Protein involved in nicotine synthesis, DNA encoding, and use of sense and antisense DNAs corresponding thereto to affect nicotine content in transgenic tobacco cells and plants

Nicotine acid sequences encoding a tobacco protein involved in nicotine synthesis are described. These sequences, when inserted in to sense or anti-sense orientation, affect nicotine synthesis in transgenic tobacco plants.

TECHNICAL FIELD OF THE INVENTION 
The present invention relates to a highly purified tobacco protein involved 
in photosynthesis, to a novel process for its purification, and to its 
antisense and sense genes. In particular, this invention relates to the 
use of the sense and antisense genes encoding this protein to create 
transgenic tobacco plants having genetically altered nicotine levels. Such 
transgenic plants are useful in the production of cured tobacco for use in 
the tobacco industry. 
BACKGROUND OF THE INVENTION 
Various processes have been employed for the removal of nicotine from 
tobacco. Most of those processes, however, are not sufficiently selective 
for nicotine. They remove other ingredients from the tobacco, thereby 
adversely affecting its flavor and aroma. In addition, such processes are 
typically complex and expensive. 
Nicotine, and biologically synthesized compounds in general, are formed 
through sequences of biochemical reactions, wherein each reaction is 
catalyzed by a different enzyme. The particular reaction sequence leading 
to a given compound is known as a pathway. One approach for inhibiting the 
operation of a pathway, and thus output of its end product, is reducing 
the amount of a required enzyme in the pathway. If the enzyme's abundance, 
relative to the other enzymes of the pathway, is normally low enough to 
make that enzyme rate-limiting in the pathway's operation, then any 
reduction in the enzyme's abundance will be reflected in lowered 
production of the end product. If the enzyme's relative abundance is not 
normally rate limiting, its abundance in the cell would have to be reduced 
sufficiently to make it rate-limiting, in order for the pathway's output 
to be diminished. Similarly, if the enzyme's relative abundance is rate 
limiting, then any increase in its abundance will result in increased 
production of the pathway's end product. 
Nicotine is formed primarily in the roots of the tobacco plant and 
subsequently is transported to the leaves, where it is stored (Tso, 
Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden, 
Hutchinson & Ross, Stroudsburg, Pa. (1972)). The nicotine molecule is 
comprised of two heterocyclic rings, a pyridine moiety and a pyrrolidine 
moiety, each of which is derived from a separate biochemical pathway. The 
pyridine moiety of nicotine is derived from nicotinic acid. The 
pyrrolidine moiety of nicotine is provided through a pathway leading from 
putrescine to N-methylputrescine and then to N-methylpyrroline. An 
obligatory step in nicotine biosynthesis is the formation of 
N-methylputrescine from putrescine (Goodwin and Mercer, Introduction to 
Plant Biochemistry, pp. 488-91, Pergamon Press, New York, (1983)). 
Conversion of putrescine to N-methylputrescine is catalyzed by the enzyme 
putrescine N-methyltransferase ("PMT"), with S-adenosylmethionine serving 
as the methyl group donor. PMT appears to be the rate-limiting enzyme in 
the pathway supplying N-methylpyrroline for nicotine synthesis in tobacco 
(Feth et al., "Regulation in Tobacco Callus of Enzyme Activities of the 
Nicotine Pathway", Planta, 168, pp. 402-07 (1986); Wagner et al., "The 
Regulation of Enzyme Activities of the Nicotine Pathway in Tobacco", 
Physiol. Plant., 68, pp. 667-72 (1986)). 
A relatively crude preparation of PMT (30-fold purification) has been 
subjected to limited characterization (Mizusaki et al., "Phytochemical 
Studies on Tobacco Alkaloids XIV. The Occurrence and Properties of 
Putrescine N-Methyltransferase in Tobacco Plants", Plant Cell Physiol., 
12, pp. 633-40 (1971)). The purification steps leading to that preparation 
were limited to ammonium sulfate precipitation from the initial extract 
and gel filtration chromatography. Id. 
Antisense RNA technology allows the production of plants characterized by 
levels of an enzyme (or other protein) that are significantly lower than 
those normally contained by the plants. Ordinarily, transcription of a 
gene coding for a target enzyme gives rise to a single-stranded mRNA, 
which is then translated by ribosomes to yield the target enzyme. An 
antisense RNA molecule is one whose nucleotide sequence is complementary 
to some portion of the target mRNA molecule. The antisense RNA molecule, 
thus, will undergo complementary base pairing (hybridization) with the 
target mRNA molecule, rendering the target mRNA molecule unavailable for 
translation, more susceptible to degradation, or both. The ability of the 
cell to produce the specific enzyme coded for by the target mRNA is thus 
inhibited. 
Antisense technology has been employed in several laboratories to create 
transgenic plants characterized by lower than normal amounts of specific 
enzymes. For example, plants with lowered levels of chalcone synthase, an 
enzyme of a flower pigment biosynthetic pathway, have been produced by 
inserting a chalcone synthase antisense gene into the genome of tobacco 
and petunia. These transgenic tobacco and petunia plants produce flowers 
with lighter than normal coloration (Van der Krol et al., "An Anti-Sense 
Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation", 
Nature, 333, pp. 866-69 (1988)). Antisense RNA technology has also been 
successfully employed to inhibit production of the enzyme 
polygalacturonase in tomatoes (Smith et al., "Antisense RNA Inhibition of 
Polygalacturonase Gene Expression in Transgenic Tomatoes", Nature, 334, 
pp. 724-26 (1988); Sheehy et al., "Reduction of Polygalacturonase Activity 
in Tomato Fruit by Anti-sense RNA", Proc. Natl. Acad. Sci. USA, 85, pp. 
8805-09 (1988)), and the small subunit of the enzyme ribulose bisphosphate 
carboxylase in tobacco (Rodermel et al., "Nuclear-Organelle Interactions: 
Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme 
Levels in Transformed Tobacco Plants", Cell, 55, pp. 673-81 (1988)). 
Alternatively, transgenic plants characterized by greater than normal 
amounts of a given enzyme may be created by transforming the plants with 
the gene for that enzyme in the sense (i.e., normal) orientation. 
Genetic engineering of tobacco plants to lower nicotine content has not 
previously been possible because a cloned gene encoding the subject 
protein which is involved in nicotine synthesis had not previously been 
available. Also, a means for purifying said protein had not been known 
prior to the present invention.

SUMMARY OF THE INVENTION 
The present invention provides, a highly purified protein isolated from 
tobacco which is involved in nicotine synthesis, and a novel process for 
its purification. 
The purification process of this invention comprises the step of applying a 
tobacco plant extract to an anion exchange medium, wherein the application 
temperature and the pH and composition of the extract are such that the 
subject protein is retained by the anion exchange medium. The subject 
protein is then eluted from the anion exchange medium with an elution 
buffer comprising an effective amount of a polyamine, wherein the elution 
temperature and the pH and chemical composition of the elution buffer are 
such that but for the polyamine, the subject protein would be retained by 
the anion exchange medium. 
In a preferred embodiment, the eluate of the anion exchange medium is 
concentrated by directly applying the eluate to a chromatography medium 
having an affinity for the subject tobacco protein in the presence of the 
anion exchange medium elution buffer, and then eluting the bound material. 
Most preferably, the outlet from the anion exchange column is connected to 
the inlet of an omega-aminohexyl agarose column, on which dilute the 
subject tobacco protein from the anion exchange column is collected, for 
subsequent elution in a more concentrated form. 
The protein of this invention has a molecular weight of between about 55 
and 65 kilodaltons, a native isoelectric point of between about pH 5.0 and 
6.0, an apparent K.sub.m for putrescine of between about 300 .mu.M and 500 
.mu.M, and an apparent K.sub.m for S-adenosylmethionine of between about 
100 .mu.M and 150 .mu.M. In a preferred embodiment, the protein of the 
invention comprises a sequence of 17 amino acids selected from the amino 
acid sequences identified in the Sequence Listing as SEQ ID NO:1, SEQ ID 
NO:2, and SEQ ID NO:3. 
The present invention also provides sense and antisense recombinant DNA 
molecules encoding the subject tobacco protein, and vectors comprising 
those recombinant DNA molecules, as well as transgenic tobacco cells and 
plants transformed with those DNA molecules and vectors. The transgenic 
tobacco cells and plants of this invention are characterized by different 
nicotine content than untransformed control tobacco cells and plants. 
DETAILED DESCRIPTION OF THE INVENTION 
Purification of the Subject Tobacco Protein 
Starting material for purification of the subject tobacco protein consists 
of tobacco roots. Preferably, the roots are harvested from hydroponically 
grown tobacco plants. Hydroponic cultivation facilitates growth of the 
plants under highly controlled, reproducible conditions, and it allows 
efficient harvest of the extensive, filamentous root system in a clean, 
intact condition. 
Tobacco seeds are allowed to germinate at or near the surface of a moist 
plant potting mixture. Most preferred conditions are about 80.degree. F. 
and 60% relative humidity. About two weeks after seed germination, 
seedlings are thinned (removed) to leave sufficient room for unhindered 
growth of the remaining seedlings to a stage at which they are about six 
inches tall, and have about six leaves. When the seedlings reach a height 
of about six inches they are typically transplanted, with root system and 
pellet of potting material intact, into a hydroponic device containing a 
suitable nutrient solution and a means for aeration (oxygenation) of the 
nutrient solution. The hydroponic device also should provide for 
replenishment of the dissolved nutrients, and should be of a size 
sufficient to accommodate a fully grown tobacco plant. 
It is well known in the art that removal of the flower head (topping), a 
standard practice in commercial tobacco cultivation, increases root growth 
and increases nicotine content of the leaves. Therefore, plants to be used 
as a starting material for purification of the subject protein normally 
are topped at an appropriate stage of development. The appropriate 
interval separating planting and topping depends on several factors 
including, inter alia, plant variety, light intensity, photoperiod, soil 
and air temperatures, soil moisture, and mineral nutrition. Typically, 
however, the roots are harvested 3 to 7 days after topping. The optimal 
time for topping a given tobacco variety cultivated under a given set of 
growing conditions can readily be determined by one of ordinary skill in 
the art. 
Preferably, the harvested roots are washed with cold water, and then 
residual water is removed by aspiration in a Buchner funnel. The washed 
roots are then either used fresh, or frozen at -80.degree. C. immediately 
after harvesting. The frozen roots are stored at about -80.degree. C. 
until use. 
For a typical purification procedure for the subject tobacco protein, 
between about 400 and 600 g of frozen root tissue per liter of extraction 
buffer is homogenized in a high speed blender. The extraction buffer 
typically contains effective amounts of one or more buffering agents, one 
or more reducing agents, one or more heavy metal chelating agents, one or 
more water activity modifying agents, and one or more protease inhibitors. 
Preferably, the extraction buffer also will contain an effective amount of 
one or more phenolic compound adsorbing agents. The effective amounts of 
these agents depends on the particular agents used; however, amounts used 
generally will be chosen from among the typical amounts used during 
purification of plant proteins. The choice of agents and their effective 
amounts is, thus, well within the skill of the ordinary worker. 
The pH of the extraction buffer should be between about 7.2 and 8.3, and 
preferably about 7.5. Any water-soluble compound that has a dissociation 
constant (pK.sub.a) giving it effective pH buffering capacity at the 
desired pH may be used. Preferred buffering agents are also essentially 
transparent to ultraviolet light. Suitable buffering agents include, inter 
alia, tris(hydroxymethyl)aminomethane ("Tris"), imidazole, phosphate, 
N-morpholinopropane sulfonic acid ("MOPS"), 
N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid ("TES"), 
triethanolamine, and N-tris(hydroxymethyl)-methyl-glycine ("Tricine"). 
Tris buffer is preferred. 
Reducing agents are added to the extraction buffer in order to inhibit 
possible oxidation of protein sulfhydryl groups, and possible oxidation of 
plant phenolic compounds to reactive free radicals, both of which events 
might inactivate the subject purified tobacco protein. Suitable reducing 
agents include, inter alia, dithiothreitol ("DTT"), dithioerythritol, 
2-mercaptoethanol, thioglycolate, glutathione, cysteine, and ascorbate. 
DTT and ascorbate are preferred. 
Heavy metal chelating agents are added to the extraction buffer in order to 
prevent activation of proteases and possible inactivation of the subject 
protein by heavy metals through direct interaction with the subject 
protein or through promotion by the metals of oxidation of phenolics to 
species that inactivate the subject protein. The preferred heavy metal 
chelating agent is ethylene diaminetetraacetic acid ("EDTA"), but other 
conventional chelating agents, such as ethylene glycol bis(beta-aminoethyl 
ether) N,N,N',N'-tetraacetic acid ("EGTA") and citrate, may be used. 
Water activity modifying agents are added to the extraction buffer in order 
to stabilize the subject protein against possible denaturation and other 
more subtle conformational changes that might result in protein 
inactivation. Such water activity modifying agents are non-ionic, 
hydrophilic compounds that lower the water activity of an aqueous solution 
to which they are added. Glycerol, ethylene glycol, and low molecular 
weight polyethyleneglycol (e.g., "PEG 400") are preferred, but glucose, 
sucrose, fructose, and sorbitol are examples of other compounds useful as 
water activity modifying agents. 
Protease inhibitors usually are added to the extraction buffer in order to 
prevent possible inactivation of the subject tobacco protein through 
proteolytic cleavage by proteolyric enzymes that may be released during 
tissue homogenization. Useful protease inhibitors include, inter alia, 
phenylmethylsulfonyl fluoride ("PMSF"), leupeptin, aprotinin, chymostatin 
and pepstatin. PMSF and leupeptin are preferred. 
A phenolic compound adsorbing agent preferably is added to the extraction 
buffer to remove phenolic plant compounds that might, if present, 
inactivate or precipitate the subject protein following their oxidation 
when the plant cells are broken. Typically, insoluble polyvinylpyrrolidone 
("PVPP") and Amberlite XAD-4 are suspended in the extraction buffer to 
adsorb phenolic compounds. Other materials that remove or inactivate 
phenolic compounds without significant harm to protein activity could be 
included with or substituted for PVPP or Amberlite XAD-4. 
Prior to addition of the root tissue, the extraction buffer is cooled to 
between about -15.degree. and -20.degree. C. to form a frozen slurry. 
During the homogenization process, the temperature of the homogenate 
should not be allowed to rise above about 3.degree. to 5.degree. C. 
As will be appreciated by those of ordinary skill in the art, the quantity 
of root tissue used in the process can be varied, but the approximate 
weight of the tissue used should be measured, and the amounts of other 
components used adjusted accordingly. 
After homogenization, insoluble material (including PVPP with adsorbed 
phenolics) preferably is removed from the homogenate. Preferably, this is 
accomplished by sedimentation for between about 30 to 90 minutes at about 
4.degree. C., in a refrigerated centrifuge set at about 10,000 to 
20,000.times.g. The soluble extract (i.e., the supernatant) is decanted 
after sedimentation of the insoluble material. The final protein 
concentration of the soluble extract is generally about 2.5 to 3.5 mgl. 
The soluble extract is subjected to ammonium sulfate fractionation, and a 
40% to 65% ammonium sulfate fraction (precipitate) is collected from the 
soluble extract according to standard methods (Scopes, Protein 
Purification Principles and Practice, pp. 48-52, Springer-Verlag, New York 
(1982)). That fraction then is dissolved in about 0.1 to 0.4 ml of a 
dissolution buffer per g of root weight. 
The preferred buffer for dissolution of the 40% to 65% ammonium sulfate 
fraction contains effective amounts of a buffering agent, a heavy metal 
chelating agent, a reducing agent, a water activity-modifying agent, and 
protease inhibitors. The most preferred dissolution buffer contains Tris 
buffer (pH about 7.5) (about 10 to 20 mM), EDTA (about 1 to 10 mM), 
glycerol (about 10 to 30%), DTT (about 1 to 10 mM), PMSF (about 0.2 to 
10.0 mg/l), and leupeptin (about 0.2 to 10.0 mg/l). These buffer 
components are included for the purposes described above for the analogous 
components in the extraction buffer. The skilled worker will appreciate 
that these components may be substituted with others of similar function. 
The ammonium sulfate fraction may then be desalted by standard 
techniques--e.g., dialysis or sieving chromatography--and the desalted 
fraction directly subjected to anion exchange chromatography, as described 
below. In a preferred embodiment, however, the ammonium sulfate fraction 
first is subjected to hydrophobic interaction chromatography. 
Before the dissolved ammonium sulfate fraction is subjected to hydrophobic 
interaction chromatography, salt is added to give a salt concentration 
that is high enough to ensure that the subject tobacco protein binds to 
the hydrophobic interaction medium. The preferred concentration of added 
salt is 1.5N. The preferred added salt is NaCl. Another useful salt is 
ammonium sulfate. 
The preferred hydrophobic interaction medium comprises approximately 
spherical particles of crosslinked agarose gel, of a size suitable for 
chromatography, bearing covalently bonded phenyl groups. Such 
phenylagarose is commercially available as "phenyl-Sepharose CL-4B" 
(Pharmacia-LKB, Inc., Piscataway, N.J., Cat. No. 17-0810-01). 
Hydrated phenylagarose is packed into a suitable chromatography column 
using standard procedures, and is equilibrated at about 4.degree. to 
8.degree. C. with a high salt equilibration buffer having a pH of from 
between about 7.2 to 8.3, and preferably about 7.5. The preferred high 
salt equilibration buffer contains effective amounts of a buffering agent, 
a heavy metal chelating agent, a water activity modifying agent, a 
reducing agent, and salt at a concentration of between about 1.5 to 2.0M. 
The most preferred high salt equilibration buffer solution contains about 
10 mM Tris (pH about 7.5), about 1.5M NaCl, about 1 mM EDTA, about 2 mM 
DTT, and about 20% (v/v) glycerol. 
A sample of the salt-adjusted soluble extract (about 0.5 to 2.0 ml of 
extract per ml phenylagarose packed bed volume) is loaded onto the 
equilibrated phenylagarose column, and the column is washed with the 
equilibration buffer until the eluate becomes essentially free of 
proteinaceous material. If the buffering agent is transparent to 
ultraviolet light, this may be determined by measuring ultraviolet light 
absorbance at around 280 nm. Generally, the phenylagarose column is washed 
with about 5 to 7 column volumes of equilibration buffer. The subject 
tobacco protein remains bound to the hydrophobic interaction medium. 
Proteins still adsorbed to the phenylagarose matrix (including the subject 
protein) are then eluted at 4.degree. to 8.degree. C. with between about 4 
to 6 column volumes of an elution buffer containing a linear salt gradient 
decreasing from the load salt concentration (preferably about 1.5M) to 
about 0.0M, followed by an additional 2 to 3 column volumes of elution 
buffer without salt. Preferably, the elution buffer will include Tris 
(about 10 mM) (pH about 7.5), DTT (about 2 mM), and EDTA (about 1 mM), and 
glycerol (about 20% v/v). 
Fractions of between about 0.01 to 0.03 column volumes are collected and 
assayed for apparent PMT activity as described below and for absorbance at 
280 nm. Typically, the pooled eluate fractions have a volume of between 
about 1 to 2 column volumes, and a protein concentration of between about 
0.4 and 2.5 mg/ml. 
It will be understood that salts other than the preferred NaCl may be used 
in the foregoing buffers. Such salts include potassium chloride and 
ammonium sulfate. 
The critical step of the purification process of this invention is a novel 
anion exchange chromatography step, performed as described below. In order 
to perform this step, however, the tobacco plant extract applied to the 
column (e.g., preferably, the phenylagarose eluate) must have a pH and 
chemical composition such that the subject protein content in the extract 
will bind to the anion exchange medium. That is, the extract should have a 
pH of between about 7.2 and 8.3, comprise between about 0.0 and 10 mM 
salt, and preferably should further comprise the following: between about 
5 and 15 mM of a buffering agent, between about 1 and 10 mM of a reducing 
agent, between about 10 and 30% (v/v) of a water activity modifying agent, 
and between about 1 and 5 mM of a heavy metal chelating agent. Most 
preferably, the tobacco plant extract comprises 10 mM Tris/HCl, pH 7.5, 2 
mM DTT, 1 mM EDTA and 20% (v/v) glycerol. 
The skilled worker will, of course, appreciate that the pH and salt 
concentration of the tobacco plant extract may be varied in concert from 
the values recited above, resulting in a load condition at which the 
subject protein will still bind to the anion exchange medium. In 
particular, it is well known that an increase in salt concentration 
generally will decrease the binding of a protein to an anion exchange 
medium and an increase in pH generally will increase binding of a protein 
to an anion exchange medium. The skilled worker, therefore, could easily 
determine various combinations of salt concentration and pH, other than 
those recited above, at which the subject tobacco protein will bind to the 
anion exchange medium. The only constraint on the possible pH/salt 
concentration combinations is that the pH may not be so high as to 
denature and inactivate the subject tobacco protein. Generally, the 
subject protein should not be exposed for significant periods of time to a 
pH above about 9. 
From the foregoing, it is clear that if the tobacco root extract to be 
applied to the anion exchange medium is an ammonium sulfate fraction or 
the eluate from the above-described hydrophobic interaction chromatography 
step, then it must be desalted into an appropriate buffer. This may be 
accomplished by any standard technique. For example, gel filtration 
chromatography (using, e.g., Sephadex G-25) or dialysis may be employed, 
using well known procedures. Preferably, such desalting will be 
accomplished by dialysis. 
In a preferred process, the pooled eluate from the above hydrophobic 
interaction chromatography step (or another high salt tobacco root 
extract) is dialyzed at about 4.degree. to 8.degree. C. against a dialysis 
buffer with about 15 to 25 ml dialysis buffer per ml of pooled eluate or 
extract, for about 8 to 20 hours. Preferably, the dialysis buffer will be 
stirred. A dialysis membrane having a 10,000 kD cut-off is preferred. The 
chemical composition and pH of the dialysis buffer is chosen so that the 
subject tobacco protein in the dialyzed fraction will be retained by the 
anion exchange medium, as described above. 
The anion exchange medium should consist of relatively rigid particles 
(e.g., crosslinked agarose), of a size suitable for chromatography, that 
bear one or more functional anion exchange moieties. Such anion exchange 
moieties may be selected, inter alia, from the group consisting of 
diethylaminoethyl, polyethyleneimino, tertiary amino, quaternary amino, 
p-aminobenzyl, and diethyl-(2-hydroxypropyl)aminoethyl. Such media are 
commercially available. An anion exchange medium bearing diethylaminoethyl 
("DEAE") moieties is preferred. DEAE-agarose ("DEAE-Sepharose, Fast Flow", 
Pharmacia-LKB, Inc., Piscataway, N.J., Cat. No. 17-0709-01) is most 
preferred. 
The anion exchange medium is equilibrated to the pH and salt condition of 
the equilibration buffer, according to standard procedures. 
The equilibrated anion exchange medium then is packed according to standard 
procedures into a column (i.e., a hollow tube) having at its bottom a 
means of retaining the medium (e.g., a sintered glass disk) and an outlet 
tube. The top of the column is then covered and connected to an inlet 
tube. Then, preferably, equilibration buffer should be run through the 
column, and the pH and conductivity of the flowthrough monitored, to 
ensure that the medium is properly equilibrated. 
The column should contain enough anion exchange medium so that the proteins 
in the tobacco plant extract to be applied would occupy no more than about 
50% of the medium's capacity if they all were to bind. For example, when 
the tobacco plant extract to be applied is the above-described dialyzed 
phenylagarose eluate, the column should contain about 0.04 to 0.10 ml 
(packed bed volume) of DEAE-agarose per ml of dialyzed phenylagarose 
eluate. 
Preferably, the column is packed and the medium equilibrated at the same 
temperature at which the tobacco plant extract is to be applied. If the 
column is to be washed or eluted at a warmer temperature than that at 
which the tobacco plant extract is applied, then the slurry containing the 
anion exchange matrix may be degassed prior to packing the column. 
As described above for the tobacco plant extract to be applied to the anion 
exchange medium, the anion exchange medium equilibration buffer must have 
a pH and chemical composition such that the subject tobacco protein is 
retained by the medium. Similarly, the skilled worker easily may determine 
suitable pHhemical composition combinations. The preferred equilibration 
buffer contains essentially no added salt and has a pH of between about 
7.2 to 8.3, most preferably 7.5. A more preferred equilibration buffer 
contains effective amounts of a buffering agent, a heavy metal chelating 
agent, a reducing agent, and a water activity modifying agent. The most 
preferred equilibration buffer contains 10 mM Tris/HCl (pH 7.5), 1 mM 
EDTA, 2 mM DTT, and 20% (v/v) glycerol. 
Preferably, the tobacco plant extract, the equilibration buffer and the 
anion exchange medium are all at a temperature of between about 2.degree. 
to 10.degree. C., and most preferably between about 4.degree. to 8.degree. 
C. before and during equilibration, loading, and washing of the column. 
The tobacco plant extract is applied at a flow rate of between about 0.1 to 
0.3 column volumes/min. The flowthrough from the tobacco plant extract 
application contains practically none of the subject tobacco protein, and 
is discarded. The column is then washed with equilibration buffer until 
elution of proteinaceous material stabilizes at a low level. If the 
equilibration buffer does not contain a buffering agent that absorbs at 
280 nm, the column is washed with elution buffer until the UV absorbance 
at 280 nm stabilizes at a low level. Typically, the anion exchange medium 
is washed with 5 to 12 column volumes of equilibration buffer with 10 mM 
NaCl, and then another 3 to 10 column volumes of equilibration buffer 
without NaCl. The subject tobacco protein is retained by the anion 
exchange medium during the washing step. 
After washing, the subject tobacco protein is eluted from the anion 
exchange medium with an elution buffer comprising an effective amount of a 
polyamine, wherein the elution temperature and the pH and chemical 
composition of the elution buffer are such that but for the polyamine, the 
subject protein would be retained by the anion exchange medium. 
The polyamine in the elution buffer is selected from the group consisting 
of putrescine, N-methylputrescine, spermine, spermidine, agmatine, 
cadaverine, and mixtures thereof. Putrescine is the preferred polyamine. 
The polyamine should be present in the elution buffer at a concentration 
of between about 0.5 to 50 mM, preferably 1 to 10 mM, and most preferably 
at about 5 mM. 
The elution buffer preferably further comprises effective amounts of a 
buffering agent, a heavy metal chelating agent, a reducing agent, and a 
water activity modifying agent. Those components are as described above 
for the extraction buffer. The effective amounts of these components may 
be determined without undue experimentation by the skilled worker. The pH 
of the elution buffer should be between about 7.2 and 8.3, preferably 
about 7.5. The anion exchange medium equilibration buffer, when 
supplemented with a polyamine, is a suitable elution buffer. A preferred 
elution buffer contains 10 mM Tris/HCl (ph 7.5), 1 mM EDTA, 20% (by 
volume) glycerol, 2 mM DTT, and 5 mM putrescine (1,4-diaminobutane) (Sigma 
Chemical Co., St. Louis, Mo., Cat. No. P7505). 
Elution of the subject tobacco protein from the anion exchange column is 
preferably carried out at between about 18.degree. to 26.degree. C. (i.e., 
room temperature). The elution buffer and the anion exchange column should 
be at the chosen elution temperature before elution is commenced. 
To elute the subject tobacco protein from the column, elution buffer is 
applied at a flow rate of between about 0.02 to 0.10 column volumes/min, 
and fractions are collected from the bottom of the column. The eluate also 
may be collected into a single eluate pool. In the most preferred elution 
process, approximately one column volume of elution buffer is applied to 
the column, and the flow is then stopped. The anion exchange medium is 
left in contact with that aliquot of elution buffer for between about 1 to 
6 hours, preferably about one hour. Application of elution buffer is then 
recommenced. 
The subject tobacco protein elutes from the anion exchange medium very 
gradually. Typically, the anion exchange medium is eluted with between 
about 40 and 70 column volumes of elution buffer, and most preferably at 
least 50 column volumes of elution buffer. Apparent PMT activity of eluted 
fractions is assayed, as described below, to monitor the subject tobacco 
protein elution. 
As the subject protein is recovered in a relatively dilute form and in a 
relatively large volume, it is desirable to concentrate the anion exchange 
eluate. The eluate may, for example, be applied to any chromatography 
medium which has an affinity for the subject protein in the presence of 
the anion exchange medium elution buffer, and from which the bound 
material can be eluted with good yield in a relatively concentrated form. 
Alternatively, the subject tobacco protein may be precipitated. In a 
preferred process of this invention, the outlet from the anion exchange 
column, during elution, is connected to the inlet of the concentration 
column. In this way, the eluted protein runs out of the anion exchange 
column and directly onto the concentration column, where it is adsorbed. 
After elution of the subject protein from the anion exchange column is 
complete, the outlet of that column is disconnected from the concentration 
column, and the subject tobacco protein is eluted from the concentration 
column. 
The preferred concentration column utilizes omega-aminohexyl agarose 
("omega-aminohexyl-Sepharose 4B", Sigma Chemical Co., St. Louis, Mo., Cat. 
No. A8894) ("AHS"), with a bed volume 10 to 30% that of the anion exchange 
column. The subject tobacco protein is eluted from this column with an 
elution buffer comprising a relatively high concentration of salt, 
preferably 1.5M NaCl. Preferably, the elution buffer further comprises 
effective amounts of a buffering agent, a heavy metal chelating agent, a 
reducing agent, and a water activity modifying agent. The most preferred 
elution buffer comprises 1.5M NaCl, 10 mM Tris/HCl (ph 7.5), 1 mM EDTA, 
20% (v/v) glycerol, and 2 mM DTT. The concentration column is preferably 
loaded and eluted at 4.degree.-8.degree. C. 
The first 4 to 8 column volumes of eluate from the concentration column 
contains the majority of the PMT activity. This fraction is further 
concentrated, preferably in an ultra-filtration device (such as the 
"Centricon 30", available from Amicon Corp., Danvers, Mass.). After 
ultrafiltration the sample typically has a protein concentration of 
between about 0.04 and 0.70 mg/ml. Typically the subject 
protein-containing fractions from several such concentration columns are 
pooled and further concentrated. 
The subject protein obtained after the anion exchange and sample 
concentration steps is further purified by preparative scale isoelectric 
focussing. Isoelectric focussing involves placing the sample mixture in a 
stabilized pH gradient, across which a voltage is then applied. Each 
protein species migrates electrophoretically toward the point in the pH 
gradient at which the net electrical charge of that protein species is 
zero. The pH at which a protein has a net electric charge of zero is 
called that protein's isoelectric point. 
Various pH gradient stabilizing media, including, inter alia, sucrose 
solutions and polyacrylamide gels, can be used. Similarly, various methods 
of fractionating the pH gradient to recover proteins after isoelectric 
focussing can be employed. The pH gradient fractionation method should be 
chosen so as to be compatible with the gradient stabilizing medium. 
The preferred pH gradient stabilizing medium is a sucrose solution (density 
gradient) contained in a glass tube. Most preferably, the sucrose density 
gradient contains a pH gradient ranging from about pH 5 to about pH 6. The 
preferred gradient fractionation method is precisely controlled liquid 
flow through a stopcock. Fractions collected are tested for pH and 
apparent PMT activity. Apparatuses, chemicals, and protocols for 
isoelectric focussing are available from several commercial sources. 
The tobacco protein isolated by the process of this invention is 
substantially free of other tobacco proteins, in that the subject protein 
is the predominant protein in the preparation. The few contaminating 
tobacco proteins in the preparation are separated from the subject tobacco 
proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis 
("SDS-PAGE"), according to standard techniques. In this way, sufficiently 
pure protein for amino acid sequence analysis is obtained. 
Characterization of the Subject Tobacco Protein 
The subject tobacco protein is characterized by a molecular weight of 
between about 55 and 65 kilodaltons, as determined by SDS-PAGE, and a 
native isoelectric point of between about pH 5.0 and 6.0, as measured by 
isoelectric focussing. 
The subject tobacco protein is further characterized by the apparent 
ability to catalyze the transfer of the methyl group of 
S-adenosylmethionine to the delta amino group of putrescine, and by 
apparent high substrate specificity for putrescine. 
The Michaelis-Menten constant (K.sub.m) is defined as the substrate 
concentration at which the initial reaction velocity is equal to one half 
of the maximal velocity of the reaction. K.sub.m values vary widely, even 
for separate enzyme species that catalyze the same reaction. K.sub.m 
measurements are thus useful "identity markers" for enzymes. Partially 
purified tobacco protein according to the invention is characterized by an 
apparent K.sub.m for putrescine of between about 300 and 500 .mu.M. Highly 
purified tobacco protein of the present invention is characterized by an 
apparent K.sub.m for S-adenosylmethionine of between about 100 and 150 
.mu.m. 
Determination of Partial Amino Acid Sequence of the Subject Tobacco Protein 
In preparation for amino acid sequence analysis, the standard technique of 
SDS-PAGE is used to separate the subject protein from the few 
contaminating proteins that remain after the anion exchange, sample 
concentration, and isoelectric focussing steps. Detailed protocols for 
SDS-PAGE are found in Laemmli, "Cleavage of Structural Proteins During the 
Assembly of the Head of Bacteriophage T4", Nature, 227, pp. 680-85 (1970); 
and in manuals supplied by manufacturers of electrophoresis equipment. By 
well known techniques, bands containing individual proteins are 
transferred electrophoretically (electroblotted) onto thin sheets or 
membranes, where they are retained and visualized. In one well-known 
method, protein bands are electroblotted onto glass microfiber sheets 
coated with a hydrophobic polycation, such as 
poly(4-vinyl-N-methylpyridinium)iodide, and visualized by a non-anionic 
agent such as fluorescamine. Another method involves electroblotting of 
proteins onto polyvinylidene difluoride membranes ("Immobilon-P", 
Millipore, Bedford, Mass.) and visualization of bands by an anionic dye 
such as amido black (Bauw et al, "Alterations in the Phenotype of Plant 
Cells Studied by NH.sub.2 -Terminal Amino Acid Sequence Analysis of 
Proteins Electroblotted from Two Dimensional Gel-Separated Total 
Extracts", Proc. Natl. Acad. Sci. USA, 84, pp. 4806-10 (1987); A Practical 
Guide to Protein and Peptide Purification for Microsequencing, Paul T. 
Matsudaira (ed.), Academic Press, New York, (1989)). 
The aforementioned techniques for transferring isolated proteins from 
electrophoretic gels and visualizing the transferred proteins are 
preferred. However, it will be appreciated by those skilled in the art 
that variations in materials and procedures used to prepare 
electrophoretically isolated proteins for sequence analysis are not 
excluded from the present invention. 
The bands constituting purified tobacco protein according to the invention 
are identified by apparent molecular weight (i.e., about 60 kD). Following 
transfer of the protein bands from electrophoresis gel to membrane, and 
visualization of the transferred bands, the pieces of membrane bearing the 
individual bands of the subject protein are cut out precisely, so as to 
avoid contamination from any adjacent protein band. 
The protein bands (isolated as described above) constituting the subject 
purified tobacco protein are subjected to amino terminal sequence analysis 
by standard automated methods. Tobacco proteins according to the invention 
comprise an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, 
and SEQ ID NO:3. 
SEQ ID NO:1 is from the "a1" band (FIG. 5). SEQ ID NO:2 is from the "a2" 
band (FIG. 5). SEQ ID NO:3 is the consensus sequence of SEQ ID NO:1 and 
SEQ ID NO:2. 
Highly homologous sequences from closely adjacent purified protein bands 
suggest the existence of multiple forms of the subject tobacco protein. 
Such multiple forms of the subject tobacco protein may arise from 
post-translational modification of a single gene product, or from multiple 
forms of genes encoding the subject tobacco protein. 
Cloning Of DNA Sequences Encoding the Subject Tobacco Protein 
The partial amino acid sequences (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) of 
the tobacco proteins isolated according to the invention are used to 
design a set of oligonucleotides, one or more of which selectively 
hybridizes with DNA sequences encoding the absent tobacco protein in a 
tobacco root cDNA library. This selective hybridization is used to 
identify cDNA clones containing sequences encoding part or all of the 
subject tobacco protein. A description of the design of oligonucleotide 
probes from amino acid sequences is presented in Chapter 11 of Sambrook et 
al. Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Press 
(1989). Synthesis of such oligonucleotide probes is carried out routinely 
with commercially available, automated equipment. 
Construction of cDNA libraries is now a routine task in molecular biology 
laboratories. See generally Chapter 8 of Sambrook et al., supra. 
Similarly, screening of cDNA libraries with oligonucleotide probes, to 
identify clones containing sequences of interest, is now commonplace and 
well within the capability of those of skill in the art. A description of 
the use of oligonucleotides for screening cDNA libraries is found in 
Chapter 11 of the laboratory manual by Sambrook et al., supra. The cDNA 
clones selected on the basis of hybridization with oligonucleotide probes 
are characterized as to size, presence of restriction sites, and 
nucleotide sequence. Such methods of DNA analysis are well described in, 
inter alia, publications by Sambrook et al., supra, and Ausubel et al., 
Short Protocols in Molecular Biology, Green Publishing Associates and 
Wiley Interscience, New York (1989). Any cDNA clone obtained in this way 
can itself be used as a probe for identification of additional cDNA clones 
corresponding to the subject tobacco protein. 
A tobacco (Nicotiana tabacum L. var. NK326) genomic library is commercially 
available (Clonetech Laboratories, Inc., Palo Alto, Calif.). Such a 
genomic library is screened according to protocols supplied by the vendor, 
to obtain the chromosomal gene(s) encoding the subject tobacco protein. 
In addition to the aforementioned methods, one of skill in the art may 
advantageously employ polymerase chain reaction ("PCR") methods to obtain 
the desired cDNA clones. The basic principle of PCR is rapid amplification 
of a segment of DNA that lies between two regions of known sequence. 
Various PCR techniques have been developed for use in the detection, 
analysis and construction of specific DNA molecules. Equipment, chemicals 
and protocols for numerous applications of PCR technology are commercially 
available. For a general discussion of PCR methods, see chapter 14 of 
Sambrook et al. (supra). 
A PCR method preferred in the practice of the present invention is known as 
RNA PCR. There are two basic steps in RNA PCR: (1) reverse transcriptase 
synthesis of a first cDNA strand using RNA as a template in the presence 
of a reverse primer, and (2) Taq polymerase synthesis of the complementary 
cDNA strand, followed by Taq polymerase amplification of both strands, in 
the presence of both the forward and reverse primers. 
A cDNA molecule encoding the subject tobacco protein may be obtained by 
application of the RNA PCR method, using tobacco root poly(A.sup.+) RNA as 
a template with oligonucleotide primers based on known PMT partial amino 
acid sequences. 
Accordingly, this invention provides recombinant DNA molecules encoding the 
subject purified tobacco proteins. 
Production Of Transgenic Tobacco Cells and Plants Stably Transformed With 
DNA Sequences Encoding The Subject Protein In The Sense Or Antisense 
Orientation 
This invention also provides transgenic tobacco cells and plants stably 
transformed with recombinant DNA molecules, operably linked to regulatory 
sequences, that encode the subject tobacco proteins and that encode 
antisense RNA molecules corresponding to said tobacco proteins. 
To produce a tobacco plant having lower nicotine content than an 
untransformed control tobacco plant, a tobacco cell is transformed with an 
artificial antisense transcriptional unit comprising a partial cDNA 
sequence, a full-length cDNA sequence, a partial chromosomal sequence, or 
a full-length chromosomal sequence corresponding to the subject purified 
protein, cloned in the antisense orientation, with appropriate operably 
linked regulatory sequences. Appropriate regulatory sequences include a 
transcription initiation sequence ("promoter"), and a 
polyadenylation/transcription termination sequence. 
Expression of antisense sequences in transgenic tobacco plants typically 
utilizes the Cauliflower Mosaic Virus (CaMV) 35S promoter. See, e.g., 
Cornelissen et al., "Both RNA Level and Translation Efficiency are Reduced 
by Anti-Sense RNA in Transgenic Tobacco", Nucleic Acids Res., 17, pp. 
833-43 (1989); Rezaian et al., "Anti-Sense RNAs of Cucumber Mosaic Virus 
in Transgenic Plants Assessed for Control of the Virus", Plant Molecular 
Biology, 11, pp. 463-71 (1988); Rodermel et al., "Nuclear-Organelle 
Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate 
Carboxylase Enzyme Levels in Transformed Tobacco Plants", Cell, 55, pp. 
673-81 (1988); Smith et al., "Antisense RNA Inhibition of 
Polygalacturonase Gene Expression in Transgenic Tomatoes", Nature, 334, 
pp. 724-26 (1988); Van der Krol et al., "An Anti-Sense Chalcone Synthase 
Gene in Transgenic Plants Inhibits Flower Pigmentation", Nature, 333, pp. 
866-69 (1988). Use of the CaMV 35S promoter for expression of the subject 
protein in the transformed tobacco cells and plants of this invention is 
preferred. Use of the CaMV promoter for expression of other recombinant 
genes in tobacco roots has been well described (Lam et al., "Site-Specific 
Mutations Alter In Vitro Factor Binding and change Promoter Expression 
Pattern in Transgenic Plants", Proc. Natl. Acad. Sci. USA, 86, pp. 7890-94 
(1989); Poulsen et al., "Dissection of 5' Upstream Sequences for Selective 
Expression of the Nicotiana plumbaginifolia rbcS-8B Gene", Mol. Gen. 
Genet., 214, pp. 16-23 (1988)). 
While use of the CaMV 35S promoter is preferred, it should be appreciated 
that other promoters are successfully used for expression of foreign genes 
in tobacco plants, and the use of promoters other than the CaMV 35S 
promoter falls within the scope of the present invention. 
Various transcription termination sequences are known. The source and 
identity of the transcription termination sequence is primarily a matter 
of convenience. For example, the nopaline synthase ("NOS"), octopine 
synthase ("OCS"), and CaMV polyadenylation/transcription termination 
sequences are used for expression of foreign genes in transgenic tobacco 
plants, and would be useful for expression of sequences encoding the 
subject protein. See, e.g., Rezian et al., supra, and Rodermel et al., 
supra. 
Standard techniques, such as restriction mapping, Southern blot 
hybridization, and nucleotide sequence analysis, are then employed to 
identify clones bearing sequences encoding the subject tobacco protein in 
the antisense orientation, operably linked to the regulatory sequences 
(i.e., promoter and polyadenylation/transcription termination sequences). 
There is a well-developed technology applicable for introduction of 
exogenous DNA into the genome of tobacco cells so as to produce transgenic 
tobacco cells, stably transformed with the exogenous DNA. Any of the 
numerous known methods of tobacco cell transformation can be used in 
practicing the present invention. Methods for tobacco cell transformation 
are conveniently classified on the basis of whether or not they utilize 
components of the Agrobacterium system. 
Agrobacterium tumefasciens is a gram negative bacterium that harbors a 
plasmid with nucleotide sequences called "T-DNA" (for transferred DNA), 
that are efficiently transferred and integrated into chromosomes of 
dicotyledonous plants (including tobacco) in nature, causing tumor growth 
on infected plants. This naturally-occurring vector system for integration 
of foreign DNA into plant chromosomes has been extensively studied, 
modified, and exploited for plant genetic engineering. (Deblaere et al., 
"Efficient Octopine Ti Plasmid-Derived Vectors for Agrobacterium-Mediated 
Gene Transfer to Plants", Nucleic Acids Research, 13, pp. 4777-88 (1985)). 
Naked recombinant DNA molecules comprising DNA sequences corresponding to 
the subject purified tobacco protein operably linked, in the sense or 
antisense orientation, to regulatory sequences are joined to appropriate 
T-DNA sequences by conventional methods. These are introduced into tobacco 
protoplasts by polyethylene glycol techniques or by electroporation 
techniques, both of which are standard. Alternatively, such vectors 
comprising recombinant DNA molecules encoding the subject purified tobacco 
protein are introduced into live Agrobacterium cells, which then transfer 
the DNA into the tobacco plant cells (Rogers et al., "Gene Transfer in 
Plants: Production of Transformed Plants Using Ti Plasmid Vectors", 
Methods in Enzymology, 118, pp. 627-40 (1986)). 
Although widely used in the art, Agrobacterium technology is not a 
necessary component of the present invention. Transformation by naked DNA 
without accompanying T-DNA vector sequences can be accomplished via fusion 
of tobacco protoplasts with DNA-containing liposomes or via 
electroporation. (Shillito et al., "Direct Gene Transfer to Protoplasts of 
Dicotyledonous and Monocotyledonous Plants by a Number of Methods, 
Including Electroporation", Methods in Enzymology, 153, pp. 313-36 
(1987)). Naked DNA unaccompanied by T-DNA vector sequences can also be 
used to transform tobacco cells via inert, high velocity microprojectiles 
(BIOLISTIC.TM. Particle Delivery System, DuPont, Wilmington, Del.). 
Preferably, the recombinant DNA molecules and vectors used to produce the 
transformed tobacco cells and plants of this invention will further 
comprise a dominant selectable marker gene. Suitable dominant selectable 
markers for use in tobacco include, inter alia, antibiotic resistance 
genes encoding neomycin phosphotransferase, hygromycin phosphotransferase, 
and chloramphenicol acetyltransferase. Another well-known dominant 
selectable marker suitable for use in tobacco is a mutant dihydrofolate 
reductase gene that encodes methotrexate-resistant dihydrofolate reductase 
(Deblaere et al., supra). DNA vectors containing suitable antibiotic 
resistance genes, and the corresponding antibiotics are commercially 
available. 
Transformed tobacco cells are selected out of the surrounding population of 
non-transformed cells by placing the mixed population of cells into a 
culture medium containing an appropriate concentration of the antibiotic 
(or other compound normally toxic to tobacco cells) against which the 
chosen dominant selectable marker gene product confers resistance. Thus, 
only those tobacco cells that have been transformed will survive and 
multiply. 
Transformed cells are induced to regenerate intact, fertile, tobacco plants 
through application of tobacco cell and tissue culture techniques that are 
well known in the art. The method of plant regeneration is chosen so as to 
be compatible with the method of transformation. Verification of the 
stable presence and the orientation of the subject purified protein 
encoding sequence in the genome of putatively transgenic tobacco plants is 
by Mendelian inheritance of such DNA sequence, as revealed by standard 
methods of DNA analysis applied to progeny resulting from controlled 
crosses. 
After regeneration of transgenic tobacco plants from transformed cells, the 
introduced DNA sequence is readily transferred to other tobacco varieties 
through conventional plant breeding practices and without undue 
experimentation. 
Decreased levels of nicotine in transgenic tobacco plants containing 
antisense DNA segments corresponding to the subject tobacco protein are 
detected by standard nicotine assays. 
Those familiar with the recombinant DNA methods described above will 
recognize that one could employ a full-length cDNA molecule or a 
full-length chromosomal gene, joined in the sense orientation, with 
appropriate operably linked regulatory sequences, to construct transgenic 
tobacco cells and plants. (Those of skill in the art will also recognize 
that appropriate regulatory sequences for expression of genes in the sense 
orientation include any one of the known eukaryotic translation start 
sequences, in addition to the promoter and polyadenylation/transcription 
termination sequences described above). Such transformed tobacco plants 
are characterized by increased levels of the subject tobacco protein. 
It should be understood, therefore, that use of the subject DNA sequences 
to affect levels of the subject protein, and thereby affect the nicotine 
content in tobacco plants, falls within the scope of the present 
invention. 
In order that this invention may be better understood, the following 
examples are set forth. These examples are for purposes of illustration 
only, and are not to be construed as limiting the scope of the invention 
in any matter. 
EXAMPLES 
______________________________________ 
Composition of Buffer Solutions 
______________________________________ 
Buffer A 
50 mM Tris/HCl, pH 7.5 
5 mM EDTA (free acid) 
20% (v/v) glycerol 
2 mM DTT 
0.5% (w/v) sodium ascorbate 
2% (w/v) PEG 400 
0.4 mg/l PMSF (from a 1 mg/ml stock solution) 
0.4 mg/l leupeptin (from a 1 mg/ml stock solution) 
100 g/l PVPP 
40 g/l Amberlite XAD-4 
Buffer B 
10 mM Tris/HCl, pH 7.5 
1 mM EDTA (free acid) 
20% (v/v) glycerol 2 mM DTT 
0.4 mg/l PMSF (from a 1 mg/ml stock solution) 
0.4 mg/l leupeptin (from a 1 mg/ml stock solution) 
Buffer C 
10 mM Tris/HCl, pH 7.5 
1 mM EDTA (free acid) 
20% (v/v) glycerol 2 mM DTT 
Protease Inhibitor Stock Solutions 
PMSF (1 mg/ml) was dissolved in dimethylformamide and 
stored in 2.1 ml aliquots at -20.degree. C. until use. 
Leupeptin (1 mg/ml) was dissolved in distilled water and 
stored in 2.1 ml aliquots at -20.degree. C. until use. 
______________________________________ 
Preparation of Crude Extract 
Approximately one kg of roots from hydroponically grown tobacco (Nicotiana 
tabacum L. var. Burley 21) plants was harvested at 3 days after topping. 
The harvested roots were washed with cold water and placed on a Buchner 
funnel, where water was removed by aspiration. The washed roots were 
stored frozen at -80.degree. C. The frozen roots were added to 2.5 liters 
of Buffer A that had been chilled into a frozen slurry, in a one-gallon 
Waring blender. The roots were mixed into the buffer slurry with a large 
spoon. The blender was started on a low speed setting, followed by 
additional homogenization at a medium speed setting. Care was taken to 
avoid permitting the temperature of the homogenate to rise above 
3.degree.-5.degree. C. 
The extract was dispensed into centrifuge bottles, and insoluble debris was 
pelleted by centrifugation at 13,680.times.g for 70 minutes at 4.degree. 
C. 
The supernatant was decanted, and its volume was 2.37 l. Approximately 0.77 
g of DTT was added to the extract. 
Ammonium Sulfate Fractionation 
Crystalline ammonium sulfate was slowly added to the extract in the amount 
of 22.6 g per 100 ml of extract, so as to bring the extract to 40% of 
saturation with ammonium sulfate. The extract with ammonium sulfate was 
stirred for two hours at 4.degree. C. 
The 40% ammonium sulfate precipitate was removed by centrifugation at 
27,500.times.g for 30 min at 4.degree. C. An additional 0.33 g of DTT was 
added per liter of extract. Crystalline ammonium sulfate, in the amount of 
15.3 g per 100 ml of extract, was slowly added to the extract, so as to 
increase the ammonium sulfate concentration from 40% to 65% saturation. 
The extract with 65% ammonium sulfate was stirred overnight at 4.degree. 
C. The 40-65% ammonium sulfate fraction was pelleted by centrifugation at 
27,500.times.g for 70 minutes at 4.degree. C., and the supernatant was 
discarded. 
The 40-65% ammonium sulfate precipitate was dissolved in Buffer B to yield 
a total volume of 200 ml, and then 17.53 g of NaCl was added and allowed 
to dissolve during stirring on ice. The dissolved 40-65% fraction with 
added NaCl was centrifuged at 47,800.times.g for 30 min at 4.degree. C., 
and the pellets were discarded. 
Preparation of a crude extract and ammonium sulfate fractionation were 
performed 3 more times, substantially as described above, and the 4 
resulting 40-65% ammonium sulfate fractions (200 ml each) were pooled. The 
800 ml pool thus formed represented a total of 5.239 kg of root tissue. 
Hydrophobic Interaction Chromatography 
A phenyl-Sepharose CL 4B (Pharmacia Inc., Piscataway, N.J., Cat. No. 
17-0810-01) hydrophobic interaction column (5 cm.times.20 cm) was 
equilibrated with Buffer C supplemented with 1.5M NaCl. An 800 ml pool of 
40-65% clarified ammonium sulfate fraction, representing 5.239 kg of root 
tissue, was then loaded onto the equilibrated phenyl-Sepharose column. The 
column was washed with Buffer C supplemented with 1.5M NaCl until a stable 
baseline of 280 nm absorbance was obtained, indicating that practically 
all unbound protein had been removed. The subject tobacco protein was then 
eluted with a 2 l, linear gradient of NaCl decreasing from 1.5M to 0.0M in 
Buffer C. The column was further washed with an additional 1 l of Buffer 
C. Fractions of 12 ml each were collected, and fractions (every third 
fraction in and around the apparent PMT activity peak, and every tenth 
fraction elsewhere in the gradient) were subsequently assayed for apparent 
PMT activity as described below. The hydrophobic interaction 
chromatography was carried out at 4.degree. C., with a flow rate of 4.7 
ml/min. 
Phenyl-Sepharose fractions #86 through #116, which contained apparent PMT 
activity, were pooled and the pool was dialyzed for about 18 hours, 
against 9 l of Buffer C, with constant stirring. The dialyzed sample was 
separated into 4 aliquots of 100 ml each, and stored at -80.degree. C. 
______________________________________ 
Assay of Apparent PMT Activity 
Each reaction tube contained the following: 
______________________________________ 
12.5 .mu.mol 
Tris/HCl pH 8.3 
0.25 .mu.mol 
EDTA 
1.25 .mu.mol 
2-mercaptoethanol 
0.9 .mu.mol 
putrescine 
0.15 .mu.mol 
uplabelled S-adenosylmethionine 
0.18 .mu.mol 
[.sup.14 C-methyl]S-adenosylmethionine 
(57 nCi/nmol) 
enzyme sample 
Total Volume = 0.25 ml 
______________________________________ 
The reaction was started by addition of the enzyme sample, and it was 
carried out at 30.degree. C. for 30 minutes. The reaction was stopped by 
addition of 0.5 ml of 10% (w/v) NaOH saturated with NaCl. 
The radioactive product, N-[.sup.14 C-methyl]putrescine, was separated from 
the substrate by solvent extraction into chloroform. After vortexing the 
stopped reaction mixture with 1 ml of chloroform for 90 seconds, the 
organic and aqueous phases were separated by centrifugation at 
1600.times.g.sub.av for 5 minutes. A 0.5 ml aliquot of the organic phase 
was then assayed. The 0.5 ml aliquot of the organic phase was added to 9.5 
ml of liquid scintillation cocktail (Beckman Instruments, Columbia, Md.) 
and radioactivity was measured by standard procedures with a liquid 
scintillation counter. 
One unit of apparent PMT activity is defined as one nanomole of product 
formed per 30 min., at 30.degree. C. 
Negative controls were included with all PMT assays. Negative controls 
consisted of reaction mixtures minus enzyme, or reaction stopped with NaOH 
at time zero. 
Anion Exchange Chromatography 
Two 100 ml aliquots of the phenyl-Sepharose-purified sample were thawed and 
then loaded, at 4.degree. C., at a flow rate of 1.5 ml/min, onto a 
DEAE-Sepharose "Fast Flow" (Pharmacia-LKB, Piscataway, N.J., Cat. No. 
17-070901, Lot No. OB-05854) column (1 cm.times.14.5 cm) that had been 
equilibrated at 4.degree. C. with Buffer C. 
The DEAE-Sepharose column was then washed at a flow rate of 1.5 ml/min with 
70 ml Buffer C containing 10 mM NaCl until a stable 280 nm baseline was 
obtained. The column was then re-equilibrated with 50 ml of Buffer C 
without NaCl. The column was then raised to room temperature (24.degree. 
C.), and the void volume of the column was replaced with Buffer C 
containing 5 mM putrescine (Sigma Chemical Co., St. Louis, Mo., Cat. No. 
P7505, Lot No. 39F0039). The column was held at 24.degree. C. with no flow 
for about 1 hour, and then the subject tobacco protein was eluted at 
24.degree. C. with 632 ml of Buffer C containing 5 mM putrescine, at a 
flow rate of 0.7 ml/min (15 hours). 
Concentration of the Subject Tobacco Protein by Adsorption 
The eluted composition containing the subject protein from the 
DEAE-Sepharose column was collected directly onto a column (1 cm.times.3 
cm) of omega-aminohexyl-Sepharose 4B ("AHS") (Sigma Chemical Co., St. 
Louis, Mo., Cat. No. A8894) that was maintained at 4.degree. C. The 
subject protein was eluted from the AHS column with Buffer C containing 
1.5M NaCl, at a flow rate of 1.6 ml/min. Four fractions of 12-15 ml each 
were collected and assayed for apparent PMT activity as described above. 
The first fraction (14.7 ml) contained more than 80% of the apparent total 
PMT activity recovered from the AHS concentration column. 
Ultrafiltration 
For further concentration, 13.7 ml of the first AHS fraction was divided 
into 6 aliquots and placed in "Centricon 30" (Amicon, Danvers, Mass.) 
ultrafiltration devices and concentrated about 25-fold. Concentrates from 
six such devices were pooled, diluted about 80-fold with Buffer C without 
added salt, and subjected to a second round of ultrafiltration in a single 
"Centricon 30," until the total volume was about 150 .mu.l. The 150 .mu.l 
of concentrate was stored at -80.degree. C. 
Preparative Isoelectric Focussing 
The subject tobacco protein purified through the DEAE/AHS stage (including 
concentration by ultrafiltration) was further purified by isoelectric 
focussing. Preparative scale isoelectric focussing was performed with 
commercially available ampholytes (Pharmacia-LKB, Piscataway, N.J.) in a 
sucrose density gradient (1.6 cm.times.21 cm). The pH gradient was 
prepared according to the ampholyte vendor's instructions, and spanned the 
pH range from about 5.3 to about 6.3. Focussing was carried out for about 
3 hours with application of from 1,000 to 4,000 volts (power between 1 and 
4 watts). Fractions of 1 ml each were collected after focussing, and the 
pH and apparent PMT activity of each fraction was measured. Focussing and 
fraction collection were done at 4.degree. C. 
FIG. 4 is a dual plot of relative apparent PMT activity and pH versus 
fraction number (i.e., location in the sucrose density gradient), after 
isoelectric focussing. The data from the experiment depicted in FIG. 4 
indicated the isoelectric point of the subject tobacco protein to be 
approximately 5.7. In other isoelectric focussing experiments the pI of 
the subject tobacco protein appeared to be as low as 5.0 and as high as 
5.8. Those of skill in the art will recognize that in practice, numerous 
factors affect apparent pI, and thus pI measurements normally exhibit some 
variation. 
Assessment of Relative Purity of Subject Protein 
Relative purity of the subject tobacco protein at successive steps in the 
purification process was assessed by specific activity measurements (Table 
1). The purification (fold) values shown in Table 1 are underestimates of 
the actual degree of purification from tobacco root crude extract, because 
the 40-65% ammonium sulfate fraction was taken as 100%, for activity yield 
calculations. 
TABLE 1 
______________________________________ 
Total Specific Activity 
Purifi- 
Process Protein Activity Yield cation 
Stage (mg) (units/mg) (%) (fold) 
______________________________________ 
Ammonium 4128* 47.9 100.0 1.0 
Sulfate 
Phenyl- 680 134.6 46.3 2.8 
Sepharose 
DEAE/AHS 1.76** 5203 7.7** 108.6 
______________________________________ 
*Pool of 40-65% ammonium sulfate fractions from 4 separate crude extracts 
**Represents only half of material from phenylSepharose column. 
Relative purity of the subject tobacco protein at successive steps in the 
present process was also assessed by the standard procedure of SDS-PAGE. 
FIG. 1 shows SDS-PAGE protein band patterns displayed (upon silver 
staining) by samples at each of the steps in the purification process. 
Samples on the gel were as follows: lanes 1 and 6, molecular weight 
standard proteins (listed above, in Brief Description of the Figures); 
lane 2, 40-65% ammonium sulfate fraction; lane 3, apparent PMT activity 
peak fraction from phenyl-Sepharose column; lane 4, concentrated material 
from DEAE/AHS step; lane 5, apparent PMT activity peak fraction from 
isoelectric focussing of concentrated material from DEAE/AHS step. It 
should be noted that the protein band corresponding to the purified 
protein of the number (indicated by arrow) that is prominent in the 
DEAE/AHS-purified material (lane 4) is barely visible in the material from 
the preceding hydrophobic interaction step (lane 3). 
Molecular Weight of Subject Tobacco Protein 
The apparent molecular weight of the subject tobacco protein was measured 
in an experiment that involved isolation of the subject protein on a 
non-denaturing electrophoresis gel loaded with the subject protein 
material that had been through the ammonium sulfate, phenyl-Sepharose, and 
DEAE/AHS/ultrafiltration stages of purification. The non-denaturing 
stacking gel buffer contained 0.27M Tris/HCl (pH 6.8), 10% (v/v) glycerol, 
and 20 mM 2-mercaptoethanol. The nondenaturing 12.5% polyacrylamide 
resolving gel buffer contained 0.38M Tris/HCl (pH 8.8), 10% (v/v) 
glycerol, and 12 mM 2-mercaptoethanol. 
A single lane from the non-denaturing gel was excised, cut in half along 
its length, and then cut into 3 mm slices. One half of each gel slice was 
placed directly into the standard PMT assay mixture, and the corresponding 
half of each gel slice was subjected to SDS-PAGE. 
The non-denaturing gel slice that displayed the highest apparent PMT 
activity (FIG. 2) contained essentially a single protein with an apparent 
molecular weight of about 60 kD (FIG. 3). 
Apparent Enzymatic Activity of The Subject Tobacco Protein 
Substrate specificity tests were carried out with the highly purified 
tobacco protein of the present invention. 1,3-Diaminopropane and 
1,5-diaminopentane (chemical analogs of putrescine) 
phosphatidylethanolamine (a methyl group acceptor), and N-methylputrescine 
(the normal product of PMT), were compared with putrescine 
(1,4-diaminobutane) for ability to serve as an apparent substrate for PMT. 
When 1,3-diaminopropane, 1,5-diaminopentane, and the 
phosphatidylethanolamine were substituted for putrescine in the standard 
PMT assay (described above), no detectable amount of radioactive product 
was formed. When N-methylputrescine was substituted for putrescine in the 
PMT assay, radioactive product formation was less than 6% of that observed 
with putrescine. 
Apparent K.sub.m values for the two PMT substrates, putrescine and 
S-adenosylmethionine, were determined by measuring PMT activity (as 
described above) at various rate-limiting concentrations of one substrate, 
while the other substrate was present in excess. The apparent K.sub.m of 
partially purified tobacco protein according to the invention for 
putrescine was about 400 gM. The apparent K.sub.m of the highly purified 
tobacco protein of the present invention for S-adenosylmethionine was 
about 125 .mu.M. The apparent K.sub.m values found for putrescine, with 
partially purified tobacco protein according to the invention and for 
S-adenosylmethionine, with highly purified tobacco protein according to 
the invention agree closely with published values for PMT (Mizusaki et 
al., supra; Feth et al., "Determination of Putrescine N-methyltransferase 
By High Performance Liquid Chromatography", Phytochemistry, 24, pp. 921-23 
(1985)). 
Amino-Terminal Amino Acid Sequence Analysis 
The subject purified tobacco protein used for sequence analysis was 
isolated via SDS-PAGE of material that had been subjected to the 
purification steps of ammonium sulfate fracrionation, phenyl-Sepharose 
chromatography, DEAE-Sepharose chromatography with putrescine elution 
(followed by concentration via AHS and ultrafiltration), and free-flow 
isoelectric focussing. Following SDS-PAGE of the highly purified protein 
according to the invention, the protein bands were electroblotted onto a 
polyvinylidene difluoride membrane ("Immobilon-P", Millipore, Bedford, 
Mass.) and visualized with amido black, by standard procedures. The piece 
of membrane bearing the "a1" band (see FIG. 5), which was one of only two 
bands in the highly purified preparation displaying a molecular weight 
characteristic of the subject tobacco protein (see FIG. 3), was cut out so 
as to avoid the adjacent "a2" band. The protein tobacco thus isolated was 
subjected to amino terminal amino acid sequence analysis on an Applied 
Biosystems model 477A with an on-line 120A analyzer (pulse liquid phase 
sequencer), according to the manufacturer's recommended procedures. 
The sequence of the first 17 amino acids at the amino terminus of the 
subject purified tobacco protein "a1" band was found to be (SEQ ID NO:1): 
Leu Ser Xaa Asn Phe Leu Phe Gly Thr Ala Ser Ser Xaa Tyr Gln Tyr Glu. 
The "a2" band (see FIG. 5) was the second of only two bands displaying the 
molecular weight of the subject tobacco protein (see FIG. 3). When the 
"a2" band (FIG. 5) was prepared and analyzed in the same manner as the 
"a1" band, the "a2" band yielded the following partial amino acid sequence 
(SEQ ID NO:2): Leu Ser Ser Asn Phe Leu Phe Gly Thr Ala Ala Pro Tyr Tyr Gln 
Tyr Glu. 
Confirmation of Amino-Terminal Sequence of 60 kD Protein According to the 
Invention 
Additional amounts of the subject 60 kD tobacco protein were prepared for 
further amino terminal amino acid sequence analysis. On the basis of this 
further analysis, the sequence of the first 29 amino acids of the "a1" 
band was found to be (SEQ ID NO:4): Leu Ser Ser Asn Phe Leu Phe Gly Thr 
Ala Ser Ser Tyr Tyr Gln Tyr Glu Gly Ala Phe Leu Ser Asp Gly Val Gly Leu 
Ser Asn. 
Partial Amino Acid Sequences of CNBr Fragments 
To obtain partial internal (as opposed to amino terminal) amino acid 
sequences, corresponding to the subject purified tobacco protein we 
isolated electrophoretic bands of the subject tobacco protein as described 
above and subjected the purified protein to cyanogen bromide ("CNBr") 
cleavage. (CNBr cleaves polypeptides specifically at methionine residues.) 
We analyzed the CNBr reaction products by SDS-PAGE and found CNBr 
fragments with apparent molecular weights of approximately 15, 6.2 and 3 
kD. We excised the electrophoretic bands containing CNBr fragments from 
the polyacrylamide gels, electroblotted the protein from the bands and 
subjected the fragments to amino acid sequence analysis (as described 
above). 
The sequence of the first 13 amino acids of the 15 kD CNBr fragment of the 
subject purified tobacco protein was found to be (SEQ ID NO:5): Phe Ile 
Thr Glu Asn Gly Phe Ala Gly Arg Ser Gly Arg. 
The sequence of the first 15 amino acids of the 6.2 kD CNBr fragment of the 
subject tobacco protein was found to be (SEQ ID NO:6): Asn Glu Pro Xaa Phe 
Val Ala Ile Ser Gly Tyr Arg Asp Xaa Thr. 
The sequence of the first 20 amino acids of the 3 kD CNBr fragment of the 
subject purified tobacco protein was found to be (SEQ ID NO:7): Ala Asp 
Ile Glu His Tyr Ser Lys Leu Ile Asp Ala Leu Xaa Ile Lys Gly Ile Gln Phe. 
Isolation and Characterization of cDNA Clones Corresponding to the Subject 
Tobacco Protein 
Conventional RNA PCR procedures were used to isolate cDNA clones encoding 
the subject tobacco protein. The template in the RNA PCR procedures, was 
poly(A.sup.+) RNA isolated from tobacco roots. The starting material for 
poly(A.sup.+) RNA preparation consisted of roots from hydroponically-grown 
tobacco plants (N. tabacum, var. Burly 21). The first step in preparation 
of the poly(A.sup.+) RNA was preparation of total RNA. Isolation of total 
tobacco root RNA was carried out using reagents and instructions from a 
commercially-available kit for preparation of total RNA (Stratagene, 
LaJolla, Calif., Cat. No. 200345). Poly(A.sup.+) RNA was isolated from the 
total RNA preparation using a commercially-available kit for preparation 
of poly(A.sup.+) RNA (Pharmacia LKB, Piscataway, N.J., Cat. No. 
27-9258-01). The oligonucleotide PCR primers were designed according to 
standard principles, utilizing the partial amino acid sequence data 
obtained from the subject purified tobacco protein, initially. In later 
experiments, primers were designed from sequences of previously isolated 
cDNA clones. 
PCR Procedures 
The RNA PCR procedures were carried out with a commercially available kit 
("GeneAmp RNA PCR Kit", Perkin Elmer Cetus, Norwalk, Conn.). The procedure 
was essentially according to the PCR kit vendor's recommendations. The 
vendor's protocol was modified as follows: 
Reverse transcriptase reactions 
(1) reverse primer concentration was 2.5 .mu.M; 
(2) poly(A.sup.+) RNA amount was about 1 .mu.g per reverse transcriptase 
reaction; 
(3) thermal cycler program included 
42.degree. C. 60 min. 
99.degree. C. 10 min. 
4.degree. C. 10 min. 
1 cycle 
linked to 
soaker file 4.degree. C.; 
Polymerase chain reactions 
(1) primer concentrations were 2.5 .mu.M each; 
(2) Taq polymerase enzyme was added to the reaction last, after the 
98.degree. C. cDNA denaturation step; 
(3) thermal cycler program included step-cycle file 
98.degree. C. 1 min. 
60.degree. C. 10 min. 
1 cycle 
linked to 
94.degree. C. 1 min. 
50.degree. C. 2 min. 
72.degree. C. 3 min. 
30 cycles 
linked to 
step-cycle file 
72.degree. C. 10 min. 
linked to 
soaker file 4.degree. C. 
After a standard chloroform extraction step, 20% (by vol.) of each PCR 
sample was loaded onto a 2% agarose gel for electrophoretic analysis of 
PCR products. 
Cloning Procedures 
PCR products selected for further analysis were cloned into the 
commercially available vector pBluescript II (SK+) (Stratagene). An 
aliquot of the PCR product was treated with Klenow enzyme to generate 
blunt ends on the DNA. The Klenow reaction mixtures were as follows: 
approximately 1 .mu.g of PCR-generated DNA, each of the four 
deoxynucleotide triphosphates at a final concentration of 80 .mu.M each, 
2.5 .mu.l of 10.times. universal buffer (Stratagene), 5 units of Klenow 
enzyme (Stratagene), and water to yield a total volume of 25 .mu.l. The 
Klenow reactions were run for 30 min. at room temperature and stopped by 
heat-inactivation at 75.degree. C. for 10 min. 
The PCR-generated DNA fragments were then ligated into pBluescript II (SK+) 
that been digested with restriction enzyme SmaI. The ligation reaction 
mixtures were as follows: approximately 1 .mu.g of bluntended DNA, 
approximately 0.2 .mu.g of SmaI-digested pBluescript II, 3 .mu.l of 
10.times. ligation buffer (Stratagene), 12 units of T4 DNA ligase, and 
water to yield a total volume of 30 .mu.l. We ran the ligase reactions 
overnight at 4.degree. C. and stopped the reactions by heat activation at 
75.degree. C. for 10 min. 
Following each ligation reaction, the following bacterial transformation 
procedure was carried out. A suspension of commercially-obtained competent 
E. coli cells ("XL1-Blue" cells, Stratagene) was chilled on ice. 
Approximately 30 .mu.l of the suspension was added to a sample of DNA 
(ligated as described above) that had been also chilled on ice. The 
competent cells were left in the presence of the ligated DNA, on ice, for 
30 min. The cells were then heat shocked in the presence of the DNA for 2 
min., using a 42.degree. C. water bath. Following the heat shock, the 
cells and DNA were returned to an ice bath for 5 min. After addition of 1 
ml of LB culture medium, the cells were incubated at 37.degree. C. for one 
hour, with vigorous shaking. The cells were then plated on a selective 
medium. The plates were examined for colonies of transformed cells after 
an overnight incubation. For transformant selection, LB medium containing 
ampicillin at 100 mg/liter was used. 
Selected transformants were screened by PvuII restriction analysis of DNA 
prepared from transformant individual transformant colonies. This 
screening method was based on the fact that clones having the desired PCR 
product as an insert should yield a PvuII fragment approximately 0.5 kb 
larger than the PCR product. This is because there are PvuII restriction 
sites at nucleotide positions 529 and 977, flanking the pBluescript II 
multiple cloning site (MCS), into which the PCR products were cloned. (See 
map of plasmid pBluescript II in the commercial vendor's product 
literature.) Clones selected in the PvuI1 screening were further analyzed 
by DNA sequence analysis. 
Clone PMT1.2 
Clone PMT1.2 (approx. 1.2 kb) was obtained by RNA PCR and cloning as 
described above. The template was tobacco root poly(A.sup.+) RNA, and the 
forward and reverse primers were P-20 (SEQ ID NO:8) and P-22 (SEQ ID 
NO:9), respectively. The sequence design of primer P-20 was based on amino 
acids 13-20 of the subject 60 kD tobacco protein. The sequence design of 
primer P-22 was based on amino acids 1-7 of the 15 kD CNBr fragment of the 
subject purified tobacco protein. 
Preliminary DNA sequence analysis was carried out on clone PMT1.2. A 
deduced amino acid sequence encoded by one of the PMT1.2 reading frames 
showed strong homology (94%) to the N-terminal region of the subject 60 kD 
tobacco protein. A deduced amino acid sequence encoded by PMT1.2 also 
showed strong homology (95%) to the 3 kD CNBr fragment (described above) 
which represents an internal region of the subject 60 kD tobacco protein. 
Clone PMT-26 
Clone PMT-26 (approx. 1.2 kb) was obtained by RNA PCR and cloning as 
described above. The template was tobacco root poly(A.sup.+) RNA, and the 
forward and reverse primers were P-24 (SEQ ID NO:10) and P-21 (SEQ ID 
NO:11), respectively. The sequence of primer P-24 was based on amino acids 
4-10 of the subject intact 60 kD tobacco protein and included a BamHI site 
at its 5' end. The sequence of primer P-21 was based on an N-terminal 
methionine and amino acids 1-6 of the 15 kD CNBr fragment of the subject 
60 kD tobacco protein. In addition, primer P-21 included an EcoRI site at 
its 5' end. Thus, the experiment was designed to obtain a cDNA clone 
encoding the region from amino acid #4, near the N-terminus of the subject 
60 kD tobacco protein, to amino acid #6 of the 15 kD CNBr fragment of that 
protein. 
Preliminary DNA sequence analysis was carried out on cDNA clone PMT-26. 
Clone PMT-26 encoded approximately two-thirds of the subject 60 kD tobacco 
protein. The N-terminal region of a deduced amino acid sequence encoded by 
one of the PMT-26 reading frames showed strong homology to the N-terminal 
region of the subject 60 kD tobacco protein. The C-terminal region of that 
deduced amino acid sequence showed strong homology to the 3 kD CNBr 
fragment (described above) which represents an internal region of the 
subject 60 kD tobacco protein. 
Clone Q7 
Clone Q7 (approx. 0.4 kb) was obtained by RNA PCR and cloning as described 
above. The PCR template was tobacco root poly(A.sup.+) RNA, and the 
forward and reverse PCR primers were P-18 (SEQ ID NO:12) and P-23 (SEQ ID 
NO:13), respectively. The sequence of primer P-18 was based on an 
N-terminal methionine plus amino acids 1 to 7 of a 15 kD cyanogen bromide 
fragment generated from the subject 60 kD protein purified as described 
above. The sequence of reverse primer P-23 consists of a poly(dt) region 
(20 deoxythimidines), an EcoRI site, and 3 additional deoxythimidines at 
the 5' end. 
Clone PMT2-5 
Clone PMT2-5 (approx. 1.5 kb) was obtained by utilizing RNA PCR and cloning 
techniques (essentially as described above) in combination with sequence 
information from the partial clone, PMT-26. (In the RNA PCR procedure, the 
primer concentrations were 1.0 .mu.M rather than 2.5 .mu.M. In addition, 
first step cycle file specified 50.degree. C. rather than 55.degree. C., 
and the last step cycle specified 30 min. rather than 10 min.) The 
template was tobacco root poly(A.sup.+) RNA, and the forward and reverse 
primers were P-38 (SEQ ID NO:14) and P-36 (SEQ ID NO:15), respectively. 
The sequence of primer P-38 was based on a DNA sequence from the 51 region 
of clone PMT-26 (described above) encoding the amino acids 4-11 of (SEQ ID 
NO:4) the N-terminal region of the subject 60 kD tobacco protein. Primer 
P-36 was designed from the 3' region of DNA clone Q7. Primers P-38 and 
P-36 were both 100% homologous to their target sequences. 
DNA sequence analysis was carried out on the cloned PMT2-5 DNA insert. The 
PMT2-5 DNA sequence matched the PMT1.2 and Q7 DNA sequences as expected. 
This result confirmed that clones PMT1.2 and Q7 represent regions of a 
single transcript and thus encode regions of a single protein. Clone 
PMT2-5 encoded the entire 60 kD tobacco protein of the present invention 
except the N-terminal methionine and amino acids 1-3 (i.e., leu-ser-ser). 
In addition to PMT2-5, other clones having partial homologies to clones 
PMT2-5 or Q7 were obtained (data not shown). 
Clone PMT14-3 
A forward primer, P-37, was synthesized to add an NcoI restriction site and 
codons for an N-terminal methionine, an alanine and PMT amino acids 1-3 at 
the 5' end of clone PMT2-5. The sequence of primer P-37 was incorporated 
into the PMT2-5 DNA by means of standard DNA PCR techniques. The PMT2-5 
DNA served as the template for the polymerase chain reaction, and the 
forward and reverse primers were P-37 (SEQ ID NO:16) and P-36 (SEQ ID 
NO:15), respectively. The DNA products from the PCR were cloned as 
described above. Clone PMT14-3 was obtained by this method. It was 
expected that clone PMT14-3 encodes the complete 60 kD protein according 
to the invention with an additional alanine residue inserted immediately 
following the N-terminal methionine. This expected structure of clone 
PMT14-3 was confirmed by conventional sequence analysis. The sequence of 
clone PMT14-3 was found to be: (SEQ ID NO:17). What effect, if any, the 
inserted alanine residue will have on post-translational processing of the 
N-terminal methionine residue may be determined by sequence analysis of 
the subject recombinant protein produced in host cells transformed with an 
expression vector carrying the PMT14-3 DNA insert in the sense 
orientation. 
Recombinant DNA sequences prepared by the processes described herein are 
exemplified by a culture deposited in the American Type Culture 
Collection, Rockville, Md. The culture identified as Escherichia coli, 
PMT14-3 was deposited on Mar. 11, 1993 and given the ATCC Designation 
69253. 
While we have described a number of embodiments of this invention, it is 
apparent that our basic constructions can be altered to provide other 
embodiments which utilize the processes and products of this invention. 
Therefore, it will be appreciated that the scope of this invention is to 
be defined by the appended claims rather than by the specific embodiments 
which have been presented by way of example. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 17 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Nicotiana tabacum 
(B) STRAIN: Burley 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LeuSerXaaAsnPheLeuPheGlyThrAlaSerSerXaaTyrGlnTyr 
151015 
Glu 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Nicotiana tabacum 
(B) STRAIN: Burley 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LeuSerSerAsnPheLeuPheGlyThrAlaAlaProTyrTyrGlnTyr 
151015 
Glu 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Nicotiana tabacum 
(B) STRAIN: Burley 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LeuSerXaaAsnPheLeuPheGlyThrAlaXaaXaaXaaTyrGlnTyr 
151015 
Glu 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Nicotiana tabacum 
(B) STRAIN: Burley 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LeuSerSerAsnPheLeuPheGlyThrAlaSerSerTyrTyrGlnTyr 
151015 
GluGlyAlaPheLeuSerAspGlyValGlyLeuSerAsn 
2025 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
PheIleThrGluAsnGlyPheAlaGlyArgSerGlyArg 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Nicotiana tabacum 
(B) STRAIN: Burley 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AsnGluProXaaPheValAlaIleSerGlyTyrArgAspXaaThr 
151015 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: internal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Nicotiana tabacum 
(B) STRAIN: Burley 21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AlaAspIleGluHisTyrSerLysLeuIleAspAlaLeuXaaIleLys 
151015 
GlyIleGlnPhe 
20 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 18 
(D) OTHER INFORMATION: /note= "Deoxyinosine was used at 
this position instead of mixed nucleotides." 
(ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- feature 
(B) LOCATION: 21 
(D) OTHER INFORMATION: /note= "Deoxyinosine was used at 
this position instead of mixed nucleotides." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TAYTAYCARTAYGARGGNGCNTT23 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AANCCRTTYTCNGTDATRAACAT23 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ATCGGATCCAAYTTYTTGTTYGGNACHGC29 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
ATGGAATTCCRTTYTCNGTDATRAACAT28 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ATGTTYATHACNGARAAYGGNTT23 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
TTTGAATTCTTTTTTTTTTTTTTTTTTTT29 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AATTTCTTGTTCGGGACAGCCTCT24 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AGGCTGAACAATTATAGTATGATT24 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CATGCCATGGCTCTTTCTTCTAATTTCTTGTTCGGGACAGCCTCT45 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1545 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
ATGGCTCTTTCTTCTAATTTCTTGTTCGGGACAGCCTCTTCATATTACCAGTATGAAGGA60 
GCTTTCCTCAGTGATGGGAAAGGCCTCAGCAACTGGGACGTTTTTACCCATGAAGCTGGT120 
CATGTTAAGGATGGAAGCAATGGAGATGTGGCTGTTGATCACTACCATCGTTATTTGGAG180 
GACATCAAACTCATGGCAGATATGGGTGTGAATAGCTTTCGTTTCTCTATCTCATGGGCA240 
AGAATTCTGCCCAAGGGAATATTTGGAGAAGTTAATATGGCCGGAATTGAGCACTACAGT300 
AAGCTCATTGATGCACTCCTACAGAAAGGGATCCAGCCGTTTGTCACATTAACACATTTT360 
GACATACCACAAGAACTTGAGGACAGATATGGTGGTTGGCTAAGTTCACAGATACGGGAT420 
GATTTCAGCTATTTCGCAAACATATGCTTCAAATACTTGGGAGATAGAGTTAAATACTGG480 
GTAACGATGAATGAGGCTAACTTCGTGGCCATTAGTGGCTATAGAGATGGGACTTGCCCT540 
CCAACTCGATGCTCTGGTATATTTGGGAATTGTAGTGCTGGGGATTCAGAAAGGGAACCC600 
TTCATTGCAGCTCACAATATGATCCTATCTCATGCAGATGCTGTCAGCATTTACCGCACC660 
AGATATCAGAAAAGTCAAGGAGGCATGATTGGCATTACTATGGGTTTCGAATGGTATGAA720 
CCGTTAAGCAATTCCTCAGAAGACATAGCTGCAACTCATAGAGCTCGATCATTCTATGAC780 
AGTTGGTTTTTAGACCCTATTATATTAGGAAGATATCCTGAAGAAATGGCACAAATTTTG840 
GGATCTAATCTTCCAGAATTTTCAGTGAGTGATTTGAGAATGTTGAGTTATGGCCTAGAT900 
TTCATTGGCATCAATCATTATTCAGCTGTTTATATCAAAGATTGCTTATATTCTGCCTGT960 
GAACATGGAAACTCTTGGTCAGAGGGTTCTTATTTAACGACTACACAAAGAGACGGTGTC1020 
TACATCGGGGAACCTGGGGAAGTGGACTGGCAATTTGTGTATCCACAAGGGATTGAAAAA1080 
GTTGTGATGTATATAAAGGACAGATTCAACAATACTCCTATGTTTATCACTGAAAATGGC1140 
TTTGCTGGGAACAGTTCTTCTATAGAGGATGCCTTGAACGATGTTCATAGAGTGAAATAC1200 
ATGCATAGCTACTTAAATTCATTGGCAAATGCAATCAGGAAAGGTGCAGATGTAAGGGGG1260 
TACTTTGCTTGGTCCCTTCTTGATAACTTTGAGTGGCTAGATGGATATACCATAAGATTT1320 
GGACTTTACTATGTCAACTACACAAATCTCCAGAGAACTCCAAAACTATCAGCCACTAAG1380 
TATCCAGAGCTCATGTGTAACTTTCACATAGAGCTTGAAGCACATACTGCCCAGAAATAG1440 
CGTAAGAAGACGGTGCATATGTGGAGGCTTGTTGAAGATTTTTTTATTTAGTTCTCTATT1500 
GTTGGAAGGCAATTACTGAGCAATCATACTATAATTGTTCAGCCT1545 
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