous findings that octopine synthase activity is abolished when the activator is separated from the promoter by 608 bp (the palindrome is 754 bp from the TATA box; Leisner and Gelvin, 1988). Various UAS SubfragmentsFunction Differently in Shoots and in Calli We obtained transgenic shoots from tobacco calli containing pENl, pENRl , pAlulO6, pAIRa45, and pPAL16. For each construction, we pooled leaves from 10 plants and tested them for octopine synthase activity. Leaves were tested at two separate times during development: When they were 1 cm to 2 cm high, and when they were 8 cm to 12 cm high and had formed roots. Table 2 shows the activity of the ocs element in calli, the leaves of small shoots, and the leaves of rooted plants. If the construction was designated as ocs-positive, the pooled leaves contained 20% or more of the octopine synthase activity of the construction pEN1 in that tissue. ocs-Negative plants contained 2% or less of the octopine synthase activity of the construction pEN1 in that tissue. All of the constructs that are active in calli are also active in the leaves of small shoots and the leaves of rooted plants except for pPAL16. This construction is active in calli and the leaves of small shoots but is inactive in the leaves of rooted plants. Table 2. Activity of UAS Constructions in Calli, Small Shoots, and Rooted Plants ~ pENl pENRl pAlulO6 pAIRa45 pPALl6 pOCSA2 Activity in Calli" Activity in Leaves of Small Shoots" + + + + + + + + 4- - + - Activity in Leaves of Rooted Plants* + + + + - + indicates that the pooled plant tissue contained 20% or more of the octopine synthase activity of the construction pENl in that tissue. - indicates that the pooled plant tissue contained 2% or less of the octopine synthase activity of the construction pENl in that tissue. The activity of the construction pENl in calli, the leaves of small shoots, and the leaves of rooted plants was 0.83 pmol, 0.17 pmol, and 0.23 pmol of octopine made per hour per microgram of protein, respectively. Do Upstream Regions of Other T-DNA Genes That Have Homology to the Palindrome also Contain Upstream Activator Sequences That Can Activate the ocs Promoter? Because the 16-bp palindrome is an essential motif in the octopine synthase UAS (Ellis et al., 1987b), we scanned the 5'-flanking sequences of other T-DNA genes for homology to the palindrome. The 5'-flanking sequences of both a gene involved in mannopine biosynthesis(mas) and the gene implicated in octopine and nopaline secretion (ons) contain sequences homologous to the ocs palindrome. Figure 5A shows schematically the regions containing these sequences homologous to the palindrome used in this study. Figure 58 shows the DNA sequences of these regions as determined by Barker et al., 1983 and DeGreve et al., 1983. In a previous study of the 5'-flanking sequence of the mannopine synthase gene, we hypothesizedthe existence of a transcriptional activating element located between -31 8 bp and -1 30 bp from the transcription start site (DiRita and Gelvin, 1987). This region also contains 7 contiguous base pairs homologous to the promoter-dista1 portion of the palindrome (ACGTAAG). To determine whether this element had the properties of an upstream activator, we cloned a subfragment of this region from -318 to -213, containing sequences homologous to the palindrome, onto the ocs promoter in both orientations. We subsequently mobilized these constructions into tobacco cells and assayed octopine synthase activity. We observed octopine synthase activity for both of these constructions. The mas element activates the ocs gene to a greater extent in the correct orientation than in the reverse orientation (Figure 5A). Neither orientation of the mas element functions as well as does the ocs activator to stimulate the ocs gene. These results indicate that an upstream region containing homology to the palindrome from another T-DNA gene functions as an upstream activator of a heterologous (ocs) promoter. It does not, however, prove that the 7 bp containing homology to the ocs palindrome are responsible for the activity of the mas activator. The 5' end of the ons gene also has some homology (6 bp) to the promoter-dista1 portion of the ocs palindrome (CGTAAG). We cloned a fragment (-363 bp to -170 bp upstream from the ATG) from the ons 5'-flanking sequence onto the ocs promoter. This ons sequence did not activate the ocs promoter in tobacco calli (Figure 5A). a DlSCUSSlON We have examined the octopine synthase upstream activator sequence in detail. Deletion analysis indicated that an essential sequence was located between 222 bp and ocs Upstream Activator Sequence Domains A Onwne Relalive Synlhase Octoptne AcawIy Synlhase (unW Acln~ly - -__ 6-- 3 4 m' 2 ACAGGCCMA ITCGCTCTTA G C C G T A C M T ATTACTCACC GGTCCGATGC EB1: enr CCCCCATCGT AGGTGMGGT G G M A T T M T GATCCATCTT GAGACCACAG G C C C A C M C A ............................................................ .......... - - - T T A G T T l CCACGAMGT A T T A T G T T M A T T I T A M K T TTTCGATGTA CCTACCAGTT T C C T C M G G G TCCACCAAM ACCThAGCGC TTACCIACAT GGTCGATMG - A C A l T l l T C MATCAGTGC GCMGACGTG W T A T M T G T G G C T A T M T T G T M M A T M A C T A T-TGT T CTGACCTAGT TTTTATTTTT CCGTCTTATG TATMTTTGT A A M C G C M T TTGTAGATCI T M C A T C C M CCTCCCTTTC A G G G A T C - - - .......... C T A C T M T T T CCTCCTTTAT TTCGGCCTGT ACGACATCCC M C C C G G - - - .......... C T M A T G T T T M T A T A T A T C ATAGMCGCA A T M A T A T T A MTATAGCGC T T T T A T C M A ..................................... TATMATACA TCATTACMG TTGTTTATAT TTCGGGT Figure 5. Activity of Putative Upstream Activator Sequences from Severa1T-DNA Genes. (A) Schematic diagram of the regions from several T-DNA genes containing sequences homologous to the palindrome and their orientation with respect to the ocs gene. Box with x in it represents the ocs promoter beginning at -116 bp relative to the transcriptionstart site; arrow with ocs in it represents the octopine synthase structural gene, and the arrow indicates the direction of transcription. The rectangle with the thick arrow represents the different putative upstream activator sequences; the direction of the arrow indicates the orientation of the activator. The ocs element extends from -333 bp to -1 16 bp relative to the transcription start site, the mas element extends from -318 bp to -21 3 bp relativeto the transcriptionstart site, and the ons element extends from 363 bp to 170 bp upstream from the translation start site. The picomoles of octopine made during a 2-hr incubation per microgram of protein and the relative leve1 of octopine synthase activity (normalized to pEN1 as 100%) are indicated as in Figure 3. ( 8 ) The DNA sequences of upstream regions containing sequences homologous to the palindrome. The ocs element extends from -333 bp to -1 16 bp upstream from the transcription initiation site. The mas element extends from -318 bp to -213 bp upstream from the transcription initiation site. The ons element extends from -363 bp to -1 70 bp upstream from the translation initiation site. The palindrome homologies, including the palindrome itself for ocs, are underlined. The three sequences are arranged so that the palindrome homologies are aligned. 931 177 bp upstream from the transcription initiation site. This region contains a 16-bp palindrome (ACGTAAGCGCTTACGT), between -1 92 and -1 77, that can autonomously activate the ocs promoter in tobacco calli. This palindrome can also activate the maize Adhl promoter in transient expression assays (Ellis et al., 1987b). Many other eukaryotic genes, such as the c-fos gene (Greenberg, Siegfried, and Ziff, 1987), are also regulated by palindromic sequences. Sequences exhibiting dyad symmetry are believed to interact with dimeric proteins and, in the case of c-fos, proteins are known to bind to the palindrome. The ocs palindrome also interacts with a nuclear protein (Singh et al., 1989). The ocs palindrome must be intact to function. The element functions well when present in several locations from 91 bp to 146 bp from the TATA box (Table 1). Therefore, it most likely does not need to be on a particular face of the DNA helix with respect to the TATA box. When the palindrome is moved greater than 200 bp from the TATA box, its activity diminishes. Although the palindrome is the only motif in the ocs activator that can function autonomously, other sequences also play a subtle role in modulating the activity of the palindrome. In tobacco calli, the palindrome (pPAL16) is about one-thirdas active as is the larger element contained in pEN1. Because sequences between -222 and -1 77 (pAIRa45) activate the ocs promoter to wild-type levels, these data indicate that sequences between -222 and -193 augment the activity of the palindrome. Within this 5' PAS is an 8-bp sequence (CCTCAAGG) that is also found between the TATA box of the ocs gene and the transcription initiation site. Whether or not this sequence plays a role in the regulation of transcription of the ocs gene remains to be determined. If the sequences between -222 and -168 (pAITaR54) are cloned onto the ocs promoter, activity greater than that of the entire element is observed. In addition to the palindrome and the 5' PAS, this fragment contains an intact, potential Z-DNA-forming sequence. A potential ZDNA-forming sequence is an essential part of the nos upstream activator sequence (An, 1987; Mitra and An, 1989). It is possible that the potential Z-DNA-forming sequence may mark the DNA backbone in such a way as to allow proteins to bind to the palindrome more efficiently (An, 1987). It is also possible that the alternating purine and pyrimidine tract may not form Z-DNA but the 3' PAS may also be a protein binding site. The pSaul33 fragment (-249 to -1 16) activates the ocs promoter to wild-type levels but pAlulO6 (-222 to -1 16) directs greater than wild-type activity. These data suggest that sequences between -249 and -222 may be inhibitory.To observe greater than wild-type activity, these putative inhibitory sequences must be removed, but the 3' PAS must also be present in addition to the palindrome. These data indicate a complex interaction between the putative 5' inhibitory sequences and the 3' PAS. There- 932 The Plant Cell fore, the octopine synthase UAS may be regulated by interactions between both positive and negative elements. The pea rbcS3A gene and a soybean leghemoglobin gene are also regulatedby positive and negative elements (Kuhlemeier et al., 1987; Stougaard et al., 1987). The 16-bp palindrome activates the ocs promoter in both calli and the leaves of small shoots, but is inactive in the leaves of rooted plants. These results suggest that the activity of the palindrome is sensitive to the differentiated state of the tissue in which it resides. In addition, the larger the fragment harboring the 16-bp palindrome, the more active are the constructions in rooted plants. The ST-LS7 gene from potato also requires more 5’-flanking sequence for expression in differentiated tissues than in calli (Stockhaus et al., 1987). These observations may indicate that the regulation of gene expression in differentiated tissues is more complex than in calli. We have shown that either half of the palindrome by itself is not able to stimulate the ocs promoter. This result is interesting because the promoter-dista1 7 bp of the palindrome (ACGTAAG) is found within the 5’-flanking sequences of severa1 other genes, such as the cauliflower mosaic virus 35s promoter, the potato protease inhibitor II gene, the soybean lectin Lel gene, and the mas and ons genes (Barker et al., 1983; Vodkin, Rhodes, and Goldberg, 1983; Odell, Nagy, and Chua, 1985; Thornburg et al., 1987),that are expressed in plants. This sequence is found within a portion of the mannopine synthase (mas) upstream activating sequence essential for activity of the mas promoter (Figure 5). A 100-bp fragment of the mas upstream region harboring the ACGTAAG sequence can, in either orientation, stimulate an ocs promoter. The situation with the ocs UAS may be similar to that of the serum response element of the c-fos gene. The essential components of both the ocs UAS and the c-fos serum regulatory elements are palindromes. Neither of the two activates its own promoters if only half of the palindrome is present, whereas half of the palindrome sequence can be found in essential upstream regions of other genes (Elsholtz et al., 1986; Greenberg, Siegfried, and Ziff, 1987). It is also possible, however, that the presence of sequences homologous to a portion of the ocs palindrome is fortuitous and that these sequences play no role in the activity of the other genes. We tested an upstream portion from the ons gene for its ability to stimulate the ocs promoter. This fragment also has homology to a portion of the palindrome (CGTAAG). The ons fragment does not activate the ocs gene in either orientation. These data suggest that the ACGTAAG sequence, if important in activating the mas and ons genes, may need to be within the proper sequence context in order to augment the ocs promoter. It is also possible that the ons upstream fragment does not interact synergistically with the ocs promoter. Although the ocs element can activate the maize Adh 7 promoter in transient expression assays, it cannot activate the 780 gene promoter in sunflower tumors (Ellis et al., 1987a, 1987b; Bruce, Bandyopadhyay, and Gurley, 1988). These data indicate that not all promoter-activator pairs function in a productive manner. This has also been shown for the SV40 enhancer; although it stimulates the ,8-globin promoter by at least 100-fold, it cannot stimulate the a-globin promoter (Khoury and Gruss, 1983). In a previous study of the ocs element, Ellis et al. (1987b) indicated that the 16-bp palindrome was sufficient for activity in transient expression assays. These authors tested the effects of ocs activator fragments on the maize Adh7 promoter using chloramphenicol acetyltransferase as a reporter of gene activity. We studied the effects of ocs activator fragments on the ocs promoter in stable expression experiments using the ocs gene as a reporter of gene activity. Ellis et al. (1987b) did not test the role of sequences flanking the palindrome, nor the ability of the palindrome to function in rooted plants. We have found that sequences flanking the palindrome play a subtle role in the stable expression of the ocs gene in tobacco calli. The palindrome is also not sufficient for expression in the leaves of rooted plants. We are currently analyzing the various ocs activator fragments for tissue-specificexpression and determining at which stage of development they function or are inactive. METHODS Materials Restriction endonucleases were purchased from Bethesda Research Laboratories and used according to the manufacturer’s specifications. DNA polymerase I Klenow fragment and T4 DNA ligase were obtained from Pharmacia LKB Biotechnology, Inc. and used according to the manufacturer’s specifications. o~-~*PdCTP was purchased from Amersham. DNA fragments were labeled with an Amersham nick translation kit. Reagents for the octopine synthase assay and antibiotics were purchased from Sigma. Reagents for determining protein concentrations were purchased from Bio-Rad and used according to the manufacturer’s specifications. Strains and Culture Conditions fscherichia coli strains were grown in LB medium (Maniatis, Fritsch, and Sambrook, 1982). Agrobacterium strain LBA4404 (Hoekema et al., 1983) was grown in AB minimal medium (Lichtenstein and Draper, 1986). Antibiotic concentrations,when used, were for E. coli: kanamycin, 50 pg/mL; ampicillin, 100 gg/mL; for Agrobacterium tumefaciens: kanamycin, 100 gg/mL; rifampicin, 1 O pg/mL. These experimentswere conducted under P1 containment conditions as specified by the National lnstitutes of Health Recombinant DNA Guidelines. ocs Upstream Activator Sequence Domains Construction of Plasmids Harboring Deletions of the Octopine Synthase Activator All plasmids were constructed using basic recombinant DNA techniques (Maniatis, Fritsch, and Sambrook, 1982). We have previously described the construction of the plasmids pEN1, pENRl , pSaul33, pAlulO6, pRsa61, pSau84, and pOCSA2 (Leisner and Gelvin, 1988). pAluRlO6 was constructed by cleaving pAlulO6 with BamHI, religating the fragments, and screening colonies for the insert in the reverse orientation. All of the other ocs activator subfragments except the synthetic oligonucleotides were derived from either pSLl , pG1, pAl , or pX1, as described in Figure 2. pUX13 was constructed by converting the Smal site of pUCl3 to Xhol by linker addition(Figure 2C). We have described the construction of pSLl previously (Leisner and Gelvin, 1988). The BamHl to Sau3a fragment from pSL1 (Figure 2A) was cloned into the BamHl site of pUX13 (Figure 2C), resulting in the plasmid pG1 (Figure 28). pAl (Figure 2B) was constructed by converting the Alul site of the Alul to BamHl fragment from pSLl (Figure 2A) to BamHl by linker addition. This fragment was subsequently cloned into the BamHl site of pUC13. The BamHl insert of pSLl (Figure 2A) was cleaved with HinPl , the fragment ends were filled in with Klenow fragment and converted to Xhol sites by linker addition. This fragment was then cloned into the Xhol site of pUX13 (Figure 2C), resulting in pX1 (Figure 28). The BamHl insert of pAl (Figure 28) was cleaved with HinPl , the fragment ends were filled in with Klenow fragment, and the HinPl sites of both fragments convertedto Bglll by linker addition. Both halves of the pAl fragment were subsequently cloned into the BamHl site of pOCSA2 (Figure 2C), to yield pHinR70 and pHinR38 (Figure 3). The BamHl insert of pAl (Figure 28) was cleaved with HinPl , the ends were filled in with Klenow fragment, and the HinPl site converted to Xhol by linker addition. This 70bp fragment was cloned into the BamHl and Xhol sites of pUX13 (Figure 2C). The resulting plasmid was cleaved with BamHl and Xhol. and the 70-bp fragment was cloned into the BamHl and Sal1 sites of pOCSA2 (Figure 2C), yielding pHin70 (Figure 3). The Xhol insert of pX1 (Figure 2B) was cleaved with Alul, and the 38-bp fragment was cloned into the Hincll and Xhol sites of pUX13 (Figure 2C). The resulting plasmid was cleaved with Hindlll and Xhol, and the 38-bp fragment was cloned into the Hindlll and Sal1 sites of pOCSA2 (Figure 2C), yielding pHin38 (Figure 3). The Xhol insert of pX1 (Figure 28) was cleaved with Sau3a, and the 69-bp fragment was cloned into the Xhol and BamHl sites of pUX13 (Figure 2C). The resulting plasmid was cleaved with Hindlll and Xhol, and the 69-bp fragment was cloned into the Hindlll and Sal1 sites of pOCSA2 (Figure 2C), resulting in pSaHi69 (Figure 3). The BamHl insert of pSLl (Figure 2A) was cleaved with Rsal, and the 133-bp fragment was cloned into the Hincll site of pUXl3 (Figure 2C). The resulting plasmids were screened for the Rsal fragment in both orientations. A plasmid harboring the Rsal fragment in the correct orientation and a plasmid harboring the Rsal fragment in the reverse orientation were cleaved with Hindlll and BamHI, and the 131-bp fragments were cloned separately into the Hindlll and BamHl sites of pOCSA2 (Figure 2C), resulting in pRsal31 and pRsaR131 (Figure 3). respectively. The BamHl insert of pAl (Figure 28) was cleaved with Taql, and the 54-bp fragment was cloned into the BamHl and Accl sites of pUX13 (Figure 2C). The resulting plasmid was cleaved with Hindlll and BamHI, and the 54-bp fragment was cloned into the Hindlll and 933 BamHl sites of pOCSA2 (Figure 2C), resultingin pAITaR54 (Figure 3). The BamHl insert of pAl (Figure 28) was cleaved with Rsal, Xhol linkers were attached to the Rsal site, and the 45-bp fragment was cloned into the BamHl and Xhol sites of pUX13 (Figure 2C). The resulting plasmid was cleaved with either Hindlll and Xhol, or BamHl and Xhol. These fragments were cloned separately into the Hindlll and Sall, or the BamHl and Sal1 sites of pOCSA2 (Figure 2C), generating the plasmids pAIRa45 and pAIRaR45 (Figure 3), respectively. The plasmid pSauR84 (Figure 3) was constructed by cutting the plasmid pG1 (Figure 28) with BamHl and Xhol, and cloning the 84-bp fragment into the BamHl and Sal1 sites of pOCSA2 (Figure 2C). The plasmid pG1 was cleaved with Hindlll and Xhol, and the 84-bp fragment was cloned into the Hindlll and Sal1 sites of pAIRa45 and pRsa61 (Figure 3), yielding pSAl and pSRl (Figure 3), respectively. The BamHl to Xhol activator fragment of pG1 (Figure 2B) was cleaved with Mspl, and both halves were purified by preparative gel electrophoresis (Maniatis, Fritsch, and Sambrook, 1982). The Mspl fragment end was filled in with Klenow fragment, converted into an Xhol site by linker addition for the promoter-dista1half of the pG1 (Figure 28) fragment, or to BamHl for the promoter-proximalhalf, and the resulting BamHl to Xhol fragments were cloned individually into the BamHl and Xhol sites of pUX13 (Figure 2C). The resulting plasmids harboring the promoter-dista1 and promoter-proximal halves of pG1 were cleaved with Hindlll and Xhol, and the excised fragments were subsequently cloned separately into the Hindlll and Sal1 sites of pOCSA2 (Figure 2C), resulting in pMsp42 and pMpSa42 (Figure 3), respectively. Construction of Plasmids Harboring Oligonucleotides Five different oligonucleotides were synthesized and cloned into pOCSA2 (Figure 2C). The oligonucleotides were as follows: (1) 5’-TCGAACGTAAG-3 ’ 3 ’ - TGCATTCCTAG- 5 ’ (2) (3) 5’-TCGACTTACGT- 3 ‘ 3 ’ - GAATGCACTAG- 5 ’ (4) (5) 5’-GATCACGTAAGCGCTTACGT- 3 ‘ (5) 3 ‘ - TGCATTCGCGAATGCACTAG-5’ Oligonucleotide pair 1 and 2 was cloned into the Sal1 and BamHl sites of pOCSA2 (Figure 2C). This resulted in the loss of the Sal1 and Xbal sites but retentionof the BamHl site. These sites allowed us to demonstrate the presence of the oligonucleotides in the new plasmid pHPPD (Figure 3). The oligonucleotide pair 3 and 4 was also cloned into the Sal1 and BamHl sites of pOCSA2 (Figure 2C), resulting in the loss of the BamHl and Xbal sites but the retention of the Sal1 site. These sites allowed us to demonstrate the presence of the oligonucleotides in the resulting plasmid pHPPP (Figure 3). Oligonucleotide number 5 was annealed to itself and cloned into the BamHl site of pOCSA2 (Figure 2C), thus destroying the BamHl site. pOCSA2 derivatives that did not cut with BamHl (and possibly contained the oligonucleotide) were digested with Xbal, the fragment ends filled in with Klenow fragment and d2P-dCTP, and the fragments cleaved with Dral. pOCSA2 was treated in the same manner. The resulting labeled DNA was subjected to electrophoresis through a 4% polyacryl- 934 The Plant Cell amide gel, and the gel was autoradiographed. pOCSA2 (Figure 2C) yielded a 100-bp labeled Xbal to Dral fragment, but pOCSA2 containing the oligonucleotide yielded a 121-bp fragment. A plasmid containing the 121-bp Xbal to Dral fragment was designated pPALl6 (Figure 3). Constructionof Plasmids Harboring Upstream Fragments from the mas and ons Genes The upstream region of the mas gene, -31 8 to -213 relative to the transcription initiation site [base pairs 20513 to 20407 (Barker et al., 1983)] was isolated from the plasmid pKan2-318 (DiRita and Gelvin, 1987) by digestion with Xhol and Haelll, the ends were filled in with Klenow fragment, and Xhol linkers were a! tached. The resulting fragment was cloned into the Sal1 site of pOCSA2 in both orientations, yielding pMASl and pMASRl shown in Figure 5A. BamHl fragment 17 [base pairs 13774 to 9062 (Barker et al., 1983)] was cleaved with Ddel and Rsal [base pairs 9634 to 9836 (Barker et al., 1983)l. This fragment contains the upstream region of the ons gene from 363 bp to 170 bp upstream of the translation start site. (The transcription start site of this gene is not known.) The ends of this 203-bp fragment were filled in with Klenow fragment, and this fragment was cloned into the Hincll site of pUX13, resulting in the plasmid p03. pONSRl (Figure 5A) was constructed by cleaving p03 with Hindlll and BamHI, and cloning the 203-bp fragment into the Hindlll and BamHl sites of pOCSA2. p03 was also cleaved with Hindlll, the fragment ends were filled in with Klenow fragment, and the Hindlll site was converted to Bglll by linker addition. The resulting plasmid, called p03', was cleaved with Bglll and Xhol, and the 203bp fragment was cloned into the BamHl and Sal1 sites of pOCSA2, resulting in pONS1 (Figure 5A). Conjugation of Plasmids into A. tumefaciens Plasmids were mobilized into A. tumefaciens strain LBA4404 by triparental mating (Bevan, 1984) using the E. coli strain harboring the recombinant plasmid, E. coli strain MM294/pRK2013 (Ditta et al., 1980), and A. tumefaciens strain LBA4404. Agrobacterium transconjugants were selected on AB minimal plates containing 0.5% glucose, 1O pg/mL rifampicin, and 1O0 pg/mL kanamycin. lnfectionof Plants, Octopine Synthase Assays, and RNA Gel Blot Analysis Tobacco leaf discs were infected with the Agrobacterium strains harboring recombinant plasmids (Horsch et al., 1985), and kanamycin-resistant calli were assayed for octopine synthase activity (Otten and Schilperoort, 1978) with the following modifications. Twenty different calli of equal weight were pooled for each construction and were ground in a small mortar and pestle with grinding buffer (Otten and Schilperoort, 1978). The tissue extract was centrifuged at 1800 x g (4000 rpm in a SA600 rotor) for 5 min. The supernatant solution was transferred to a 1.5-mL microcentrifuge tube and centrifuged in a microcentrifuge for 10 sec. The supernatant solution was then divided into two separate microcentrifuge tubes. One tube was used to determine the protein concentration (Bradford, 1976). Ten microliters of the supernatant solution from the other tube was mixed with 10 pL of reaction mix containing arginine, pyruvate, and NADH (Otten and Schilperoort, 1978). Eight microliters of this mixture was immediately spotted onto a sheet of Whatman 3MM paper, and a second 8-pL sample was spotted after 2 hr of incubationat 25OC. The papers were dried, subjected to electrophoresis, and stained as previously described (Leisner and Gelvin, 1988). The papers were photographed using Polaroid type 55 film at an exposure time of 4 min. The negative was prepared according to instructions from Polaroid. The negative was scanned with the densitometer attachment of a Beckman mcdel DU8 spectrophotometer, the peaks cut out of the chart recorder paper, and weighed. The amount of octopine in each spot was determined by comparison with known octopine standards, and this value was normalized to the protein concentrationof the original tissue extract. The octopine synthase assay is linear in the protein concentration range utilized, and the intensity of the image on the photograph is within the linear response range (data not shown). One unit of octopine synthase activity is equivalent to 0.83 pmol of octopine made per hour per microgram protein. Assay samples contained approximately 15 pg of protein. Seven different pools of calli were tested for the construction pENl and the variation was minimal: the average was 89.9 f 13.2 units. Only numbers greater than 2 SD from the mean (i.e., <64 or >116) were considered as significantly different from the activity of pENl . Calli containing pENl synthesized approximately 2500,pmol of octopine per assay. The value for pENl was arbitrarily set at 100%. For transgenic plants and shoots, the leaves from 1O plants harboring the same construction were pooled and tested for octopine synthase activity as described above. The second leaf from the top of the 2-cm shoots was pooled, and leaf discs were cut out by a No. 6 size cork borer from the rooted (8 cm to 12 cm tall) plants. Octopine synthase-positive plants contained 20% or more of the octopine synthase activity of the construction pENl in that tissue. Octopine synthase-negative plants contained 2% or less of the octopine synthase activity of the construction pENl in that tissue. RNA was isolated from 20 pooled calli for each construction, and RNA gel blot analysis was done as described by Karcher, DiRita, and Gelvin (1984). The autoradiogram was scanned with a densitometer. ACKNOWLEDGMENTS We thank Marlaya Wyncott, Lillian Habeck, and Yu-Mei Wang for their technical assistance; Dr. Avtar Handa and Denise Tieman for assistance with the densitometer; Brad Goodner for critical reading of the manuscript; and Wilma Foust for help in the preparationof the manuscript.This work was supported by grants from the U.S. Department of Agriculture (87-CRCR-1-2443) and a National Science Foundation Presidential Young lnvestigator Award (DMB8351152) to S.B.G., with matching funds from Agrigenetics Research Associates. S.M.L. held a Proctor and Gamble Research Fellowship during part of the course of this work. Received May 19, 1989; revised July 17, 1989. ocs Upstream Activator Sequence Domains REFERENCES An, G. (1987). A potential 2-DNA-forming sequence is an essential upstream element of a plant promoter. BioEssays 7, 211-214. Barker, R.F., Idler, K.B., Thompson, D.V., and Kemp, J.D. (1983). 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S M Leisner and S B Gelvin Plant Cell 1989;1;925-936 DOI 10.1105/tpc.1.9.925 This information is current as of August 11, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw153229 8X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY