Proc. Natl. Acad. Sci. USAVol. 86, pp. 3733-3737, May 1989Genetics

Saturation mutagenesis of the octopine synthase enhancer:Correlation of mutant phenotypes with bindingof a nuclear protein factor

(gene regulation/plant promoter element/point mutation analysis/DNA-binding protein)

KARAMBIR SINGH*, JAMES G. TOKUHISA, ELIZABETH S. DENNIS, AND W. JAMES PEACOCKDivision of Plant Industry, Commonwealth Scientific and Industrial Research Organization, G.P.O. Box 1600, Canberra, ACT 2601, Australia

Communicated by 0. H. Frankel, February 7, 1989 (receivedfor review December 15, 1988)

ABSTRACT A 16-base-pair palindrome from theAgrobac-terium tumefaciens octopine synthase gene functions as aconstitutive enhancer in plant protoplasts. Degenerate oligo-nucleotide mutagenesis provided single base substitutions atevery position in the element and a number of multiple basesubstitutions. The effects of these changes were determined intransient expression assays with tobacco and maize protoplasts.The majority of single and double base changes had little effecton the activity of the octopine synthase enhancer, but nearly allmutants with more than two base changes had low to essentiallyno activity. There were five positions where particular singlebase changes resulted in a 4- to 10-fold loss in enhancer activity.The distribution of these positions within the palindrome wasasymmetric. Single base deletions had essentially no activity,demonstrating that the octopine synthase enhancer cannottolerate internal changes in spacing. We find a strong corre-lation between mutant phenotype and reduced binding of aprotein factor, suggesting that the DNA-protein complex isresponsible for the transcriptional enhancement; the function-ally active form of the DNA-protein complex probably involvesmore than a single protein molecule. The mutants exhibitsimilar phenotypes in protoplasts of both tobacco and maize,implying conservation of the DNA-protein interactions of theocs enhancer sequence in monocotyledonous and dicotyledon-ous plants.

Transcriptional control of genes transcribed by RNA poly-merase II is mediated through upstream promoter elementsand/or enhancers. These cis-acting DNA sequences havebeen well characterized in yeast and animal systems (see refs.1-4) and have been shown to be highly specific binding sitesfor trans-acting regulatory proteins. A number of cis-actingregulatory DNA sequences have also been identified in plants(see ref. 5).One of the best characterized plant regulatory sequences is

the octopine synthase (ocs) enhancer element (6). The ocsgene is found on the tumor-inducing plasmid of the plantpathogen Agrobacterium tumefaciens (7). The ocs gene is notexpressed in Agrobacterium, but it is expressed after inte-gration into the plant genome. Since the ocs gene is wellexpressed in a variety of tissues in transgenic plants, thetranscriptional signals of the ocs gene must function asendogenous plant sequences controlling constitutive expres-sion (7). Deletion analysis of the ocs promoter upstreamsequences and the use of synthetic oligonucleotides identifieda 16-base-pair palindrome, ACGTAAGCGCTTACGT, as theocs enhancer element, able to enhance the expression ofheterologous promoters in transient expression assays inplant protoplasts (6).

We have attempted to define the nucleotide sequencerequirements of the ocs enhancer element. Recent resultsfrom yeast and animal systems demonstrate that trans-actingfactors such as AP1 and GCN4, which have different regu-latory functions, recognize the same or similar DNA se-quences (8). We have used degenerate oligonucleotides tomutate each base of the ocs enhancer element and haveisolated a number of single and multiple point mutationsthroughout the element. The phenotypes of these mutantenhancers have been determined in transient expressionassays using protoplasts from both tobacco (a dicot) andmaize (a monocot). We have also shown that the transcrip-tional phenotypes of a number of the mutants correlate withtheir ability to bind an ocs-specific protein factor present innuclear extracts of tobacco cells.

MATERIALS AND METHODSOligonucleotide Synthesis and Cloning. The plasmid

p5O5GN (see Fig. 1), which contains bases -100 to +106 ofthe maize alcohol dehydrogenase 1 (Adhl) promoter (9)linked by a BamHI site to the coding region of the bacterial3-glucuronidase gene, and the 3' processing region of thenopaline synthase gene from the plasmid pBI101 (10), andcloned into the polylinker of pUC118, was constructed bystandard DNA manipulation procedures (11). The synthesisand cloning strategy for the degenerate oligonucleotides wasbased on the procedure of Derbyshire et al. (12). Initially, a26-base oligonucleotide, with a 5' end complementary to the5' overhang of a HindIll site, and a 3' end complementary tothe 3' overhang of a Pst I site (see Fig. 1), was synthesizedon an Applied Biosystems DNA synthesizer. The central 16bases corresponding to the ocs palindrome (see Fig. 1,boldface type) were synthesized with 92.5% of the wild-typebase and 2.5% of each of the other three bases. The pool ofsingle-stranded oligonucleotides was cloned directly into theHindIII/Pst I sites of p505GN by the sequential ligationprocedure described by Derbyshire et al. (12). The finalligation mixture was digested with Sph I prior to transfor-mation of JM101 cells. Single-stranded templates were gen-erated from the transformants using the M13K07 helperphage and sequenced by the Sanger procedure (13). Anumber of single and multiple point mutants were isolatedfrom the first degenerate oligonucleotide synthesis. How-ever, there was a pronounced bias to certain nucleotidechanges and six additional degenerate HindIII/Pst I ocsoligonucleotides were synthesized before single base changeswere obtained at every position in the ocs enhancer element.

Transient Expression Analysis. Nicotiana plumbaginifoliaprotoplasts were isolated from the cell suspension culture lineNpT5 as described (14). The protoplasts were electroporatedwith 20 jig of plasmid DNA, incubated for 20 hr at 26°C, and

*To whom reprint requests should be addressed.

3733

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Proc. Natl. Acad. Sci. USA 86 (1989)

then lysed by sonication (14). The amount of protein in theprotoplast extracts was measured by the method of Spector(15). The fluorometric glucuronidase assay was performed as

described by Jefferson et al. (10) with 4-methylumbelliferylglucuronide as the substrate. In a typical assay, 1 ,ug of totalprotein was incubated in 200 ,ul of the assay buffer (10) for 20min at 370C before the reaction was stopped by the additionof 1 ml of 0.2 M Na2CO3 and the fluoresence was measured.Maize protoplasts were isolated from a cell suspensionculture of the Black Mexican Sweet variety and electropo-rated with 100 ttg of plasmid DNA as described (16). In theglucuronidase assay, 10 ,ug of total protein was incubated inthe assay buffer (10) for 40 min at 372C.

Binding Studies. Nuclear extracts from N. plumbaginifoliasuspension culture cells were prepared by two differentmethods and gave the same results. In the first method, nucleiwere prepared essentially as described by Willmitzer andWagner (17). Crude nuclear extracts were prepared as

described by Green et al. (18). Extracts were enriched forDNA binding proteins by passage over a DNA-Sepharoseaffinity column containing linearized pUC19 DNA. Thecolumn was prepared and used as described by Kadonagaand Tjian (19). In the second method, nuclei were preparedas described by Watson and Thompson (20) and crudenuclear extracts were prepared as described by Green et al.(18). The 123-base-pair Sal I/Pvu II probe fragments (Fig. 1)from the wild type and seventeen mutant ocs palindromicsequences were end-labeled at the Sal I site with the Klenowfragment of DNA polymerase I and purified by polyacryl-amide gel electrophoresis. Gel retardation experiments were

performed as described by Garner and Revzin (21) and Friedand Crothers (22), using 4% polyacrylamide gels and 22 mMTris borate, pH 8.3/0.5 mM EDTA as the electrophoresisbuffer. The binding reactions contained 1-3 fmol of end-labeled probe (10w cpm), 1.0 ,ug of poly[d(I-C)], 0.75 jtg ofnuclear extract in 20 IlI of binding buffer [25 mM Hepes/50mM KCl/1 mM EDTA/5 mM 2-mercaptoethanol/10% (vol/vol) glycerol].

RESULTS AND DISCUSSIONTo determine the nucleotide sequence requirements of theocs enhancer element, we used the procedure of Derbyshireet al. (12) to synthesize a pool of degenerate ocs oligonucle-otides and clone them immediately upstream of a crippledAdhl promoter fragment linked to the 3-glucuronidase re-

porter gene (Fig. 1). The crippled Adhl promoter, whichprovides a TATA box and a cap site, has extremely lowactivity in transient assays in both tobacco and maizeprotoplasts (6, 9). Placement of the wild-type ocs palindromeupstream of this crippled Adhl promoter results in a >100-

5' 3'

AG CTT ACGTAAGCGCTTACGT CTGCAHind III Pst

PvHSpPS B Ss E

FIG. 1. Structure of the plasmid p5O5GN. The plasmid p5O5GN(5540 base pairs) contains bases -100 to +106 of the maize Adhipromoter (open box), the ,8-glucuronidase coding region (solid box),and the 3' sequence from the nopaline synthase gene (hatched box).Plasmid sequences for pUC118 are shown by the dashed line.Restriction endonuclease sites for Pvu 11 (Pv), HindI11 (H), Sph I(Sp), Pst I (P), Sal I (S), BamHI (B), Sst I (Ss), and EcoRI (E) areshown. The sequence of the HindIII/Pst I oligonucleotide is shownwith the bases corresponding to the ocs enhancer element in boldfacetype.

fold increase in promoter activity (6). The effect of basesubstitution on ocs enhancer activity was measured in atransient expression assay by electroporating the varioushybrid promoter/,B-glucuronidase plasmid constructs intotobacco protoplasts.

Effect of Single Base Substitutions on ocs Enhancer Activity.We have obtained single base changes at every positionwithin the ocs palindrome (Table 1). Fourteen of the 20 singlebase substitutions have >30% activity, with 9 having 50% ormore of the wild-type activity. There are 6 single substitu-tions, in five "critical" positions with activity <25% of wildtype. Mutants 1.3A, 1.3T, 1.8A, and 1.14A retain between18% and 24% of wild-type activity, while mutants 1.6T and1.7C have 10o and 12% of wild-type activity, respectively.

In three positions in the palindrome, we obtained morethan one base substitution. Phenotypes of these mutantsdemonstrate that position per se is not the only factor ofimportance; type of nucleotide change is also significant(Table 1, compare 1.3T and 1.3A vs. 1.3C, 1.6T vs. 1.6G, and1.8A vs. 1.8T). In both positions six and eight, mutantsresulting from nucleotide transitions retain high activity whiletransversions cause a 4- to 10-fold loss of activity. Since wehave only obtained 20 of the possible 48 single base changes,it is possible that other critical positions exist. Saturationmutagenesis studies on the TN10 tet operator (23) and theyeast his3 TATA element (24) also showed that both nucle-otide identity and position were important.The distribution of mutant phenotypes in the palindrome is

asymmetric. Five of the six mutants with <25% of wild-typeactivity are in the 5' half of the palindrome (Table 1).Conversely, six of the nine mutants with 50% or more of

Table 1. Effect of single base substitutions on ocsenhancer activity

Mu- Activity, % WTtant Sequence Tobacco MaizeWT A C G T A A G C G C T T A C G T 100 (SD) 1001.1T T - - - - - - - - - - - - - - - 46 (25) 361.2T - T - - - - - - - - - - - - - - 34 (16) 37t1.3A - - A - - - - - - - - - - - - - 18*(10) 38t1.3C - - C - - - - - - - - - - - - - 46 (11) 391.3T - - T - - - - - - - - - - - - - 21* (7) 251.4G - - - G - - - - - - - - - - - - 40 (22) 761.5T - - - - T - - - - - - - - - - - 65 (29) 961.6G - - - - - G - - - - - - - - - - 98*(29) 871.6T - - - - - T - - - - - - - - - - 10* (5) 181.7C ------ C--------- 12* (8) 41t1.8A - - - - - - - A - - - - - - - - 21* (14) 331.8T - - - - - - - T - - - - - - - - 67*(24) 631.9A - - - - - - - - A - - - - - - - 109 (32) 761.10G - - - - - - - - - G - - - - - - 50*(26) 411.11A - - - - - - - - - - A - - - - - 72 (17) 721.12C.---------- C---- 52 (18) 731.13G - - - - - - - - - - - - G - - - 40 (19) 711.14A - - - - - - - - - - - - - A - - 24* (15) 191.15A - - - - - - - - - - - - - - A - 53 (11) 731.16A - - - - - - - - - - - - - - - A 59 (24) 49

The single mutations are designated with the number of mutationsfirst followed by the position of the mutation in the palindrome andthe nucleotide present in the mutant. The 5' end of the palindromeis designated position 1. The mutant sequences are listed below thewild-type (WT) sequence. Only the mutated base is shown and ahyphen denotes wild-type sequence. The activity of the mutants isexpressed as a percentage of wild-type activity for both the tobaccoand maize data. For the tobacco data, the means ± SD of 5-10independent electroporation experiments are shown.*Two separate plasmid DNA preparations were used; no significantdifference was found.tAverages of five experiments; remaining maize data are the resultof a single experiment.

3734 Genetics: Singh et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 3735

wild-type activity are in the 3' half of the palindrome (Table1). Since we have not obtained all possible nucleotidesubstitutions, the above results must be interpreted withcaution. However, the phenotypes of the symmetrical mu-tants we did obtain demonstrate the asymmetry of the ocsenhancer. In six of eight positions in the 5' half of thepalindrome, we have obtained the corresponding change inthe 3' half. For positions 1 (A to T, 46%) vs. 16 (T to A, 59%),2 (C to T, 34%) vs. 15 (G to A, 53%), 3 (G to T, 21%) vs. 14(C to A, 24%), and 8 (C to T, 67%) vs. 9 (G to A, 109%), thephenotypes of the symmetrical mutants are similar. In con-trast, at positions 6 (A to T, 10%) vs. 11 (T to A, 72%) and7 (G to C, 12%) vs. 10 (C to G. 50%), the symmetrical changesresult in different phenotypes.The above results suggest that in the context of our assay

the 5' half of the palindrome is more important for enhanceractivity. One possibility is that sequences immediately flank-ing the palindrome contribute to ocs enhancer activity, or therelative importance of each half of the palindrome may bedependent on their position with respect to other regulatorysequences such as the TATA box.

Effect of Multiple Base Substitutions on ocs EnhancerActivity. The eight double mutants all have >25% of wild-type activity (Table 2). Nearly all mutants with more than twobase changes have low activity. Five of the seven triplemutants have <5% of wild-type activity; mutant 3.5 has 10%of wild-type activity, while mutant 3.7 still retains 33% ofwild-type activity. All three changes in mutant 3.7 wereamong the single base changes isolated, and in each case thesingle mutant retained >50% of wild-type activity. All theother triple mutants have at least one change that was notpresent among the single mutants. Mutant 4.1 has four basechanges but still retains significant activity (20% of wildtype); none of the four changes in mutant 4.1 was among thesingle mutants. Mutants 5.1 and 6.1, which have five and sixbase changes, respectively, are essentially inactive (2% orless of wild type).Phenotype of the Mutants in Tobacco vs. Maize. The ocs

enhancer has been shown to be active in maize (6). To

Table 2. Effect of multiple base substitutions on ocsenhancer activity

A C G T A AT- - -

- A- -

- T- -

- -_

- - - G - -

-- - - G- -

Activity, % WT

Sequence Tobacco MaizeG C G C T T A C G T 100 (SD) 100- - - - - - - - - A 43 (17) 79- - - - G - - - - - 29 (6) 16.C-- - - G - - - - 35 (18) 18

- - - - - - - - A - 31 (5) 30A - - - - - - - - 35* (9) 36

--T - - - - - - - 29 (14) 50--A - - C - - - - 44 (14) 30- - - G - - - - A - 27 (10) 16

G - - - - C- - - - - - T - - - 4 (1)T - - - - C- - - - - - - - - A 3 (4)- A -C - - - G - - - - - - - - 4 (1)- A T- - - C - - - - - - - - - 2 (3)- - - G - C - A - - - - - - - 10* (4)- - - G - - - A T - - - - - - - 2 (1)- - - - - - - T A - - C - - - - 33*(13)

4.1 - A - - - C - - - - - - - - T C5.1 G - - A C T - - - - - - T - - -

6.1 - - - - - - - - T T G C - T - C

20 (10)1 (0)2 (2)

55248

lot20t

1621

determine, at single nucleotide resolution, the extent ofsimilarity in ocs enhancer activity between tobacco andmaize, maize protoplasts were isolated and electroporatedwith all 38 single and multiple mutants. Almost all themutants exhibited a similar phenotype in maize and tobacco(Tables 1 and 2). Of the 38 mutants tested, only 3 consistentlyshowed a 2-fold difference in activity between the twospecies; mutant 1.3A was just over 2-fold more active inmaize than in tobacco, mutant 1.7C was almost 3.5-fold moreactive in maize than in tobacco, and mutant 3.6 was 5-foldmore active in maize than in tobacco. These results implyextensive conservation in the sequence requirements of theocs enhancer between maize and tobacco.

Effect of Single Base Deletions on ocs Enhancer Activity. Weisolated two mutants, D.6 with a single base change and D.7with two base changes, which also contained a single deletionwithin the palindrome (Table 3). Both mutants were found tobe essentially inactive. Mutant D.6 differed from mutant1.4G, which had the same base substitution as D.6 butretained significant activity (40%), suggesting that single basedeletions could inactivate the ocs enhancer. To test thispossibility further, a number of oligonucleotides with dele-tions within the palindrome were isolated and assayed (Table3). The position of the deletion within the palindromedetermines the effect it will have on enhancer activity. Inmutant D.1, where the deletion is near the end of thepalindrome in position 2, some activity (15%) is retained.Mutants D.2 and D.3 with deletions at positions 4 and 5/6,respectively, have very low activity (5% or less of wild type).Mutants D.4 and D.5, which have more than one deletion, areessentially inactive (2% or less of wild type).Although a deletion only removes a single base, it also in

effect creates base changes in all positions between thedeletion and the nearer end of the palindrome. In the case ofmutant D.1, these additional base changes are minimal; D.1can be viewed as a double mutation at positions one and two,with the new base at position one being a T. Mutants D.2 andD.3 could be considered to have four and five base changes,respectively, and we can thus expect the effect of thesechanges on enhancer activity to be more severe. It is clearfrom the deletion analysis that while the ocs enhancer cantolerate the majority of single and double base changes, itcannot tolerate changes in spacing caused by single basedeletions within the palindrome. This sensitivity to spacingbetween the bases in the ocs enhancer is also demonstratedby insertions, since both 4- and 10-base-pair insertionsinactivate the ocs element (ref. 6; unpublished observation).

Binding of an ocs-Specific Protein Factor to Mutant ocsEnhancers. Definitive proofthat a binding activity constitutesa transcription factor can only be obtained from an in vitro

Table 3. Effect of single base deletions on ocs enhancer activity

Tobaccoactivity,

Mutant Sequence % WT

WT A C G T A A G C G C T T A C G T 100 (SD)D.1 - A - - - - - - - - - - - - - - 15 (7)D.2 - - - A- - - - - - - - - - - - 3 (3)D.3 - - - -(A)- - - - - - - - - - 5 (1)D.4 - - -A(A)- - - - - - - - - - 2 (1)D.5 A - -(A)- - - - - - - - - - 2 (4)D.6 - - - G --A - 2 (1)D.7 G - - -(A)- - - - - - - - - A 3 (2)

The notations are as in Table 1 except that the deletion mutants aredesignated with D first; the second number was arbitrary. For thetobacco data the means ± SD offour electroporation experiments areshown. The position of the deletion is shown as A; (A) is used atpositions five and six since it is not possible to determine whichposition has been deleted.

Mu-tant

WT2.12.22.32.42.52.62.72.8

3.13.23.33.43.53.63.7

The notations are as in Table 1 except that the multiple mutants aredesignated with the number of mutations first; the second numberwas arbitrary. For the tobacco data, the mean ± SD of between threeand eight independent electroporation experiments are shown.

Genetics: Singh et al.

Proc. Natl. Acad. Sci. USA 86 (1989)

transcription system. However, in the absence of such asystem in plants, the mutagenesis approach we have under-taken enables us to ask whether a given DNA-proteininteraction is biologically significant. We have compared thein vivo transcriptional phenotype of the mutants with theirability to bind an ocs-specific protein factor in vitro.

End-labeled DNA fragments containing the wild-type and17 mutant ocs sequences were used in qualitative bindingstudies with nuclear extracts from tobacco suspension cells(Fig. 2). In gel retardation assays using tobacco or maizenuclei extracts, two specific retarded bands are observedwith a wild-type ocs DNA probe (lane 1; data not shown).Mutants with single base changes and 50% of wild-typetranscriptional activity in vivo showed wild-type levels ofbinding in vitro (lanes 2 and 3). Three mutants with singlebase changes and <25% of wild-type transcriptional activityshowed reduced upper band binding (lanes 4-6), although inthe case of mutant 1.6T (lane 5) there was only a smallreduction in binding. We tested all seven available triplemutants. Mutant 3.7, which still retained 33% wild-typetranscriptional activity, exhibited significant upper bandbinding (lane 13). Of the six remaining triple mutants that had10% or less of wild-type transcriptional activity, only mutant3.6 (lane 12) had detectable upper band binding. All but oneof the seven triple mutants exhibited varying amounts oflower band binding, including four mutants with <5% ofwild-type transcriptional activity. Mutant 5.1 with five basechanges distributed over both halves of the palindrome and1% of wild-type transcriptional activity showed no detectablebinding at either band position (lane 14). Mutant 6.1, with 2%of wild-type transcriptional activity and six base changesconfined to the 3' half of the palindrome, showed no detect-able upper band binding but did have strong lower bandbinding (lane 15). Two of the three deletion mutants testedexhibited no detectable upper band binding (lanes 16 and 17),although mutant D.2 did show strong lower band binding(lane 16). Mutant D.6 exhibited low levels of both upper andlower band binding (lane 18).The results show a strong correlation between in vivo

transcriptional activity and in vitro binding for the upperretarded band (Fig. 2, all lanes) but not for the lower band(lanes 4, 6, 8, 10, 15, and 16). This suggests that the bindingfactor we have identified is critical for ocs enhancing activityand that the upper retarded band represents the functionallysignificant binding activity. A likely explanation based on thebinding characteristics of other DNA binding proteins thatrecognize symmetric sequences is that the lower band resultsfrom monomer binding and the upper band results from dimerbinding (25). Our binding results with mutant 6.1, which hassix base changes confined to the 3' half of the palindrome andno detectable upper band binding but strong lower bandbinding, support a monomer/dimer explanation since the

H H(LOD H HED

lower band binding activity is capable of binding to what isessentially a single half site.

Tolerance of the ocs Enhancer to Base Substitutions. Wehave found the ocs enhancer to be tolerant to base substitu-tion. The majority of single and double base changes retain>25% of wild-type activity, and while most of the mutantswith more than two base changes are inactive we haveobtained mutants with three or four base changes that retain20% or more of wild-type activity. This high degree oftolerance of the ocs enhancer to base substitution is incontrast to the results of mutagenesis studies on othereukaryotic DNA transcription elements such as the mouse,3-globin promoter (26), the human p-interferon promoter(27), the yeast his3 regulatory site (28), and the yeast his3TATA element (24). In these elements, many if not all singlepoint mutations within core sequences, such as the CCAATbox in the /-globin promoter (26) or the 9-base-pair yeast his3regulatory site (28), significantly reduce the transcriptionalactivity of the respective sequence.The only palindromic-like sequence among the above

examples is the yeast his3 regulatory sequence, which is thebinding site for the yeast transcription factor GCN4 (29). TheGCN4 protein increases the transcription of a number ofunlinked genes in response to starvation by any one of severalamino acids (see ref. 30). In 16 genes examined, at least onepromoter sequence with close homology to the palindromicconsensus GCN4 binding sequence ATGACTCAT wasfound. Twelve different GCN4 binding sequences werefound, with seven sequences containing one base change andthe remaining five sequences containing two base changesfrom the consensus sequence. The GCN4 protein, like theocs protein factor is clearly able to tolerate a number of singleand double base substitutions in its recognition sequence.Furthermore, a HeLa cell octamer binding protein (OBP100)has been shown recently to bind a wide range of octamermotifs through interactions with flanking sequences (31).When a degenerate octamer was converted to a perfectoctamer motif, there was no longer a requirement for specificflanking sequences, suggesting that the sum of many inde-pendent contacts is required for effective binding and thatfew residues are obligatory.The reasons for the tolerance of the ocs enhancer to

sequence change are unknown. Tolerance in the bindingrequirements ofboth the GCN4 and ocs protein factors couldbe the result of selection. The GCN4 protein is involved in thecoordinate regulation ofa number ofgenes involved in aminoacid biosynthesis. The ocs factor appears to be involved inthe expression of at least two other Agrobacterium-encodedopine synthase genes, the nopaline synthase and the Agro-bacterium rhizogenes mannopine synthase genes (D.Bouchez and J.G.T., unpublished data); the promoters ofthese genes contain ocs palindrome-related sequences that

C' Ce).LO. ( 6LO (06 6

UB-E QaLB--do a w a -.%lo a a

FP a * Z a FP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

FIG. 2. The ocs protein factor binding to the wild-type (lane 1) and 17 mutant (lanes 2-18) ocs enhancer elements. The gel retardation assayswere carried out with tobacco nuclear extracts and contained 1-3 fmol of end-labeled probe (104 cpm), 1.0 Ag of poly[d(I-C)], and 0.75 ,ug ofnuclear extract in 20 ,Au of binding buffer. After incubation at 30°C for 30 min, the free and bound DNAs were resolved on a polyacrylamidegel and visualized by autoradiography. The positions of the free probe (FP), the, lower retarded band (LB), and the upper retarded band (UB)are shown.

- UB- LB

3736 Genetics: Singh et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 3737

bind the ocs factor in vitro. Like the ocs gene, these otherbacterial opine synthase genes are only expressed in infectedor transgenic plants, suggesting that there must be at least oneand possibly a number of endogenous plant genes whoseexpression is under the control of the plant-encoded ocstranscription factor. Divergence of the controlling sequencesof coordinately regulated genes could exert selection for abinding factor that can accommodate changes. Tolerance tosequence change could also result from a number of closelyrelated transcription factors that recognize similar but notidentical ocs binding sites. For example, in both Saccharo-myces cerevisiae and the fission yeast Schizosaccharomycespombe, an AP-1-like factor with overlapping but distinctbinding characteristics to GCN4 has been identified (8).

CONCLUSIONAlthough we have demonstrated that the ocs enhancer istolerant to the majority of single and double base substitu-tions, we have identified a minimum of five critical positionswhere certain single base substitutions cause a 4- to 10-foldloss in enhancer activity. In contrast, nearly all mutants withmore than two base changes, as well as mutants with singledeletions, have low to essentially no activity. The activity ofthe mutants in vivo correlates well with their ability to bindan ocs-specific protein factor in vitro, demonstrating atranscriptional role for this factor. The ocs enhancer activityhas been extensively conserved between maize (a monocot)and tobacco (a dicot), although these species diverged >150million years ago. Although the function of the ocs bindingfactor in plants remains unknown, the ubiquity of the factorsuggests an essential role in regulating some aspect of themetabolism of many if not all higher plants.

We thank S. Skinner and S. Stops for excellent technical assist-ance; D. Llewellyn for assistance with the tobacco nuclear extracts;G. Mayo and P. Whitfeld for the synthesis of the degenerateoligonucleotides; and D. Llewellyn, J. Ellis, A. Ashton, T. Close,and J. Haseloff for many helpful suggestions. K.S. was supported bya Queen Elizabeth II Fellowship. This research was supported in partby Agrigenetics Research Corporation.

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Genetics: Singh et al.