Proc. Nati Acad. Sci. USA Vol. 79, pp. 86-90, January 1982 Biochemistry

Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the of Agrobacterium tumefaciens (recombinant plasmid/hybridization/mRNA size/in vitro translation/immunoprecipitation) NORIMOTO MURAI AND JOHN D. KEMP Department of Plant Pathology, University ofWisconsin, and Plant Disease Research Unit, ARS, USDA, Madison, Wisconsin 53706 Communicated by Folke Skoog, September 14, 1981 ABSTRACT We have shown that the structural gene for oc- families have been identified. The enzymes responsible for the topine synthase (a crown gall-specific enzyme) is located in a cen- synthesis of the first two have been purified and characterized tral portion ofthe T-DNA that came from the Ti plasmid ofAgro- (17, 18). bacterium tumefaciens and is expressed after it has been trans- The particular family present in a crown gall cell cor- ferred to the plant cells. Polyadenylylated RNA was prepared relates with a particular Ti plasmid rather than with the host from polysomes isolated from an octopine-producing crown gall plant (19, 20). Analysis ofdeletion mutants of an octopine-type callus and purified by selective hybridization to one offive recom- Ti plasmid has shown that a gene controlling octopine produc- binant plasmids. Each such plasmid contained a different frag- tion is located on a 14.6-kbp fragment ofthe T-DNA generated ment ofT-DNA ofpTi-15955 (octopine-type Ti plasmid). Purified by Sin 1 (21) (Fig. 1). Detailed examination ofthe physical or- mRNA was translated in vitro in rabbit reticulocyte lysates, and ganization of T-DNA in octopine-type tumor lines has shown the translation products were immunoprecipitated with antibody that octopine production is correlated with the presence ofthe against octopine synthase. Total and immunoprecipitated prod- left portion ofa 2.8-kbp fragment generated by EcoRI (5). These ucts were characterized by their molecularweights. A polypeptide production of Mr 40,000 (the same as authentic octopine synthase) was syn- results indicate that the gene controlling octopine thesized in vitro by crown gall mRNA selectively hybridized to is located on the T-DNA contained in the left half of the 14.6- three of the five fragments of T-DNA and precipitated with an- kbp Sin I fragment. However, the question remains as to tibody against octopine synthase. This polypeptide was not im- whether or not the controlling gene is the structural gene for munoprecipitated with normal rabbit antibody nor was it synthe- octopine synthase. sized when mRNA from the habituated callus was substituted. A The demonstration that the controlling gene on the T-DNA mRNA 1500 bases long was detected when total mRNA was frac- is the structural gene for octopine synthase is of importance if tionated on an agarose gel, transferred to nitrocellulose, and used the Ti plasmid is to be developed as a vector for genetic mod- for hybridization to three of the five 32P-labeled T-DNA frag- ification of crop plants. In this report, we show that the struc- ments. This apparent mRNA for octopine synthase hybridized to tural gene for octopine synthase is located in a central portion the same three fragments of T-DNA as the mRNA for the Mr of the T-DNA ofA. tumefaciens Ti plasmid and expressed as a 40,000 polypeptide and was not detected in the habituated callus. 1500-base mRNA and a polypeptide of Mr 40,000 in sunflower crown gall callus. Virulence of Agrobacterium tumefaciens (the causal organism ofcrown gall tumors) (1) is correlatedwith the presence ofa large MATERIALS AND METHODS tumor-inducing (Ti) plasmid (2). During tumorigenesis, a 12- to 23-kilobase pair (kbp) portion (T-DNA) of the Ti plasmid is Isolation of Sunflower Polyadenylylated RNA. The estab- transferred to plant cells (3-5) and covalently linked to their lishment and maintenance ofthe primary sunflower (Helianthus nuclear DNA (6, 7). Specific sections ofthe integrated T-DNA annuus cv. Mammoth Russian) crown gall tissue PSCG-15955 are transcribed to RNA by tobacco (8) and sunflower (9) tumor (22) and ofhabituated sunflower stem section culture HSSS (12) cell cultures. Furthermore, T-DNA transcripts are translated have been described. PSCG-15955 was isolated from a primary in vitro into polypeptides (10), but the polypeptides have not crown gall tumor incited by A. tumefaciens (E. F. Sm. and been identified as crown gall-specific proteins. Therefore, there Town.) Conn (1) strain 15955. PSCG-15955 synthesizes octo- is still a question as to whether expression ofthe T-DNA genes pine synthase (17), accumulates octopine (12), and contains the to functional proteins in the plant cell is a requirement for the T-DNA (unpublished data). induction and maintenance of crown gall tumors. Polysomes were prepared from 3-week-old tissue cultures by In addition to the presence ofT-DNA, crown gall tissues are a modification ofthe method ofSun et aL (23). Harvested tissues characterized by their ability to grow in culture in the absence were frozen instantly with liquid N2 and ground to afine powder of auxin and cytokinin (11) and by their ability to accumulate in a Waring blendor. Ground tissues were homogenized in 2 crown gall-specific (12). These opines are in turn used vol of 150 mM Tris acetate, pH 8.6/200 mM sucrose/20 mM for nutrition by A. tumefaciens (13). The hormone autonomy KC1/5 mM Mg (OAc)2/5 mM 2-mercaptoethanol/0.4% Noni- of crown gall tissue presumably results from the activation of det P40 (British Drug House, Poole, England). The homoge- auxin and cytokinin biosynthesis. However, little is known nates were centrifuged at 30,000 X g for 20 min (4°C). The su- about individual enzymes involved in the biosynthesis ofthese pernatant was layered onto 1.5 M sucrose-containing 50 mM compounds. On the other hand, opine biosynthesis is well char- Tris acetate, pH 8.5/20 mM KC1/5 mM Mg (OAc)2 (5 ml per acterized for crown gall tissues. Three families of opines, the tube) and centrifuged at 218,000 x g for 2 hr (4°C). The sedi- octopine (12, 14), the (12, 15), and the agropine (16) mented polysomes were rinsed three times with sterile H20 and suspended in 100 mM Tris-HC1, pH 9.0/0.5% NaDodSO4 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: T-DNA, portion ofTi plasmid that is transferred to plant ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. cells; kbp, kilobase pairs. 86 Downloaded by guest on September 27, 2021 Biochemistry: Murai and Kemp Proc. Natl. Acad. Sci. USA 79 (1982) 87

0.23/0335 with ethanol and dissolved in sterile water for in vitro Eco RI IS 113IK69/o- 2.8$/'55 w.0 translation. Banm HI , /, 4 8.X/ 6.8 In Vitro Translation and Immunoprecipitation. Rabbit re- Hind II 132 !261201049 12.3 0.69-1 94 ticulocyte lysate (Green Hectares, Madison, WI) was treated qmSin 2.12.5 62 L 14.6 with micrococcal nuclease (Worthington) for preparation ofthe _ ~~~~~~~~~_ mRNA-dependent in vitro translation system (30). Poly(A)+RNA CONSERVED REGON Os was translated in vitro at 30'C for 90 min in 50 1.l ofa mixture (31) containing 25 pl ofrabbit reticulocyte lysate and 30-70 Ci C c,o 2022 ofL-[3S]methionine (1000 Ci/mmol; 1 Ci = 3.7 X 1010 becque- o 203_ rels; Amersham). Polysomes that formed were removed from 3303 the translation mixture by centrifuging at 106,000 X g for 2 hr o 403 (40C). The supernatant fraction was then assayed for octopine synthase by immunoprecipitation. FIG. 1. Restriction endonuclease map of the T-DNA region of pTi- Antibody against octopine synthase (anti-octopine synthase 15955 and location of cloned fragments. Each fragment is designated IgG) was prepared as described (32) and purified by chroma- by its size in kilobase pairs. Solid bars, T-DNA fragments contained in pBR322 recombinants; shaded areas, region of T-DNA common to tography on a protein A-Sepharose 4B column. Antibody from most Ti plasmids (4) and location of the octopine synthase (OS) gene normal rabbit serum was purified as above. as deduced by this study. The order of the BamHI 1.1- and 0.49-kbp Total products from in vitro translation were immunoprecip- fragments has not been determined. itated with anti-octopine synthase IgG and Staphylococcus au- reus strain-Cowan I (33). Forty micrograms each lot of anti-oc- (1 ml per tube). The suspensions were deproteinized twice by topine synthase IgG was added to the supernatant oftranslation mixing with buffer-saturated phenol followed by centrifugation mixtures and the mixture was incubated at 370C for 1 hr and for 5 min at 12,000 X g. The recovered aqueous phases were then at 40C overnight. A 20-,ul aliquot of10% (vol/vol) S. aureus twice mixed with chloroform/isoamyl alcohol (24:1) followed by cell suspension was then incubated with the above mixture at centrifugation as above. Polysomal RNA was precipitated from 25°C for 30 min. Immunocomplex bound to S. aureus cells was the aqueous phase by the addition of ethanol, dissolved in 0.4 purified by centrifuging the cells through 15% (wt/vol) sucrose M NaOAc, pH 6.0, reprecipitated, and finally dissolved in 10 at 5000 x g for 5 min (4°C) followed by washing as described mM Tris HCl, pH 7.5/1 mM EDTA. by Kessler (33). Finally, the immunocomplexes were eluted Poly(A)+RNA was purified from polysomal RNA by two pas- from the cells by incubating at 100°C for 2 min in 50,ul ofLaem- sages through an oligo(dT)-cellulose column (Collaborative Re- mli's application buffer (34). search, Waltham, MA) (24). Total products from in vitro translation and immunoprecip- Recombinant Plasmid DNA. T-DNA fragments ofpTi-15955 itates were separated by electrophoresis in a vertical polyacryl- generated by several restriction endonucleases were originally amide/NaDodSO4 slab gel containing the discontinuous buffer cloned in bacteriophage A Charon 3A and 4A (9). Fragments of system with 5 and 12.5% (wt/vol) acrylamide in the stacking and 7.7 and 4.8 kbp generated by BamHI and fragments of6.9, 2.8, separating gels, respectively (34). and 5.5 kbp generated by EcoRI were subcloned into the Esch- Gels were prepared for fluorography as described (35) and erichia coli plasmid pBR322, and recombinant plasmids were exposed to Kodak X-Omat R film at -80°C. designated as p101, p203, p202, p303, and p403, respectively RNA Fractionation and Transfer to Nitrocellulose. The (see Fig. 1). Detailed procedures of subcloning and of con- procedure of Thomas (36) was essentially followed. Total structing a physical map ofthe T-DNA region ofpTi-15955 will poly(A)+RNA and rRNA standards were denatured with glyoxal be published elsewhere. Plasmid DNA was isolated essentially and dimethyl sulfoxide (37). Denatured RNA (20-38 Mg) was as described (25, 26). All experiments involving recombinant applied to each lane of a 1.5% (wt/vol) agarose gel having six DNA were conducted under PI/EK1 containment in accord- or seven lanes and separated by electrophoresis at 40 mA for ance with the revised National Institutes of Health guidelines 8 hr. Fractionated RNAs were transferred to a nitrocellulose (1980). sheet by blotting for 20 hr. The nitrocellulose sheet was cut into Immobilization ofPlasmid DNA on Cellulose. Plasmid DNA strips so that each contained one lane offractionated RNA. The (150 ,g) was digested with BamHI or EcoRI (New England strips were baked at 80°C for 2 hr to immobilize the RNA. BioLabs) under conditions recommended by the supplier. The Labeling DNA and Hybridization. Plasmid DNA was la- enzymes were inactivated by incubating at 370C for 15 min in beled with [a-32P]dATP (Amersham, 600 Ci/mmol) to a high 1% NaDodSOJ10 mM EDTA and removed by deproteinizing specific activity (5-10 x 107 cpm/,ug ofDNA) using anick trans- as above. DNA fragments were desalted on a Sephadex G-50 lation kit (Amersham). One microgram of labeled DNA was column and precipitated with ethanol. hybridized with 20-38 Mg of fractionated RNA. Plasmid DNAs were immobilized on diazotized m-amino- The procedure of Wahl et al. (38) was used for hybridization benzyloxy methyl-cellulose as described (27) and modified (28). and washing ofnitrocellulose strips, except that dextran sulfate An average 77% of plasmid DNA was bound to the cellulose. was omitted from the hybridization solution. The final product contained 6-10 Ag of DNA/mg dry weight Washed and dried nitrocellulose strips were exposed at of cellulose. -80°C to Kodak X-Omat AR film in Kodak X-O Matic cassettes Purification ofPoly(A)+RNA by Hybridization. Poly(A)+RNAs with intensifying screens. (50-75 ug) were hybridized at 370C for 20 hr with 10 4g of plasmid DNA immobilized on cellulose (29). Then, the DNA- RESULTS cellulose was washed by two incubations at 370C (1 hr each) in Isolation and Characterization of Poly(A)+RNA. Polysome 200 ,ul of 95% formamide (vol/vol)/10 mM Tris-HCl, pH 7.5/ preparations were isolated from both sunflower crown gall tis- 1 mM EDTA/0.2% NaDodSOgwheat germ tRNA/(50 jig/ sue culture (PSCG-15955) and habituated sunflower tissue cul- ml). Bound RNA was eluted from the DNA-cellulose by incu- ture (HSSS). The preparations from crown gall callus contained bating at 100TC for 2 min in 200 A.l of H20 containing wheat 36% monosomes and 64% polysomes with recognizable aggre- germ tRNA at 125 Ag/ml. The eluted RNA was precipitated gates up to decatomers (Fig. 2). The average yield ofpolysomal Downloaded by guest on September 27, 2021 88 Biochemistry: Murai and Kemp Proc. Nad Acad. Sci. USA 79 (1982)

kbp 9.6-

u4 1:~ 6.8- 4.5-

4 6 8 10 Bottom 2.3- Relative gradient depth 1.9-

FIG. 2. Absorbance profile of a polysome preparation isolated from crown gall tissue culture (PSCG-15955). A total of 8.8 A265 units of a polysome preparation was layered on an 11-ml linear sucrose gradient [12.5-50.0% (wt/vol)] in 50 mM Tris'acetate, pH 8.5/200 mM KCl/5 mM Mg (OAc)2. Polysomes were sedimented at 272,400 x gfor 65 min 1 2 3 4 5 6 (4WC). Absorbance was recorded on an ISCO model 640 fractionator and model UA-5 monitor. FIG. 3. Separation of the five T-DNA fragments generated by re- striction endonucleases from the recombinant plasmids. Fragments wereseparatedby electrophoresis in a horizontal 0.7% agarose slab gel. RNA was 120 of fresh weight of crown gall tissue. This Ag/g DNA (0.5 ,Ag) was digested with BamH (lanes 1, 2, and 4) or EcoRI represented =80% of the total extractable RNA in this tissue. (lanes 3, 5, and 6). Lanes: 1, pBR322; 2, plO1; 3, p202; 4, p2O3; 5, p3O3; Poly(A)+RNA was prepared by passing polysomal RNA twice 6, p403. DNA molecular weightmarkers areHindll digests of A DNA. through an oligo(dT)-cellulose column. Poly(A)+RNA repre- Arrow indicates position of pBR322. sented <1% of the polysomal RNA. Similar recoveries were obtained from the habituated callus. of in vitro protein synthesis was directly proportional to the Total poly(A)+RNA from either the crown gall callus or the RNA concentration at 10-80 Ag/ml. With exogenous RNA at habituated callus served as an active template for in vitro trans- 80 ug/ml, in vitro translation was stimulated to >80-fold the lation by mRNA-dependent rabbit reticulocyte lysates. The rate endogenous yield.

B

Mr Mr (x 1 0 (x i )

_# 66 66

=.-!it __

...... 45 W~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 45- o Os Os- #* _. w e ..- 35 35 t E SG

_ .;.e.'..;.;;i;.t 24 24- -

1g .:,.... 18 }, ,)S, l .4 18

T 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10

FIG. 4. Electrophoretic separation and fluorographic analyses of total and immunoprecipitated products from in vitro translation encoded by crown gall mRNA. (A) Total translation products encoded by total mRNA (lane T) and RNA purified by hybridization to plasmid DNA p101 (lane 1), p203 (lane 2), and p303 (lane 3). Lanes 4, 5, and 6, immunoprecipitates of the preparations run in lanes 1, 2, and 3, respectively. Samples, 50 Mg each, of mRNAs were hybridized to lug10 of DNA; 4.7% of total products and 90% of immunoprecipitates were subjected to electrophoresis in a 12.5% (wt/vol) polyacrylamide gel. The gel was fluorographed for 80 hr. (B) Total translation products encoded by mRNA purified by hybridization to plasmid DNA p101 (lane 1), p202~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.(lane 2), p203 (lane 3), p303 (lane 4), and p403 (lane 5). Lanes 6-10, immunoprecipitates of the preparations run in lanes 1, 2, 3, 4, and 5, respectively. Samples, 60 ug each, of mRNAs were used for hybridization. The gel was fluorographed for 120 hr. Mr markers (Sigma): ,B-lactoglobulin (18,000), trypsinogen (24,000), pepsin (35,000), ovalbumin (45,000), bovine albumin (66,000). Arrows, positions of plasmid DNA-specific polypeptides; OS, octopine synthase. Downloaded by guest on September 27, 2021 Biochemistry: Murai and Kemp Proc. Natd Acad. Sci. USA 79 (1982) 89 Selective Hybridization of Crown Gall mRNA. The five re- Mr polypeptides, particularly one ofMr 25,000 encoded by both combinant plasmids (p1O0, p202, p203, p303, and p403) (Fig. p202- and p203-selected mRNA, were enriched (A, lane 2; B, 3) contained different fragments of T-DNA generated by two lanes 2 and 3). The mRNA for the Mr 25,000 polypeptide ap- restriction endonucleases and these fragments represent the peared to be the most abundant message among the purified entire T-DNA region ofpTi415955 (Fig. 1). Crown gall mRNA mRNAs. was purified from total poly(A)+RNA by selective hybridization On the other hand, distribution ofimmunoprecipitated poly- to individual recombinant plasmid immobilized on cellulose. peptides was entirely different. A prominent polypeptide that Assuming that the rate of in vitro translation is directly pro- corresponds to the molecular weight ofsunflower octopine syn- portional to the quantity of RNA, the amount of hybridized thase, 40,000, was immunoprecipitated with anti-octopine syn- mRNA varied from 0.5% (p403) to4.9% (p202)ofthepoly(A)+RNA thase IgG from translation products encoded by p303-selected originally used for hybridization, with the remaining mRNA mRNA (Fig. 4 A, lane 6; B, lane 9). Polypeptides ofMr 40,000, preparations in the 1-2% range. 25,000, 23,000 and 19,000 (Fig. 4, arrows) were detected in im- Between 2 and 7% oftotal products from in vitro translation munoprecipitates ofboth p202- and p203-selected mRNA prod- encoded by the purified mRNA were immunoprecipitated by ucts (A, lane 5; B, lanes 7 and 8). No specific polypeptide but antibody against octopine synthase, with a <2-fold difference contaminating polypeptides of Mr. 54,000 and 35,000 were ob- between the mRNA samples. These results indicate that neither served in the immunoprecipitates from protein products of purification by selective hybridization norimmunoprecipitation mRNA hybridized to p101 and p403 (A, lane 4; B, lanes 6 and alone gave sufficient specificity but the combination resulted 10). The presence ofcontaminating polypeptides may have been in 2000-fold purification of the translation products. due to the nonspecific binding ofabundant translation products Electrophoretic Separation of Total and Immunoprecipi- to anti-octopine synthase IgG or S. aureus cells. tated Products From In Vitro Translation. Total products from When normal rabbit IgG was substituted for anti-octopine in vitro translation of crown gall mRNA were separated ac- synthase IgG, no polypeptide band OfMr40,000, 25,000, 23,000 cording to molecular weight by electrophoresis through 12.5% or 19,000 was detected in immunoprecipitates from eitherp203- (wt/vol) polyacrylamide/NaDodSO4 gels. Radioactive polypep- or p303-selected mRNA products (Fig. SA). tides were visualized by fluorography of the gels. Distribution Habituated sunflower callus has been reported to be free ofpolypeptides encoded by the selectively hybridized mRNAs from T-DNA and octopine (9, 12). Poly(A)+RNA isolated from did not vary significantly (Fig. 4A, lanes 1-3; B, lanes 1-5) but the callus was purified by hybridization to p202, p203, and did differ from those of total mRNA (lane T) in that some low p303, and total products from in vitro translation encoded by purified mRNA were immunoprecipitated with anti-octopine B synthase IgG. In each case, no polypeptide band OfMr 40,000, A 25,000, 23,000, or 19,000 was detected, indicating that all of these polypeptides may be specific to octopine-type crown gall callus (Fig. SB).

Mr 66 (XI-4)

120- .... 103- ._ ... At. *U_ :! o .:SS 45 Q o < ,, - Os, 55- w * * 35

. w ......

...... -; ::

...... E .R ,...

.. ... a 24 - ..; ... .. g, ..; .q . 1 2 T1 2 3 1 2 3 4 5 6 7 FIG. 5. Fluorograph of control immunoprecipitation and in vitro FIG. 6. Autoradiograph of crown gall mRNAs hybridized to dif- translation of habituated callus mRNA. (A) Immunoprecipitates by ferent fragments of T-DNA. Twenty-nine micrograms of denatured normal rabbit antibody of total products from in vitro translation en- mRNA from crown_Lgall callus v was''applied to each lane and subjected coded by crown gall mRNA. mRNA was purified by selective hybrid- to electrophoresis in a 1.5% (wt/vol) agarose gel. RNA was transferred ization to p203 (lane 1)*:.or p3037: (lane 2). Samples, 63 pg each, of crown to a nitrocellulose sheet, which was then cut into strips, each contain- gall mRNA were hybridized to 10 pg of plasmid DNA. The gel was ing one lane of RNA. Nitrocellulose...... strips.. were hybridized to 32P-la- fluorographed for 9 days. (B) Total products from in vitro translation beled plasmid DNA p101 (lane 1), p202 (lane 2), p203 (lane 3), p303 encoded by total mRNA from habituated callus (lane T). Immunopre- (lane 4), and p403 (lane 5). Samples (38 pg each) of habituated callus cipitates by anti-octopine synthase IgG from translation products en- mRNA were treated as above and hybridized to '2P-labeled p101 (lane coded by habituated callus mRNA purified by hybridization to p202 6) and p203 (lane 7). Mr markers: pea 25S (120 x 106) rRNA, pea 18S (lane 1), p203 (lane 2), and p303 (lane 3). Samples, 75 jig each, of (6.8 x 105) rRNA, E. coli 23S (10.3 x :;105) ! '::. rRNA, E. coli 16S (5.5 x 105) mRNA were used for hybridization. The gel was fluorographed for 9 rRNA (37). Arrows, positions of plasmid DNA-specific mRNA. Strips days. Arrows, positions of the plasmid-specific polypeptides. Mr mark- containing lanes 1, 2, and the upper and lower sections of lane 3 were ers are as in Fig. 4. exposed for 24 hr, and the remainder were exposed for 72 hr. Downloaded by guest on September 27, 2021 90 Biochemistry: Murai and Kemp Proc. Natl. Acad. Sci. USA 79 (1982)

Size of mRNA for Octopine Synthase. Total poly(A)+RNAs Vicen for photographs. This work was supported in part by U.S. De- from the crown gall and habituated callus were denatured in- partment of Agriculture Science and Education Administration Com- dividually, fractionated by electrophoresis in an agarose gel, and petitive Research Grant 5901-0410-8-0168-0. transferred to nitrocellulose sheets. Nitrocellulose strips ofim- mobilized RNAs were hybridized with each offive 32P-labeled plasmid DNAs. Autoradiography of these strips showed that crown gall 1. Smith, E. F. & Townsend, C. 0. (1907) Science 25, 671-673. mRNA contained a minimum of seven transcripts from T-DNA 2. Zaenen, I., van Larebeke, N., Tuechy, H., van Montagu, M. & of 15955 Schell, J. (1974)J. Mol. Biol. 86, 109-127. pTi (Fig. 6). Plasmid p101 hybridized to mRNAs.1800, 3. Chilton, M-D., Drummond, M. H., Merlo, D. J., Sciaky, D., 1100, and 800 bases long (lane 1). Both p202 and p203.hybrid- Montoya, .A. L., Gordon, M. P. & Nester, E. W. (1977) Cell 11, ized to other mRNAs 1500, 1000, and 800 bases long, (lanes 2 263-271. and 3), the first of which was also detected by p303 (lane 4). 4. Chilton, M-D., Drummond, M. H., Merlo, D. J. & Sciaky, D. Plasmid p403 weakly hybridized to a 1600-base-long mRNA (1978) Nature (London) 275, 147-149. (lane 5). Habituated callus mRNA showed no detectable hy- 5. Thomashow, M. F., Nutter, R., Montoya, A. L., Gordon, M. P. to & Nester, E. W. (1980) Cell 19, 729-739. bridization p101. and p203 (lanes 6 and 7). 6. Chilton, M-D., Saiki, R. K., Yadav, N., Gordon, M. P. & Que- The 1500-base-long poly(A)+RNA from crown gall callus was tier, F. (1980) Proc. NatL Acad. Sci. USA 77, 4060-4064. the only mRNA that hybridized to all three plasmids p202, 7. Yadav, N., Postle, K., Saiki, R. K., Thomashow, M. R. & Chilton p203, and p303. This message hybridized to p203 strongly but M-D. (1980) Nature (London) 287, 458-461. to p202 and p303 very weakly. 8. Drummond, M. H., Gordon, M. P., Nester, E. W. & Chilton, M-D. (1977) Nature (London) 269, 535-536. 9. Gurley, W. B., Kemp, J. D., Albert, M. J., Sutton, D. W. & DISCUSSION Callis, J. (1979) Proc. Natl Acad. Sci. USA 76, 2828-2832. 10. McPherson, J. C., Nester, E. W. & Gordon, M. P. (1980) Proc. ,Our results represent direct evidence that the structural gene Natl Acad. Sci. USA 77, 2666-2670. for octopine synthase, a crown gall-specific enzyme, is located 11. Braun, A. C. (1956) Cancer Res. 16, 53-56. on the T-DNA ofthe Ti plasmid ofA. tumefacians. The evidence 12. Kemp, J. D. (1978) Plant Physiol 62, 26-30. for this conclusion.is the identification of an in vitro translation 13. Petit, A., Delhaye, S., Tempe, J. & Morel, G. (1970) Physiol. product OfMr40,000 as octopine synthase including (i) the iden- Veg. 8, 205-213. tical molecular of the and authentic octo- 14. Menag6, A. & Morel, G. (1964) C.R. Hebd. Seances Acad. Sci. weight polypeptide 259, 4795-4796. pine synthase; (ii) immunoprecipitation ofthe polypeptide with 15. Goldman, A., Thomas, D. W. & Morel, G. (1969) C.R. Hebd. anti-octopine synthase IgG but not with, normal IgG; (iii) the Seances Acad. Sci. 268, 852-854. presence of the mRNA for the Mr 40,000 protein in the crown 16. Firmin, J. L. & Fenwick, R. G. (1977) Phytochemistry 16, gall that contains octopine synthase and its absence in habit- 761-762. uated callus; and (iv) selective hybridization of the mRNA for 17. Hack, E. & Kemp, J. D. (1980) Plant PhysioL 65, 949-955. the Mr 40,000 polypeptide to p202, p203, and p303 DNA but 18. Kemp, J. D., Sutton, D. W. & Hack, E. (1979) Biochemistry 18, not to or DNA. would further 3755-3760. p101 p403 Peptide mapping 19. Goldman, A., Tempe, J. & Morel, G. (1968) C.R. Hebd. Seances strengthen the identification of the Mr 40,000 polypeptide as Acad. Sci. 162, 630-631. octopine synthase. 20. Bomhoff, G., Klapwijk, P. M., Kester, H. C. M., Schilperoort, The mRNA for octopine synthase appears to be 1500 bases R. A., Hernalsteens, J. P. & Schell, J. (1976) Mol Gen. Genet. long and has sequence homology to restriction endonuclease 145, 177-181. fragments of pTi-15955 that map at the middle of the T-DNA 21. Koekman, B. P., Ooms, G., Klapwijk, P. M. & Schilperoort, R. and but not to those that at A. (1979) Plasmid 2, 347-357. (p202, p203, p303) fragments map 22. Merlo, D. J. & Kemp, J. D. (1976) Plant Physiol 58, 100-106. the right (p403) or left (p101) borders. Based on the molecular 23. Sun, S-M. M., Buchbinder, B. U. & Hall, T. C. (1975) Plant weight and composition of octopine synthase, the Physiol 56, 780-785. lower size limit of the octopine synthase gene is 1.1 kbp. As- 24. Bantle, J. A., Maxwell, I. H. & Hahn, W. E. (1976) AnaL suming a size of 1.1 kbp, the octopine synthase gene must be Biochem. 72, 413-427. located approximately as shown in Fig. 1 because it spans the 25. Clewell, D. B. & Helinski, D. R. (1969) Proc. Natl. Acad. Sci. end of the EcoRI and the USA 62, 1159-1166. right 6.9-kbp fragment generatedby 26. Humphreys, G. O., Willshaw, G. A. & Anderson, E. S. (1975) left end of the 2.8-kbp fragment generated by EcoRI. The Biochim. Biophys. Acta 383, 457-463. mRNA for octopine synthase should include a 1100-base-long 27. Noyes, B. E. & Stark, G. R. (1975) Cell 5, 301-310. coding region and 5'-end and 3'-end untranslated regions as 28. Park, W. D., Lewis, E. D. & Rubenstein, I. (1980) Plant Physiol well as polyadenylic acid at the 3'-end. 65, 98-106. The Mr 25,000, 23,000, and 19,000 polypeptides also appear 29. Adams, S. L., Alwine, J. C., de Crombrugghe, B. & Pastan, I. to be for crown tissue. Their (1979)J. Biol Chem. 254, 4935-4938. specific octopine-type gall mRNAs, 30. Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, including 1000- and 800-base-long RNAs, were selected by hy- 247-256. bridization to p202 and p203 but not to p101, p303, or p403 31. Scheele, G. & Blackburn, P. (1979) Proc. Natl Acad. Sci. USA 76, DNAs. Habituated callus did not contain mRNA for any of the 4898-4902. three polypeptides. Their immunoprecipitation by anti-octo- 32. Hack, E. (1980) Dissertation (Univ. Wisconsin, Madison, WI). pine synthase IgG but not by normal IgG indicates that they 33. Kessler, S. W. (1975)J. Immunol 115, 1617-1624. be related to 34. Laemmli, U. K. (1970) Nature (London) 227, 680-685. may immunologically octopine synthase. 35. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, In conclusion, we have shown that a structural gene for oc- 83-88. topine synthase is located in a central portion ofT-DNA ofpTi- 36. Thomas, P. S. (1980) Proc. Natl Acad. Sci. USA 77, 5201-5205. 15955 and is expressed after transfer to the plant cells. 37. McMaster, G. K. & Carmichael, G. G. (1977) Proc. Natl Acad. Sci. USA 74, 4835-4838. We thank Mses. Kim Jurrens and Dorothy Nesbitfor tissue culturing 38. Wahl, G. M., Stern, M. & Stark, G. R. (1979) Proc. NatL Acad. and plasmidisolation, Ms. LucyTaylor for illustrations, and Mr. Steven Sci. USA 76, 3683-3687. Downloaded by guest on September 27, 2021