U.S. patent application number 11/285537 was filed with the patent office on 2006-03-23 for modifying nicotine and nitrosamine levels in tobacco.
Invention is credited to Mark A. Conkling.
Application Number | 20060060211 11/285537 |
Document ID | / |
Family ID | 23145081 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060060211 |
Kind Code |
A1 |
Conkling; Mark A. |
March 23, 2006 |
Modifying nicotine and nitrosamine levels in tobacco
Abstract
The present invention generally relates to tobacco and tobacco
products having a reduced amount of nicotine and/or tobacco
specific nitrosamines (TSNA). More specifically, several ways to
make tobacco plants that have reduced nicotine and TSNA levels have
been discovered. Embodiments include tobacco harvested from said
tobacco plants, cured tobacco from said tobacco plants, tobacco
products made with said cured tobacco and methods of making these
compositions.
Inventors: |
Conkling; Mark A.; (Chapel
Hill, NC) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
23145081 |
Appl. No.: |
11/285537 |
Filed: |
November 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11077752 |
Mar 10, 2005 |
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11285537 |
Nov 22, 2005 |
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10729121 |
Dec 5, 2003 |
6907887 |
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11077752 |
Mar 10, 2005 |
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PCT/US02/18040 |
Jun 6, 2002 |
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10729121 |
Dec 5, 2003 |
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60297154 |
Jun 8, 2001 |
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Current U.S.
Class: |
131/364 |
Current CPC
Class: |
A61P 35/00 20180101;
A24B 15/10 20130101; A24B 15/20 20130101; A24B 15/245 20130101;
A61P 25/34 20180101; C12N 9/1077 20130101; C07K 14/415 20130101;
A24B 15/18 20130101; A24B 15/243 20130101; C12N 15/8243
20130101 |
Class at
Publication: |
131/364 |
International
Class: |
A24D 1/00 20060101
A24D001/00 |
Claims
1. A tobacco product comprising a cured tobacco that comprises a
genetic modification, wherein said tobacco product has a collective
content of N'-nitrosonomicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less
than 0.5 .mu.g/g.
2. The tobacco product of claim 1, wherein said cured tobacco is of
the genus Nicotiana.
3. The tobacco product of claim 2, wherein said cured tobacco is
selected from the group consisting of Burley, Flue, and
Oriental.
4. The tobacco product of claim 2, wherein said cured tobacco is
Burley.
5. The tobacco product of claim 2, wherein said cured tobacco is
Flue.
6. The tobacco product of claim 1, wherein said cured tobacco
comprises an exogenous nucleic acid that encodes an enzyme in the
nicotine synthesis pathway or a fragment thereof at least 25
nucleotides in length.
7. The tobacco product of claim 1, wherein said cured tobacco
comprises a fragment of a nucleic acid that encodes an enzyme in
the nicotine synthesis pathway that is at least 50 nucleotides in
length.
8. The tobacco product of claim 1, wherein said cured tobacco
comprises a fragment of a nucleic acid that encodes an enzyme in
the nicotine synthesis pathway that is at least 100 nucleotides in
length.
9. The tobacco product of claim 1, wherein said cured tobacco
comprises a fragment of a nucleic acid that encodes an enzyme in
the nicotine synthesis pathway that is at least 250 nucleotides in
length.
10. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 11 mg/g.
11. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 9 mg/g.
12. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 7 mg/g.
13. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 5 mg/g.
14. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 3 mg/g.
15. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 2 mg/g.
16. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 1 mg/g.
17. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 0.7 mg/g.
18. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 0.5 mg/g.
19. The tobacco product of claim 1, wherein the amount of nicotine
in said product is less than 0.1 mg/g.
20. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonomicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.4 .mu.g/g.
21. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonomicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.3 .mu.g/g.
22. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.2 .mu.g/g.
23. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.1 .mu.g/g.
24. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.05 .mu.g/g.
25. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.02 .mu.g/g.
26. The tobacco product of claim 1, wherein the collective content
of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is less
than 0.01 .mu.g/g.
27. The tobacco product of claim 6, wherein said enzyme in the
nicotine synthesis pathway is selected from the group consisting of
putrescine N-methyltransferase, N-methylputrescine oxidase,
ornithine decarboxylase, S-adenosylmethionine synthetase, NADH
dehydrogenase, phosphoribosylanthranilate isomerase, and quinolate
phosphoribosyl transferase.
28. The tobacco product of claim 7, wherein said enzyme in the
nicotine synthesis pathway is selected from the group consisting of
putrescine N-methyltransferase, N-methylputrescine oxidase,
ornithine decarboxylase, S-adenosylmethionine synthetase, NADH
dehydrogenase, phosphoribosylanthranilate isomerase, and quinolate
phosphoribosyl transferase.
29. The tobacco product of claim 8, wherein said enzyme in the
nicotine synthesis pathway is selected from the group consisting of
putrescine N-methyltransferase, N-methylputrescine oxidase,
ornithine decarboxylase, S-adenosylmethionine synthetase, NADH
dehydrogenase, phosphoribosylanthranilate isomerase, and quinolate
phosphoribosyl transferase.
30. The tobacco product of claim 9, wherein said enzyme in the
nicotine synthesis pathway is selected from the group consisting of
putrescine N-methyltransferase, N-methylputrescine oxidase,
ornithine decarboxylase, S-adenosylmethionine synthetase, NADH
dehydrogenase, phosphoribosylanthranilate isomerase, and quinolate
phosphoribosyl transferase.
31. The tobacco product of claim 1, wherein said tobacco product is
a cigarette.
32. The tobacco product of claim 6, wherein said tobacco product is
a cigarette.
33. The tobacco product of claim 10, wherein said tobacco product
is a cigarette.
34. The tobacco product of claim 15, wherein said tobacco product
is a cigarette.
35. The tobacco product of claim 18, wherein said tobacco product
is a cigarette.
36. The tobacco product of claim 22, wherein said tobacco product
is a cigarette.
37. The tobacco product of claim 25, wherein said tobacco product
is a cigarette.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
11/077,752, filed Mar. 10, 2005, which is a continuation of U.S.
patent application Ser. No. 10/729,121, filed Dec. 05, 2003, now
U.S. Pat. No. 6,907,887, which is a continuation of international
application number PCT/US02/18040, and claims the benefit of
priority of international application number PCT/US02/18040 having
international filing date of Jun. 06, 2002, designating the United
States of America and published in English, which claims the
benefit of priority of U.S. provisional patent application No.
60/297,154, filed Jun. 08, 2001. The disclosures of all of the
aforementioned applications are hereby expressly incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention generally relates to tobacco and
tobacco products having a reduced amount of nicotine and/or tobacco
specific nitrosamines (TSNA). More specifically, several ways to
make tobacco plants that have reduced nicotine and TSNA levels have
been discovered. Embodiments include tobacco harvested from said
tobacco plants, cured tobacco from said tobacco plants, tobacco
products made with said cured tobacco and methods of making these
compositions.
BACKGROUND OF THE INVENTION
[0003] The health consequences of tobacco consumption are known but
many people continue to use tobacco products. The addictive
properties of tobacco products are largely attributable to the
presence of nicotine. In addition to being one of the most
addictive substances known, nicotine is also a precursor for a
large number of carcinogenic compounds present in tobacco and the
body.
[0004] There is currently great interest in methods for production
of tobacco with decreased levels of noxious, carcinogenic, or
addictive substances including tar, nitrosamines, and nicotine.
Although researchers have developed several approaches to reduce
the nicotine content or the nicotine delivery of tobacco products,
many techniques result in a product that has poor taste, fragrance,
or smoking properties. Some processes, for example, reduce the
nicotine content of tobacco after it has been harvested through
microbial enzymatic degradation, chemical treatment, or high
pressure extraction. (See U.S. Pat. Nos. 4,557,280; 4,561,452;
4,848,373; 4,183,364; and 4,215,706, all of which are hereby
expressly incorporated by reference in their entireties). In view
of the foregoing, and notwithstanding the various efforts
exemplified in the prior art, there remains a need for tobacco
having reduced nicotine and TSNAs and methods of producing such
tobacco.
SUMMARY OF THE INVENTION
[0005] Embodiments of the invention concern the production of
tobacco and tobacco products having a reduced amount of nicotine
and/or tobacco specific nitrosamines (TSNAs). In addition to having
a reduced level of nicotine, some tobacco and tobacco products of
the invention have reduced amounts of N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),
4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal
(NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL),
4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or
4-(N-nitrosomethylamino)-4-(3-pyridyl)butanoic acid (iso-NNAC).
Some embodiments, for example, are substantially free of at least
one TSNA selected from the group consisting of
N'-nitrosonornicotine,
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone,
N'-nitrosoanatabine, and N'-nitrosoanabasine. The term "tobacco
products" include, but are not limited to, smoking materials (e.g.,
cigarettes, cigars, pipe tobacco), snuff, chewing tobacco, gum, and
lozenges. One embodiment, for example, includes a genetically
modified cured tobacco comprising a reduced amount of nicotine and
a collective content of NNN, NAT, NAB, and NNK of less than about
0.5 .mu.g/g, 0.4 .mu.g/g or 0.2 .mu.g/g. That is, said cured
tobacco is made from a genetically modified tobacco plant.
[0006] Another aspect of the invention concerns methods to
substantially eliminate or reduce the amount of nicotine and/or
TSNAs in tobacco. By one approach, tobacco plants are made
substantially free of nicotine by interrupting the ability of the
plant to synthesize nicotine using genetic engineering. Some
embodiments comprise cured tobacco and tobacco products wherein the
amount of nicotine is less than about 2 mg/g, 1 mg/g, 0.75 mg/g,
0.5 mg/g or desirably less than about 0.1 mg/g. By virtue of the
elimination of nicotine in these genetically modified plants,
tobacco and tobacco products made from these plants also have a
reduced amount of TSNAs. In a preferred method, transgenic tobacco
is created to have one or more TSNAs reduced including, but not
limited to, N'-nitrosonomicotine (NNN),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),
N'-nitrosoanatabine (NAT), and/or N'-nitrosoanabasine (NAB).
Tobacco products including, but not limited to, smoking materials
(e.g., cigarettes, cigars, pipe tobacco), snuff, chewing tobacco,
gum and lozenges are then prepared from said transgenic tobacco
plants using conventional techniques. Preferably these tobacco
products are manufactured from harvested tobacco leaves and stems
that have been cut, dried, cured, and/or fermented according to
conventional techniques in tobacco preparation. However, modified
techniques in curing and tobacco processing can also be implemented
to further lower the levels of TSNAs.
[0007] In some embodiments of the invention, the tobacco that is
substantially free of nicotine and TSNAs is made by exposing at
least one tobacco cell of a selected variety to an exogenous DNA
construct having, in the 5' to 3' direction, a promoter operable in
a plant cell and DNA containing a portion of a DNA sequence that
encodes an enzyme in the nicotine synthesis pathway. The DNA is
operably associated with said promoter, the tobacco cell is
transformed with the DNA construct, the transformed cells are
selected and at least one transgenic tobacco plant is regenerated
from the transformed cells. The transgenic tobacco plants contain a
reduced amount of nicotine and/or TSNAs as compared to a control
tobacco plant of the same variety. In preferred embodiments, DNA
constructs having a portion of a DNA sequence that encodes an
enzyme in the nicotine synthesis pathway may have the entire coding
sequence of the enzyme, or any portion thereof.
[0008] In some embodiments, the enzyme involved in the nicotine
synthesis pathway is putrescine N-methyltransferase,
N-methylputrescine oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase or quinolate phosphoribosyl
transferase (QPTase). In a preferred embodiment, the enzyme is
QPTase. The segment of DNA sequence encoding an enzyme in the
nicotine synthesis pathway may be in the antisense or the sense
orientation. In some embodiments, the tobacco that is made
substantially free of nicotine and/or TSNAs is prepared from a
variety of Burley tobacco (e.g., Burley 21), Oriental tobacco, or
Flue-cured tobacco. It should be understood, however, that most
tobacco varieties can be made to be nicotine and/or TSNA free using
the embodiments described herein. For example, plant cells of the
variety Burley 21 are used as the host for the genetic engineering
that results in the reduction of nicotine and/or TSNAs so that the
resultant transgenic plants are a Burley 21 variety that has a
reduced amount of nicotine and/or TSNAs.
[0009] An aspect of the invention also includes an isolated DNA
molecule comprising SEQ ID NO: 1, DNA sequences which encode an
enzyme having SEQ ID NO: 2, DNA sequences that hybridize to such
DNA and encode a quinolate phosphoribosyl transferase enzyme or a
portion of such an enzyme and DNA sequences which differ from the
above DNA due to the degeneracy of the genetic code. A peptide
encoded by such DNA is a further aspect of the invention.
[0010] A further aspect of the present invention concerns a DNA
construct comprising a promoter operable in a plant cell and a DNA
segment encoding a quinolate phosphoribosyl transferase enzyme
positioned downstream from the promoter and operatively associated
therewith. The DNA encoding the enzyme may be in the antisense or
sense direction.
[0011] A further aspect of the present invention involves a method
of making a transgenic plant cell having reduced quinolate
phosphoribosyl transferase (QPTase) expression, by providing a
plant cell of a type known to express quinolate phosphoribosyl
transferase; transforming the plant cell with an exogenous DNA
construct comprising a promoter and DNA comprising a portion of a
sequence encoding quinolate phosphoribosyl transferase mRNA. In
preferred embodiments, DNA constructs having a portion of a DNA
sequence encoding quinolate phosphoribosyl transferase may have the
entire coding sequence of the enzyme, or any portion thereof. More
preferred are tobaccos containing genetic modification comprising a
sequence corresponding to the quinolate phosphoribosyl transferase
(QPTase) gene or a fragment thereof at least 13 nucleotides in
length.
[0012] A further aspect of the present invention concerns a
transgenic plant of the species Nicotiana having reduced quinolate
phosphoribosyl transferase (QPTase) expression relative to a
non-transformed control plant. The cells of such plants comprise a
DNA construct that includes a DNA sequence that encodes a plant
quinolate phosphoribosyl transferase mRNA or some portion
thereof.
[0013] A further aspect of the present invention involves a method
for reducing expression of a quinolate phosphoribosyl transferase
gene in a plant cell by growing a plant cell transformed to contain
exogenous DNA, where a transcribed strand of the exogenous DNA is
complementary to quinolate phosphoribosyl transferase mRNA
endogenous to the cell. Transcription of the complementary strand
reduces expression of the endogenous quinolate phosphoribosyl
gene.
[0014] A further aspect of the present invention includes a method
of producing a tobacco plant having decreased levels of nicotine in
leaves of the tobacco plant by regenerating a tobacco plant from
cells that comprise an exogenous DNA sequence that encodes an RNA
that is complementary to a region of endogenous quinolate
phosphoribosyl transferase messenger RNA in the cells.
[0015] A further aspect of the invention concerns a method of
producing a tobacco plant having reduced nicotine and/or TSNAs,
which involves regenerating a tobacco plant from cells that
comprise an exogenous DNA sequence, where a transcribed strand of
the exogenous DNA sequence is complementary to a region of
endogenous quinolate phosphoribosyl transferase messenger RNA in
the cells. Related embodiments include methods of producing tobacco
products from said tobacco plant that have a reduced amount of
nicotine and/or TSNAs, said tobacco products including, but are not
limited to, cigarettes, cigars, pipe tobacco, chewing tobacco, and
may be in the form of leaf tobacco, shredded tobacco, or cut
tobacco.
[0016] A further aspect of the invention concerns the manufacture,
isolation, and/or characterization of tobacco mutants that exhibit
a mutation in a gene involved in nicotine biosynthesis that results
in a tobacco plant that has a reduced amount of nicotine and/or
TSNAs. Some embodiments, for example, have a mutation in at least
one gene involved in nicotine biosynthesis including, but not
limited to, putrescine N-methyltransferase, N-methylputrescine
oxidase, ornithine decarboxylase, S-adenosylmethionine synthetase,
NADH dehydrogenase, phosphoribosylanthranilate isomerase, or
quinolate phosphoribosyl transferase (QPTase). Natural mutants in
the above genes can be selected for reduced levels of nicotine
and/or TSNAs using techniques common to plant breeding. In some
embodiments, the tobacco mutants above are prepared from a variety
of Burley tobacco (e.g., Burley 21), Oriental tobacco, or
Flue-cured tobacco. It should be understood, however, that mutants
of genes in nicotine biosynthesis can be selected from most tobacco
varieties. These tobacco plants can also be used to prepare tobacco
products that have reduced levels of nicotine and/or TSNAs.
[0017] Additional embodiments include tobacco products that have
been carefully blended so that desired levels of nicotine and/or
TSNAs are obtained. For example, tobacco having a reduced level of
nicotine and/or TSNAs, prepared as described above, can be blended
with conventional tobacco so as to obtain virtually any amount of
nicotine and/or TSNAs. Further, two or more varieties of tobacco
having a reduced level of nicotine and/or TSNAs can be blended so
as to achieve a desired amount of nicotine and/or TSNAs. In this
manner, differences in variety, flavor, as well as amounts of
nicotine and/or TSNAs can be incrementally adjusted. These blended
tobacco products can be incorporated into tobacco use cessation
kits and programs designed to reduce or eliminate nicotine
dependence and carcinogenic potential. Such kits and programs are
also embodiments of the invention.
[0018] More embodiments of the invention concern methods to reduce
the carcinogenic potential of tobacco products, including
cigarettes, cigars, chewing tobacco, snuff and tobacco-containing
gum and lozenges. Some methods, for example involve the preparation
of tobacco having a reduced amount of nicotine and/or TSNAs and the
manufacture of tobacco products containing said tobacco.
Accordingly, the transgenic tobacco plants, described above, are
harvested, cured, and processed into tobacco products. These
tobacco products have a reduced carcinogenic potential because they
are prepared from tobacco that has a reduced amount of nicotine
and/or TSNAs.
[0019] Yet another aspect of the invention concerns the reduction
of the amount of TSNAs, preferably NNN and NNK, and metabolites
thereof in humans who smoke, consume or otherwise ingest tobacco.
This method is practiced by providing a tobacco product having a
reduced amount of TSNAs to said humans, thereby lowering the
carcinogenic potential of such product in said humans. By one
approach, for example, the carcinogenic potential of side stream or
main stream tobacco smoke in a human exposed to said side stream or
main stream tobacco smoke is reduced by providing the cured tobacco
as described above in a product that undergoes pyrolysis, wherein
pyrolysis of said product results in side stream or main stream
smoke comprising a reduced amount of TSNAs. Thus, the cured tobacco
described above can be used to prepare a tobacco smoking product
that produces a reduced amount of TSNAs in the side stream and/or
mainstream smoke and thereby reduce the amount of carcinogen in
humans who come in contact with tobacco smoke.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows the biosynthetic pathway leading to nicotine.
Enzyme activities known to be regulated by Nicl and Nic2 are QPTase
(quinolate phosphoribosyl transferase) and PMTase (putrescence
methyl-transferase).
[0021] FIG. 2A provides the nucleic acid sequence of NtQPT1 cDNA
(SEQ ID NO: 1), with the coding sequence (SEQ ID NO: 3) shown in
capital letters.
[0022] FIG. 2B provides the deduced amino acid sequence (SEQ ID NO:
2) of the tobacco QPTase encoded by NtQPT1 cDNA.
[0023] FIG. 3 aligns the deduced NtQPT1 amino acid sequence and
related sequences of Rhodospirillum rubrum, Mycobacterium lepre,
Salmonella typhimurium, Escherichia coli, human, and Saccharomyces
cerevisiae.
[0024] FIG. 4 shows the results of complementation of an
Escherichia coli mutant lacking quinolate phosphoribosyl
transferase (TH265) with NtQPT1 cDNA. Cells were transformed with
an expression vector carrying NtQPT1; growth of transformed TH265
cells expressing NtQPT1 on minimal medium lacking nicotinic acid
demonstrated that NtQPT1 encodes QPTase.
[0025] FIG. 5 compares nicotine levels and the relative
steady-state NtQTP1 mRNA levels in Nic1 and Nic2 tobacco mutants;
wild-type Burley 21 (Nicl/Nicl Nic2/Nic2); Nic1-Burley 21
(nicl/nicl Nic2/Nic2); Nic2-Burley 21 (Nicl/Nicl nic2/nic2); and
Nicl-Nic2-Burley 21 (nicl/nicl nic2/Nnc2). Hatched bars, sloping
downward and right indicate mRNA transcript levels; hatched bars,
sloping downward and left indicate nicotine levels.
DETAILED DESCRIPTION OF THE INVENTION
[0026] Several approaches to create tobacco and tobacco products
that have a reduced amount of nicotine and/or TSNAs have been
discovered. Aspects of the technology described herein are also
described in PCT/US98/11893, which is hereby expressly incorporated
by reference in its entirety. By one approach, transgenic tobacco
plants that have reduced nicotine and TSNA levels are created and
tobacco harvested from said transgenic tobacco plants is used to
prepare a variety of tobacco products. One such transgenic tobacco
plant comprises a DNA construct that encodes an antisense RNA that
complements at least a portion of the quinolate phosphoribosyl
transferase (QPTase) gene. Transcription of the complementary
strand of RNA reduces expression of the endogenous quinolate
phosphoribosyl gene, which, in turn, reduces the amount of nicotine
and, concomitantly, the amount of TSNA in the tobacco plant. Thus,
one inventive concept is that reducing the nicotine content in a
tobacco plant using genetic engineering can reduce TSNA content in
said plant. The section below provides more description on
nitrosamines and tobacco-specific nitrosamines.
[0027] Nitrosamines and Tobacco-Specific Nitrosamines
[0028] The term nitrosamine generally refers to any of a class of
organic compounds with the general formula R.sub.2NNO or RNHNO
(where R denotes an amine-containing group). Nitrosamines are
present in numerous foods and have been found to be carcinogenic in
laboratory animals. These compounds are formed by nitrosation
reactions of amines such as amino acids and alkaloids with nitrites
and/or nitrous oxides. By themselves, nitrosamines are not
carcinogenic substances, but in mammals nitrosamines undergo
decomposition by enzymatic activation to form alkylating
metabolites which appear to react with biopolymers to initiate
their tumorogenic effect. Thus, by reducing the amount of
nitrosamine intake, one has effectively reduced the carcinogenic
potential in humans.
[0029] Nitrosamines have been identified in tobacco, tobacco
products, and tobacco smoke by the use of techniques such as gas
chromatography-thermal exchange analysis (GC-TEA).Some of these
nitrosamines have been identified as tobacco-specific nitrosamines
(TSNAs). TSNAs are primarily formed by reactions between the two
most abundant alkaloids, nicotine and nornicotine, with nitrous
oxides (NOx), and they account proportionately for the highest
concentration of nitrosamines in both tobacco products and in
mainstream smoke. Of the TSNAs identified, and the subset that have
been found to be present in cigarette smoke, the most characterized
is N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(N-nitrosamine-ketone), or NNK. When injected at relatively high
doses, NNK is carcinogenic in rodents. Minimal amounts of TSNAs are
found in green tobacco, indicating that TSNA formation may occur
during processing steps such as curing, drying, fermentation,
burning or storage of tobacco.
[0030] TSNA formation is attributed to chemical, enzymatic and
bacterial influences during tobacco processing, particularly during
curing, fermentation and aging. Nitrosation of nornicotine,
anatabine, and anabasine gives the corresponding nitrosamines:
N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT) and
N'-nitrosoanabasine (NAB). Nitrosation of nicotine in aqueous
solution affords a mixture of
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and
4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal (NNA). Less
commonly encountered TSNAs include NNAL
(4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol), iso-NNAL
(4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol, 11) and iso-NNAC
(4-(N-nitrosomethylamino)-4-(3-pyridyl)butanoic acid, 12). See,
U.S. Pat. No. 6,135,121, the entire disclosure of which is hereby
expressly incorporated by reference in its entirety.
[0031] TSNA levels are particularly high in chewing tobaccos and
snuff. The partially anaerobic processes that occur during
fermentation promote the formation of TSNAs from tobacco alkaloids
by promoting increased nitrite levels; in particular,
over-fermentation can increase TSNA levels in snuff by its effects
on nitrate levels and microbial enzymatic activity. The reduction
of the nitrosamine level in snuff in recent years has been achieved
by maintaining a better control over the bacterial content in these
products.
[0032] Since the nitrate level of tobacco is important for
nitrosamine formation in cigarette smoke, a significant reduction
of nitrosamines in smoke can be achieved by low-nitrate leaf and
stem blends. However, these methods may negatively impact the
smokability or the taste of the tobacco. The nitrosamine content of
mainstream smoke can be reduced by as much as 80% by cellulose
acetate filters, and it can be reduced still further by filter
ventilation.
[0033] Air-cured tobaccos such as burley and dark-fired may have
higher levels of TSNAs than certain types of flue-cured bright,
burley, or dark tobaccos apparently because the high temperatures
associated with flue-curing can kill the micro-organisms that
transform the alkaloids into TSNAs. In air-cured types, nitrate
(N--NO.sub.3) is more abundant in the leaf (particularly in the
leaf and stems) than in flue-cured tobacco and the alkaloid content
is also much higher. This N--NO.sub.3 is reduced to nitrite
(NO.sub.2.sup.-) by microbes during curing and the NO.sub.2.sup.-
can be further reduced to NOx or react directly with alkaloids to
form TSNAs.
[0034] It is contemplated that, in addition to the techniques
described above, nitrate levels in tobacco (especially in the leaf)
can be reduced by limiting exposure to nitrosating agents or
conditions. Air-curing experiments at a higher temperature have
shown that considerably higher levels of N-nitrosamines are formed
at a curing temperature of 32.degree. C. than at 16.degree. C.,
which is associated with a rise of the nitrite level in the
tobacco, and may also be associated with a rise in microbial
enzymatic activity. Modified curing that involves faster drying
from wider spacing or from more open curing structures has been
shown to reduce TSNA levels in burley tobacco. The climatic
conditions prevailing during curing exert a major influence on
N-nitrosamine formation, and the relative humidity during
air-curing can be of importance. Stalk curing results in higher
TSNA levels in the smoke than primed-leaf curing. Sun-cured
Oriental tobaccos have lower TSNA levels than Flue and air-cured
dark tobaccos. Accelerated curing of crude tobaccos such as
homogenized leaf curing limits the ability of bacteria to carry out
the nitrosation reactions. However, many of the methods described
above for reducing TSNAs in Burley tobacco can have undesirable
effects on tobacco taste.
[0035] TSNA formation in flue-cured tobacco also results from
exposure of the tobacco to combustion gases during curing, where
nearly all of the TSNAs in flue-cured tobacco (e.g., Virginia Flue)
result from a reaction involving NOx and nicotine. The predominant
source of NOx is the mixture of combustion gases in direct-fired
barns. At present, flue-cured tobacco is predominantly cured in
commercial bulk barns. As a result of energy pressures in the U.S.
during the 1960's, farmer-built "stick barns" with heat-exchanged
flue systems were gradually replaced with more energy efficient
bulk barns using direct-fired liquid propane gas (LPG) burners.
These LPG direct-fired burner systems exhaust combustion gases and
combustion by-products directly into the barn where contact is made
with the curing tobacco. Studies indicate that LPG combustion
by-products react with naturally occurring tobacco alkaloids to
form TSNA.
[0036] In contrast to direct-fired curing, heat-exchange burner
configurations completely vent combustion gases and combustion
by-products to the external atmosphere rather than into the barn.
The heat-exchange process precludes exposure of the tobacco to LPG
combustion by-products, thereby eliminating an important source of
nitrosating agent for TSNA formation, without degrading leaf
quality or smoking quality. The use of heat exchangers reduces TSNA
levels by about 90%. Steps are being taken to reduce TSNA levels in
US tobacco by converting barns to indirect heat through the use of
a heat exchanger, but these methods are very expensive. Although
many of the approaches described in this section have significant
drawbacks, it should be understood that any or all of these
techniques can be used with other techniques, as described herein,
to make tobacco and tobacco products having reduced nitrosamines.
The section below provides more detail on nicotine and approaches
to reduce nicotine in tobacco.
[0037] Nicotine
[0038] Nicotine is formed primarily in the roots of the tobacco
plant and is subsequently transported to the leaves, where it is
stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp.
233-34, Dowden, Hutchinson & Ross, Stroudsburg, Pa. (1972)).
Classical crop breeding techniques have produced tobacco with lower
levels of nicotine, including varieties with as low as 8% of the
amount of nicotine found in wild-type tobacco. The many methods
described herein can be used with virtually any tobacco variety but
are preferably used with burley, oriental or Flue (e.g., Virginia
Flue) varieties.
[0039] Nicotine is produced in tobacco plants by the condensation
of nicotinic acid and 4-methylaminobutanal. The biosynthetic
pathway resulting in nicotine production is illustrated in FIG. 1.
Two regulatory loci (Nic1 and Nic2) act as co-dominant regulators
of nicotine production. Enzyme analyses of root tissue from single
and double Nic mutants show that the activities of two enzymes,
quinolate phosphoribosyl transferase ("QPTase") and putrescence
methyl transferase (PMTase), are directly proportional to levels of
nicotine biosynthesis. An obligatory step in nicotine biosynthesis
is the formation of nicotinic acid from quinolinic acid, a step
that is catalyzed by QPTase. QPTase appears to be a rate-limiting
enzyme in the pathway supplying nicotinic acid for nicotine
synthesis in tobacco. (See, eg., Feth et al., Planta, 168, pp.
402-07 (1986) and Wagner et al., Physiol. Plant., 68, pp. 667-72
(1986), herein expressly incorporated by reference in its
entirety). A comparison of enzyme activity in tobacco tissues (root
and callus) with different capacities for nicotine synthesis shows
that QPTase activity is strictly correlated with nicotine content
(Wagner and Wagner, Planta 165:532 (1985), herein expressly
incorporated by reference in its entirety). In fact, Saunders and
Bush (Plant Physiol 64:236 (1979), herein expressly incorporated by
reference in its entirety), showed that the level of QPTase in the
roots of low nicotine mutants is proportional to the level of
nicotine in the leaves.
[0040] The modification of nicotine levels in tobacco plants by
antisense regulation of putrescence methyl transferase (PMTase)
expression has been proposed in U.S. Pat. Nos. 5,369,023 and
5,260,205, to Nakatani and Malik, and in PCT application WO
94/28142 to Wahad and Malik, which describe DNA encoding PMT and
the use of sense and antisense PMT constructs, the entire
disclosures of each of which are hereby expressly incorporated by
reference in their entireties. Other genetic modifications proposed
to reduce nicotine levels are described in PCT application WO
00/67558, to Timko, and WO 93/05646, to Davis and Marcum; the
entire contents of each are hereby expressly incorporated by
reference in their entireties. Although many of the approaches
described in this section have significant drawbacks, it should be
understood that any or all of these techniques can be used with
other techniques, as described herein, to make tobacco and tobacco
products having reduced nicotine. The section below explains novel
approaches to reduce the amount of nicotine and TSNAs in tobacco
and tobacco products.
[0041] Reducing the Amount of Nicotine and Tobacco Specific
Nitrosamines(TSNAs)
[0042] As discussed above, TSNAs and nicotine contribute
significantly to the carcinogenic potential and addictive
properties of tobacco and tobacco products. Thus, tobacco and
tobacco products that have reduced amounts of TSNAs and nicotine
have tremendous utility. Without wishing to be bound by any
particular theory, it is contemplated that the creation of tobacco
plants, tobacco and tobacco products that have a reduced amount of
nicotine will also have reduced amounts of TSNAs. That is, by
removing nicotine from tobacco plants, tobacco and tobacco
products, one effectively removes the alkaloid substrate for TSNA
formation. It was found that the reduction of nicotine in tobacco
was directly related to the reduction of TSNAs. Unexpectedly, the
methods described herein not only produce tobacco with a reduced
addictive potential but, concomitantly, produce a tobacco that has
a lower carcinogenic potential.
[0043] It should be emphasized that the phrase "a reduced amount"
is intended to refer to an amount of nicotine and/or TSNAs in a
treated or transgenic tobacco plant, tobacco or a tobacco product
that is less than what would be found in a tobacco plant, tobacco
or a tobacco product from the same variety of tobacco, processed in
the same manner, which has not been treated or was not made
transgenic for reduced nicotine and/or TSNAs. Thus, in some
contexts, wild-type tobacco of the same variety that has been
processed in the same manner is used as a control by which to
measure whether a reduction in nicotine and/or TSNAs has been
obtained by the inventive methods described herein.
[0044] The amount of TSNAs (e.g., collective content of NNN, NAT,
NAB, and NNK) and nicotine in wild-type tobacco varies
significantly depending on the variety and the manner it is grown,
harvested and cured. For example, a cured Burley tobacco leaf can
have approximately 30,000 parts per million (ppm) nicotine and
8,000 parts per billion (ppb) TSNA (e.g., collective content of
NNN, NAT, NAB, and NNK); a Flue-Cured leaf can have approximately
20,000 ppm nicotine and 300 ppb TSNA (e.g., collective content of
NNN, NAT, NAB, and NNK); and an Oriental cured leaf can have
approximately 10,000 ppm nicotine and 100 ppb TSNA (e.g.,
collective content of NNN, NAT, NAB, and NNK). Tobacco having a
reduced amount of nicotine and/or TSNA, can have no detectable
nicotine and/or TSNA (e.g., collective content of NNN, NAT, NAB,
and NNK), or may contain some detectable amounts of one or more of
the TSNAs and/or nicotine, so long as the amount of nicotine and/or
TSNA is less than that found in tobacco of the same variety, grown
under similar conditions, and cured and/or processed in the same
manner. That is, cured Burley tobacco, as described herein, having
a reduced amount of nicotine can have between 0 and 30,000 ppm
nicotine and 0 and 8,000 ppb TSNA, desirably between 0 and 20,000
ppm nicotine and 0 and 6,000 ppb TSNA, more desirably between 0 and
10,000 ppm nicotine and 0 and 5,000 ppb TSNA, preferably between 0
and 5,000 ppm nicotine and 0 and 4,000 ppb TSNA, more preferably
between 0 and 2,500 ppm nicotine and 0 and 2,000 ppb TSNA and most
preferably between 0 and 1,000 ppm nicotine and 0 and 1,000 ppb
TSNA. Embodiments of cured Burley leaf prepared by the methods
described herein can also have between 0 and 1000 ppm nicotine and
0 and 500 ppb TSNA, 0 and 500 ppm nicotine and 0 and 250 ppb TSNA,
0 and 250 ppm nicotine and 0 and 100 ppb TSNA, 0 and 100 ppm
nicotine and 0 and 50 ppb TSNA, 0 and 50 ppm nicotine and 0 and 5
ppb TSNA and some embodiments of cured Burley leaf described herein
have virtually no detectable amount of nicotine or TSNA. In some
embodiments above, the amount of TSNA refers to the collective
content of NNN, NAT, NAB, and NNK.
[0045] Similarly, a cured Flue tobacco embodiment of the invention
having a reduced amount of nicotine can have between 0 and 20,000
ppm nicotine and 0 and 300 ppb TSNA, desirably between 0 and 15,000
ppm nicotine and 0 and 250 ppb TSNA, more desirably between 0 and
10,000 ppm nicotine and 0 and 200 ppb TSNA, preferably between 0
and 5,000 ppm nicotine and 0 and 150 ppb TSNA, more preferably
between 0 and 2,500 ppm nicotine and 0 and 100 ppb TSNA and most
preferably between 0 and 1,000 ppm nicotine and 0 and 50 ppb TSNA.
Embodiments of cured Flue tobacco, as described herein, can also
have between 0 and 500 ppm nicotine and 0 and 25 ppb TSNA, 0 and
200 ppm nicotine and 0 and 10 ppb TSNA, 0 and 100 ppm nicotine and
0 and 5 ppb TSNA and some embodiments of cure Flue tobacco have
virtually no detectable amount of nicotine or TSNA. In some
embodiments above, the amount of TSNA refers to the collective
content of NNN, NAT, NAB, and NNK.
[0046] Further, a cured Oriental tobacco embodiment having a
reduced amount of nicotine can have between 0 and 10,000 ppm
nicotine and 0 and 100 ppb TSNA, desirably between 0 and 7,000 ppm
nicotine and 0 and 75 ppb TSNA, more desirably between 0 and 5,000
ppm nicotine and 0 and 50 ppb TSNA, preferably between 0 and 3,000
ppm nicotine and 0 and 25 ppb TSNA, more preferably between 0 and
1,500 ppm nicotine and 0 and 10 ppb TSNA and most preferably
between 0 and 500 ppm nicotine and no detectable TSNA. Embodiments
of cured Oriental tobacco can also have between 0 and 250 ppm
nicotine and no detectable TSNA and some embodiments of cured
Oriental tobacco have virtually no detectable amount of nicotine or
TSNA. In some embodiments above, the amount of TSNA refers to the
collective content of NNN, NAT, NAB, and NNK.
[0047] Some embodiments comprise cured tobaccos (e.g., Burley,
Flue, or Oriental) with reduced amounts of nicotine as compared to
control varieties, wherein the amount of nicotine is less than
about 2 mg/g, 1 mg/g, 0.75 mg/g, 0.5 mg/g or desirably less than
about 0.1 mg/g, and preferably less than 0.08 mg/g, 0.07 mg/g, 0.06
mg/g, 0.05 mg/g, 0.04 mg/g, 0.03 mg/g, 0.02 mg/g, 0.01 mg/g.
Tobacco products made from these reduced nicotine and TSNA tobaccos
are also embodiments. The term "tobacco products" include, but are
not limited to, smoking materials (e.g., cigarettes, cigars, pipe
tobacco), snuff, chewing tobacco, gum, and lozenges.
[0048] In some contexts, the phrase "reduced amount of nicotine
and/or TSNAs" refers to the tobacco plants, cured tobacco, and
tobacco products, as described herein, which have less nicotine
and/or TSNAs (e.g., the collective content of NNN, NAT, NAB, and
NNK) by weight than the same variety of tobacco grown, processed,
and cured in the same way. For example, wild type cured tobacco can
have has approximately 1-4% dry weight nicotine and approximately
0.2% -0.8% dry weight TSNA depending on the manner it was grown,
harvested and cured. A typical cigarette has between 2-11 mg of
nicotine and approximately 5.0 .mu.g of TSNAs. Thus, the tobacco
plants, tobacco and tobacco products of the invention can have, in
dry weight for example, less than 0.01%, 0.015%, 0.02%, 0.025%,
0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%,
0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%,
0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%, 0.425%,
0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%,
0.9%, 0.95%, and 1.0% nicotine and less than 0.01%, 0.015%, 0.02%,
0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%,
0.07%, 0.075%, and 0.08% TSNA (e.g., collective content of NNN,
NAT, NAB, and NNK).
[0049] Alternatively, a cigarette of the invention can have, for
example, less than 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35
mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75
mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg, 1.15 mg, 1.2
mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg, 1.55 mg, 1.6
mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg, 1.95 mg, 2.0
mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg, 2.4 mg, 2.45
mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg, 2.8 mg, 2.85
mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg, 3.25 mg, 3.3
mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg, 3.65 mg, 3.7
mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg, 4.1 mg, 4.15
mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg, 4.4 mg, 4.45
mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg, 4.8 mg, 4.85
mg, 4.9 mg, 4.95 mg, 5.0 mg, 5.5 mg, 5.7 mg, 6.0 mg, 6.5 mgmg, 6.7
mg, 7.0 mg, 7.5 mg, 7.7 mg, 8.0 mg, 8.5 mg, 8.7 mg, 9.0 mg, 9.5 mg,
9.7 mg, 10.0 mg, 10.5 mg, 10.7 mg, and 11.0 mg nicotine and less
than 0.001 ug, 0.002 ug, 0.003 ug, 0.004 ug, 0.005 ug, 0.006 ug,
0.007 ug, 0.008 ug, 0.009 ug, 0.01 ug, 0.02 ug, 0.03 ug, 0.04 ug,
0.05 ug, 0.06 ug, 0.07 ug, 0.08 ug, 0.09 ug, 0.1 ug, 0.15 ug, 0.2
ug, 0.25 ug, 0.3 ug, 0.336 ug, 0.339 ug, 0.345 ug, 0.35 ug, 0.375
ug, 0.4 ug, 0.414 ug, 0.45 ug, 0.5 ug, 0.515 ug, 0.55 ug, 0.555 ug,
0.56 ug, 0.578 ug, 0.58 ug, 0.6 ug, 0.611 ug, 0.624 ug, 0.65 ug,
0.7 ug, 0.75 ug, 0.8 ug, 0.85 ug, 0.9 ug, 0.95 ug, 1.0 ug, 1.1 ug,
1.114 ug, 1.15 ug, 1.2 ug, 1.25 ug, 1.3 ug, 1.35 ug, 1.4 ug, 1.45
ug, 1.5 ug, 1.55 ug, 1.6 ug, 1.65 ug, 1.7 ug, 1.75 ug, 1.8 ug, 1.85
ug, 1.9 ug, 1.95 ug, 2.0 ug, 2.1 ug, 2.15 ug, 2.2 ug TSNA (e.g.,
collective content of NNN, NAT, NAB, and NNK).
[0050] Unexpectedly, it was discovered that several methods for
reducing endogenous levels of nicotine in a plant are suitable for
producing tobacco that is substantially free of nitrosamines,
especially TSNAs. Any method that reduces levels of other
alkaloids, including norniticotine, will likewise be suitable for
producing tobacco substantially free of nitrosamines, especially
TSNAs. As described this invention comprises a method of reducing
the carcinogenic potential of a tobacco product comprising
providing a cured tobacco as described herein and preparing a
tobacco product from said cured tobacco, whereby the carcinogenic
potential of said tobacco product is thereby reduced. Other
embodiments of the invention include the use of the cured tobacco
described herein for the preparation of a tobacco product that
contains reduced amounts of carcinogens as compared to control
varieties and/or that reduces the amount of a TSNA or TSNA
metabolite in a human that uses tobacco.
[0051] In some embodiments, for example, the tobacco smoking
products described herein reduce the carcinogenic potential of side
stream or main stream tobacco smoke in humans exposed to said side
stream or main stream tobacco smoke. By providing the genetically
modified cured tobacco described herein in a product that undergoes
pyrolysis, for example, the side stream and/or main stream smoke
produced by said product comprises a reduced amount of TSNAs and/or
nicotine. Thus, the cured tobacco described herein can be used to
prepare a tobacco smoking product that comprises a reduced amount
of TSNAs in side stream and/or mainstream smoke.
[0052] In some embodiments, for example, the collective content of
NNN, NAT, NAB, and NNK in the mainstream or side stream smoke from
a tobacco product comprising the genetically modified tobacco
described herein is between about 0-5.0 .mu.g/g, 0-4.0 .mu.g/g,
0-3.0 .mu.g/g, 0-2.0 .mu.g/g, 0-1.5 .mu.g/g, 0-1.0 .mu.g/g, 0-0.75
.mu.g/g, 0-0.5 .mu.g/g, 0-0.25 .mu.g/g, 0-0.15 .mu.g/g, 0-0.1
.mu.g/g, 0-0.05 .mu.g/g, 0-0.02 .mu.g/g, 0-0.015 .mu.g/g, 0-0.01
.mu.g/g, 0-0.005 .mu.g/g, 0-0.002 .mu.g/g, or 0-0.001 .mu.g/g. That
is, some embodiments are genetically modified Burley tobacco,
wherein the side stream or mainstream smoke produced from a tobacco
product comprising said Burley tobacco has a collective content of
NNN, NAT, NAB, and NNK in the mainstream or side stream smoke
between about 0-5.0 .mu.g/g, 0-4.0 .mu.g/g, 0-3.0 .mu.g/g, 0-2.0
.mu.g/g, 0-1.5 .mu.g/g, 0-1.0 .mu.g/g, 0-0.75 .mu.g/g, 0-0.5
.mu.g/g, 0-0.25 .mu.g/g, 0-0.15 .mu.g/g, 0-0.1 .mu.g/g, 0-0.05
.mu.g/g, 0-0.02 .mu.g/g, 0-0.015 .mu.g/g, 0-0.01 .mu.g/g, 0-0.005
.mu.g/g, 0-0.002 .mu.g/g, or 0-0.001 .mu.g/g.
[0053] Other embodiments concern genetically modified Flue tobacco,
wherein the sidestream or mainstream smoke produced from a tobacco
product comprising said Flue tobacco has a collective content of
NNN, NAT, NAB, and NNK in the mainstream or side stream smoke
between about 0-5.0 .mu.g/g, 0-4.0 .mu.g/g, 0-3.0 .mu.g/g, 0-2.0
.mu.g/g, 0-1.5 .mu.g/g, 0-1.0 .mu.g/g, 0-0.75 .mu.g/g, 0-0.5
.mu.g/g, 0-0.25 .mu.g/g, 0-0.15 .mu.g/g, 0-0.1 .mu.g/g, 0-0.05
.mu.g/g, 0-0.02 .mu.g/g, 0-0.015 .mu.g/g, 0-0.01 .mu.g/g, 0-0.005
.mu.g/g, 0-0.002 .mu.g/g, or 0-0.001 .mu.g/g.
[0054] More embodiments concern genetically modified Oriental
tobacco, wherein the sidestream or mainstream smoke produced from a
tobacco product comprising said Oriental tobacco has a collective
content of NNN, NAT, NAB, and NNK in the mainstream or side stream
smoke between about 0-5.0 .mu.g/g, 0-4.0 .mu.g/g, 0-3.0 .mu.g/g,
0-2.0 .mu.g/g, 0-1.5 .mu.g/g, 0-1.0 .mu.g/g, 0-0.75 .mu.g/g, 0-0.5
.mu.g/g, 0-0.25 .mu.g/g, 0-0.15 .mu.g/g, 0-0.1 .mu.g/g, 0-0.05
.mu.g/g, 0-0.02 .mu.g/g, 0-0.015 .mu.g/g, 0-0.01 .mu.g/g, 0-0.005
.mu.g/g, 0-0.002 .mu.g/g/, or 0-0.001 .mu.g/g.
[0055] A preferred method of producing tobacco having a reduced
amount of nicotine and TSNAs, involves genetic engineering directed
at reducing the levels of nicotine and/or nornicotine or other
alkaloids. Any enzyme involved in the nicotine synthesis pathway
can be a suitable target for genetic engineering to reduce levels
of nicotine and, optionally, levels of other alkaloids including
nornicotine. Suitable targets for genetic engineering to produce
tobacco having a reduced amount of nicotine and/or nitrosamines,
especially TSNAs, include but are not limited to putrescine
N-methyltransferase, N-methylputrescine oxidase, ornithine
decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase or quinolate phosphoribosyl
transferase (QPTase). Additionally, enzymes that regulate the flow
of precursors into the nicotine synthesis pathway are suitable
targets for genetic engineering to produce tobacco with a reduced
amount of nicotine and nitrosamines, especially TSNAs. Suitable
methods of genetic engineering are known in the art and include,
for example, the use of antisense and sense suppression technology
to reduce enzyme production, as well as use of random or targeted
mutagenesis to disrupt gene function, for example, using T-DNA
insertion or EMS mutagenesis.
[0056] By way of example, tobacco having reduced amounts of
nicotine and TSNAs is generated from a tobacco plant that is
created by exposing at least one tobacco cell of a selected tobacco
variety (preferably Burley 21) to an exogenous DNA construct
having, in the 5' to 3' direction, a promoter operable in a plant
cell and DNA containing a portion of a DNA sequence that encodes an
enzyme in the nicotine synthesis pathway or a complement thereof.
The DNA is operably associated with said promoter and the tobacco
cell is transformed with the DNA construct. The transformed cells
are selected using either negative selection or positive selection
techniques and at least one tobacco plant is regenerated from
transformed cells. The regenerated tobacco plant or portion thereof
is preferably analyzed to determine the amount of nicotine and/or
TSNAs present and these values can be compared to the amount of
nicotine and/or TSNAs present in a control tobacco plant or
portion, preferably of the same variety.
[0057] The DNA constructs having a portion of a DNA sequence that
encodes an enzyme in the nicotine synthesis pathway may have the
entire coding sequence of the enzyme a complement of this sequence,
or any portion thereof. A portion of a DNA sequence that encodes an
enzyme in the nicotine synthesis pathway or the complement thereof
may have at least 25, or preferably 50, or 75, or 100, or 150, or
250, or 500, or 750, or 1000, or 1500, or 2000, or 2500, or 5000,
or the entire coding sequence of the enzyme or complement thereof.
Accordingly, these DNA constructs have the ability to perturb the
production of endogenous enzyme in the nicotine biosynthesis
pathway through either an antisense or cosuppression mechanism. It
is contemplated that both antisense and cosuppression constructs
are effective at reducing the levels of nicotine and/or
nitrosamines in tobacco plants.
[0058] In a preferred embodiment, the enzyme involved in the
nicotine synthesis pathway can be, for example, putrescine
N-methyltransferase, N-methylputrescine oxidase, ornithine
decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase, or quinolate phosphoribosyl
transferase (QPTase). In a preferred embodiment, the enzyme is
QPTase. The segment of DNA sequence encoding an enzyme in the
nicotine synthesis pathway may be in the antisense or the sense
orientation. In a particularly preferred embodiment, the enzyme is
QPTase.
[0059] By one approach, a novel cDNA sequence (SEQ ID NO: 1)
encoding a plant quinolate phosphoribosyl transferase (QPTase) of
SEQ ID NO: 2 is used. As QPTase activity is strictly correlated
with nicotine content, construction of transgenic tobacco plants in
which QPTase levels are lowered in the plant roots (compared to
levels in wild-type plants) result in plants having reduced levels
of nicotine in the leaves. Embodiments of the invention provide
methods and nucleic acid constructs for producing such transgenic
plants, as well as, the transgenic plants themselves. Such methods
include the expression of antisense NtQPT1 RNA, which lowers the
amount of QPTase in tobacco roots.
[0060] Aspects of the present invention also concern sense and
antisense recombinant DNA molecules encoding QPTase or QPTase
antisense RNA molecules, and vectors comprising those recombinant
DNA molecules, as well as transgenic plant cells and plants
transformed with those DNA molecules and vectors. Transgenic
tobacco cells and the plants described herein are characterized in
that they have a reduced amount of nicotine and/or TSNA as compared
to unmodified or control tobacco cells and plants.
[0061] The tobacco plants described herein are suitable for
conventional growing and harvesting techniques (e.g. topping or no
topping, bagging the flowers or not bagging the flowers,
cultivation in manure rich soil or without manure) and the
harvested leaves and stems are suitable for use in any traditional
tobacco product including, but not limited to, pipe, cigar and
cigarette tobacco and chewing tobacco in any form including leaf
tobacco, shredded tobacco or cut tobacco. It is also contemplated
that the low nicotine and/or TSNA tobacco described herein can be
processed and blended with conventional tobacco so as to create a
wide-range of tobacco products with varying amounts of nicotine
and/or nitrosamines. These blended tobacco products can be used in
tobacco product cessation programs so as to slowly move a consumer
from a high nicotine and TSNA product to a low nicotine and TSNA
product. Some embodiments of the invention comprise a tobacco use
cessation kit, comprising two or more tobacco products with
different levels of nicotine and/or nitrosamines. For example, a
smoker can begin the program smoking blended cigarettes having 5 mg
of nicotine and 0.3 .mu.g of nitrosamine, gradually move to smoking
cigarettes with 3 mg of nicotine and 0.2 .mu.g of nitrosamine,
followed by cigarettes having 2 mg nicotine and 0.1 .mu.g
nitrosamine, followed by cigarettes having 1.0 mg nicotine and 0.05
.mu.g nitrosamine, followed by cigarettes having 0.05 mg nicotine
and no detectable TSNA until the consumer decides to smoke only the
cigarettes having virtually no nicotine and nitrosamines or
quitting smoking altogether. Accordingly, the blended cigarettes
described herein provide the basis for an approach to reduce the
carcinogenic potential in a human in a step-wise fashion. The
components of the tobacco use cessation kit described herein may
include other tobacco products, including but not limited to,
smoking materials (e.g., cigarettes, cigars, pipe tobacco), snuff,
chewing tobacco, gum, and lozenges.
[0062] The present inventors have discovered that the TobRD2 gene
(see Conkling et al., Plant Phys. 93, 1203 (1990)) encodes a
Nicotiana tabacum QPTase, and provide herein the cDNA sequence of
NtQPT1 (formerly termed TobRD2) and the amino acid sequence of the
encoded enzyme. Aspects of the technology described herein are also
described in PCT/US98/11893, which is hereby expressly incorporated
by reference in its entirety. Comparisons of the NtQPT1 amino acid
sequence with the GenBank database reveal limited sequence
similarity to bacterial proteins that encode quinolate
phosphoribosyl transferase (QPTase) (FIG. 3).
[0063] Quinolate phosphoribosyl transferase is required for de novo
nicotine adenine dinucleotide (NAD) biosynthesis in both
prokaryotes and eukaryotes. In tobacco, high levels of QPTase are
detected in roots, but not in leaves. To determine that NtQPT1
encoded QPTase, the present inventors utilized Escherichia coli
bacterial strain (TH265), a mutant lacking in quinolate
phosphoribosyl transferase (nadC). This mutant cannot grow on
minimal medium lacking nicotinic acid. However, expression of the
NtQPT1 protein in this bacterial strain conferred the NadC+
phenotype (FIG. 4), confirming that NtQPT1 encodes QPTase.
[0064] The effects of Nicl and Nic2 mutants in tobacco, and the
effects of topping tobacco plants, on NtQPT1 steady-state mRNA
levels and nicotine levels were determined. (Removal of apical
dominance by topping at onset of flowering is well known to result
in increased levels of nicotine biosynthesis and transport in
tobacco, and is a standard practice in tobacco production.) If
NTQPT1 is in fact involved in nicotine biosynthesis, it would be
expected that (1) NtQPT1 mRNA levels would be lower in nicl/nic2
double mutants and (2) NtQPT1 mRNA levels would increase after
topping. NtQPT1 mRNA levels in nicl/nic2 double mutants were found
to be approximately 25% that of wild-type (FIG. 5). Further, within
six hours of topping, the NtQPT1 mRNA levels in tobacco plants
increased about eight-fold. Therefore, NtQPT1 was determined to be
a key regulatory gene in the nicotine biosynthetic pathway. The
next section describes the creation of transgenic tobacco plant
cells and transgenic tobacco plants.
[0065] Transgenic Plant Cells and Plants
[0066] Regulation of gene expression in plant cell genomes can be
achieved by integration of heterologous DNA under the
transcriptional control of a promoter which is functional in the
host, and in which the transcribed strand of heterologous DNA is
complementary to the strand of DNA that is transcribed from the
endogenous gene to be regulated. The introduced DNA, referred to as
antisense DNA, provides an RNA sequence which is complementary to
naturally produced (endogenous) mRNAs and which inhibits expression
of the endogenous mRNA. Although the mechanism of antisense is not
completely understood, it is known that antisense constructs can be
used to regulate gene expression.
[0067] In some methods of the invention, the antisense product may
be complementary to coding or non-coding (or both) portions of
naturally occurring target RNA. The antisense construction may be
introduced into the plant cells in any suitable manner, and may be
integrated into the plant genome for inducible or constitutive
transcription of the antisense sequence.
[0068] As used herein, exogenous or heterologous DNA (or RNA)
refers to DNA (or RNA) that has been introduced into a cell (or the
cell's ancestor) through the efforts of humans. Such heterologous
DNA may be a copy of a sequence which is naturally found in the
cell being transformed, or fragments thereof. To produce a tobacco
plant having decreased QPTase levels, and a reduced amount of
nicotine and TSNAs, as compared to an untransformed or control
tobacco plant or portion thereof, a tobacco cell may be transformed
with an exogenous QPT antisense transcriptional unit comprising a
partial QPT cDNA sequence, a full-length QPT cDNA sequence, a
partial QPT chromosomal sequence, or a full-length QPT chromosomal
sequence, in the antisense orientation with appropriate operably
linked regulatory sequences. Appropriate regulatory sequences
include a transcription initiation sequence ("promoter") operable
in the plant being transformed, and a polyadenylation/transcription
termination sequence. Standard techniques, such as restriction
mapping, Southern blot hybridization, and nucleotide sequence
analysis, are then employed to identify clones bearing QPTase
sequences in the antisense orientation, operably linked to the
regulatory sequences.
[0069] Tobacco plants are then regenerated from successfully
transformed cells using conventional techniques. It is most
preferred that the antisense sequence utilized be complementary to
the endogenous sequence, however, minor variations in the exogenous
and endogenous sequences may be tolerated. It is preferred that the
antisense DNA sequence be of sufficient sequence similarity to the
extent that it is capable of binding to the endogenous sequence in
the cell to be regulated, under stringent conditions as described
below.
[0070] 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
Antisense RNA", Proc. NM. Acad SU 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)).
[0071] 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. Levels of nicotine in the transgenic
tobacco plants of the present invention can be detected by standard
nicotine assays. Transformed plants in which the level of QPTase is
reduced compared to untransformed control plants will accordingly
have a reduced nicotine level compared to the control; transformed
plants in which the level of QPTase is increased compared to
untransformed control plants will accordingly have an increased
nicotine level compared to the control.
[0072] The heterologous sequence utilized in the antisense methods
of the present invention may be selected so as to produce an RNA
product complementary to the entire QPTase mRNA sequence, or to a
portion thereof. The sequence may be complementary to any
contiguous sequence of the natural messenger RNA, that is, it may
be complementary to the endogenous mRNA sequence proximal to the
5'-terminus or capping site, downstream from the capping site,
between the capping site and the initiation codon and may cover all
or only a portion of the non-coding region, may bridge the
non-coding and coding region, be complementary to all or part of
the coding region, complementary to the C-terminus of the coding
region, or complementary to the 3'-untranslated region of the mRNA.
Suitable antisense sequences may be from at least about 13 to about
15 nucleotides, at least about 16 to about 21 nucleotides, at least
about 20 nucleotides, at least about 30 nucleotides, at least about
50 nucleotides, at least about 75 nucleotides, at least about 100
nucleotides, at least about 125 nucleotides, at least about 150
nucleotides, at least about 200 nucleotides, or more. In addition,
the sequences may be extended or shortened on the 3' or 5' ends
thereof.
[0073] The particular anti-sense sequence and the length of the
anti-sense sequence will vary depending upon the degree of
inhibition desired, the stability of the anti-sense sequence and
the like. One of skill in the art will be guided in the selection
of appropriate QPTase antisense sequences using techniques
available in the art and the information provided herein. With
reference to FIG. 2A and SEQ ID NO: 1 herein, an oligonucleotide of
the invention may be a continuous fragment of the QPTase cDNA
sequence in antisense orientation, of any length that is sufficient
to achieve the desired effects when transformed into a recipient
plant cell.
[0074] The present invention may also be used in methods of sense
co-suppression of nicotine production. Sense DNAs employed in
carrying out the present invention are of a length sufficient to,
when expressed in a plant cell, suppress the native expression of
the plant QPTase protein as described herein in that plant cell.
Such sense DNAs may be essentially an entire genomic or
complementary DNA encoding the QPTase enzyme, or a fragment
thereof, with such fragments typically being at least 15
nucleotides in length. Methods of ascertaining the length of sense
DNA that results in suppression of the expression of a native gene
in a cell are available to those skilled in the art.
[0075] In an alternate embodiment of the present invention,
Nicotiana plant cells are transformed with a DNA construct
containing a DNA segment encoding an enzymatic RNA molecule (i.e.,
a "ribozyme"), which enzymatic RNA molecule is directed against
(i.e., cleaves) the mRNA transcript of DNA encoding plant QPTase as
described herein. Ribozymes contain substrate binding domains that
bind to accessible regions of the target mRNA, and domains that
catalyze the cleavage of RNA, preventing translation and protein
production. The binding domains may comprise antisense sequences
complementary to the target mRNA sequence; the catalytic motif may
be a hammerhead motif or other motifs, such as the hairpin motif.
Ribozyme cleavage sites within an RNA target may initially be
identified by scanning the target molecule for ribozyme cleavage
sites (e.g., GUA, GUU or GUC sequences). Once identified, short RNA
sequences of 15, 20, 30 or more ribonucleotides corresponding to
the region of the target gene containing the cleavage site may be
evaluated for predicted structural features. The suitability of
candidate targets may also be evaluated by testing their
accessibility to hybridization with complimentary oligonucleotides,
using ribonuclease protection assays as are known in the art. DNA
sequences encoding enzymatic RNA molecules may be produced in
accordance with known techniques. See, e.g., T. Cech et al., U.S.
Pat. No. 4,987,071; Keene et al., U.S. Pat. No. 5,559,021; Donson
et al., U.S. Pat. No. 5,589,367; Torrence et al., U.S. Pat. No.
5,583,032; Joyce, U.S. Pat. No. 5,580,967; Gold et al. U.S. Pat.
No. 5,595,877; Wagner et al., U.S. Pat. No. 5,591,601; and U.S.
Pat. No. 5,622,854 (the disclosures of which are to be incorporated
herein by reference in their entirety).
[0076] Production of such an enzymatic RNA molecule in a plant cell
and disruption of QPTase protein production reduces QPTase activity
in plant cells in essentially the same manner as production of an
antisense RNA molecule: that is, by disrupting translation of mRNA
in the cell which produces the enzyme. The term `ribozyme` is used
herein to describe an RNA-containing nucleic acid that functions as
an enzyme (such as an endoribonuclease), and may be used
interchangeably with `enzymatic RNA molecule`. The present
invention further includes DNA encoding the ribozymes, DNA encoding
the ribozymes that has been inserted into an expression vector,
host cells containing such vectors and methods of decreasing QPTase
production in plants using ribozymes.
[0077] Nucleic acid sequences employed in carrying out the present
invention include those with sequence similarity to SEQ ID NO: 1,
and encoding a protein having quinolate phosphoribosyl transferase
activity. This definition is intended to encompass natural allelic
variations in QPTase proteins. Thus, DNA sequences that hybridize
to DNA of SEQ ID NO: 1 and code for expression of QPTase,
particularly plant QPTase enzymes, may also be employed in carrying
out the present invention. Multiple forms of the tobacco QPT enzyme
may exist. Multiple forms of an enzyme may be due to
post-translational modification of a single gene product, or to
multiple forms of the NtQPT1 gene.
[0078] Conditions which permit other DNA sequences which code for
expression of a protein having QPTase activity to hybridize to DNA
of SEQ ID NO: 1 or to other DNA sequences encoding the protein
given as SEQ ID NO: 2 can be determined in a routine manner. For
example, hybridization of such sequences to DNA encoding the
protein given as SEQ ID NO: 2 may be carried out under conditions
of reduced stringency or even stringent conditions (e.g.,
conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M
sodium citrate, 0.1% SDS at 60.degree. C. or even 70.degree. C.)
herein in a standard in situ hybridization assay. See J. Sambrook
et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold
Spring Harbor Laboratory)). In general, such sequences will be at
least 65% similar, 75% similar, 80% similar, 85% similar, 90%
similar, or even 95% similar or more, with the sequence given
herein as SEQ ID NO: 1, or DNA sequences encoding proteins of SEQ
ID NO: 2. (Determinations of sequence similarity are made with the
two sequences aligned for maximum matching; gaps in either of the
two sequences being matched are allowed in maximizing matching. Gap
lengths of 10 or less are preferred, gap lengths of 5 or less are
more preferred, and gap lengths of 2 or less still more
preferred.)
[0079] Differential hybridization procedures are available which
allow for the isolation of cDNA clones whose mRNA levels are as low
as about 0.05% of poly(A)RNA. See M. Conkling et al., Plant
Physiol. 93, 1203-1211 (1990). In brief, cDNA libraries are
screened using single-stranded cDNA probes of reverse transcribed
mRNA from plant tissue (e.g., roots and/or leaves). For
differential screening, a nitrocellulose or nylon membrane is
soaked in 5.times.SSC and placed in a 96 well suction manifold; 150
.mu.L of stationary overnight culture is transferred from a master
plate to each well and vacuum applied until all liquid has passed
through the filter. Approximately, 150 .mu.L of denaturing solution
(0.5M NaOH, 1.5 M NaCl) is placed in each well using a multiple
pipetter and allowed to sit about 3 minutes. Suction is applied as
above and the filter removed and neutralized in 0.5 M Tris-HCl (pH
8.0), 1.5 M NaCl. It is then baked 2 hours in vacuo and incubated
with the relevant probes. By using nylon membrane filters and
keeping master plates stored at -70.degree. C. in 7% DMSO, filters
may be screened multiple times with multiple probes and appropriate
clones recovered after several years of storage.
[0080] As used herein, the term `gene` refers to a DNA sequence
that incorporates (1) upstream (5') regulatory signals including
the promoter, (2) a coding region specifying the product, protein
or RNA of the gene, (3) downstream regions including transcription
termination and polyadenylation signals and (4) associated
sequences required for efficient and specific expression. The DNA
sequence of the present invention may consist essentially of the
sequence provided herein (SEQ ID NO: 1), or equivalent nucleotide
sequences representing alleles or polymorphic variants of these
genes, or coding regions thereof. Use of the phrase "substantial
sequence similarity" in the present specification and claims means
that DNA, RNA or amino acid sequences which have slight and
non-consequential sequence variations from the actual sequences
disclosed and claimed herein are considered to be equivalent to the
sequences of the present invention. In this regard, "slight and
non-consequential sequence variations" mean that "similar"
sequences (i.e., the sequences that have substantial sequence
similarity with the DNA, RNA or proteins disclosed and claimed
herein) will be functionally equivalent to the sequences disclosed
and claimed in the present invention. Functionally equivalent
sequences will function in substantially the same manner to produce
substantially the same compositions as the nucleic acid and amino
acid compositions disclosed and claimed herein.
[0081] DNA sequences provided herein can be transformed into a
variety of host cells. A variety of suitable host cells, having
desirable growth and handling properties, are readily available in
the art. Use of the phrase "isolated" or "substantially pure" in
the present specification and claims as a modifier of DNA, RNA,
polypeptides or proteins means that the DNA, RNA, polypeptides or
proteins so designated have been separated from their in vivo
cellular environments through the efforts of human beings.
[0082] As used herein, a "native DNA sequence" or "natural DNA
sequence" means a DNA sequence that can be isolated from
non-transgenic cells or tissue. Native DNA sequences are those
which have not been artificially altered, such as by site-directed
mutagenesis. Once native DNA sequences are identified, DNA
molecules having native DNA sequences may be chemically synthesized
or produced using recombinant DNA procedures as are known in the
art. As used herein, a native plant DNA sequence is that which can
be isolated from non-transgenic plant cells or tissue. As used
herein, a native tobacco DNA sequence is that which can be isolated
from non-transgenic tobacco cells or tissue.
[0083] DNA constructs, or "transcription cassettes," of the present
invention include, 5' to 3' in the direction of transcription, a
promoter as discussed herein, a DNA sequence as discussed herein
operatively associated with the promoter, and, optionally, a
termination sequence including stop signal for RNA polymerase and a
polyadenylation signal. All of these regulatory regions should be
capable of operating in the cells of the tissue to be transformed.
Any suitable termination signal may be employed in carrying out the
present invention, examples thereof including, but not limited to,
the nopaline synthase (nos) terminator, the octapine synthase (ocs)
terminator, the CaMV terminator or native termination signals,
derived from the same gene as the transcriptional initiation region
or derived from a different gene. See, e.g., Rezian et al. (1988)
supra, and Rodermel et al. (1988), supra.
[0084] The term "operatively associated," as used herein, refers to
DNA sequences on a single DNA molecule that are associated so that
the function of one sequence is affected by the other. Thus, a
promoter is operatively associated with a DNA when it is capable of
affecting the transcription of that DNA (i.e., the DNA is under the
transcriptional control of the promoter). The promoter is said to
be "upstream" from the transcribed DNA sequence, which is in turn
said to be "downstream" from the promoter.
[0085] The transcription cassette may be provided in a DNA
construct that also has at least one replication system. For
convenience, it is common to have a replication system functional
in Escherichia coli, such as ColEl, pSC101, pACYC184, or the like.
In this manner, at each stage after each manipulation, the
resulting construct may be cloned, sequenced, and the correctness
of the manipulation determined. In addition, or in place of the E.
coli replication system, a broad host range replication system may
be employed, such as the replication systems of the P-1
incompatibility plasmids, e.g., pRK290. In addition to the
replication system, there will frequently be at least one marker
present, which may be useful in one or more hosts, or different
markers for individual hosts. That is, one marker may be employed
for selection in a prokaryotic host, while another marker may be
employed for selection in a eukaryotic host, particularly the plant
host. The markers may be protection against a biocide (such as
antibiotics, toxins, heavy metals or the like), provide
complementation by imparting prototrophy to an auxotrophic host
and/or provide a visible phenotype through the production of a
novel compound in the plant.
[0086] The various fragments comprising the various constructs,
transcription cassettes, markers and the like may be introduced
consecutively by restriction enzyme cleavage of an appropriate
replication system and insertion of the particular construct or
fragment into the available site. After ligation and cloning, the
DNA construct may be isolated for further manipulation. All of
these techniques are amply exemplified in the literature as
demonstrated by J. Sambrook et al., Molecular Cloning, A Laboratory
Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).
[0087] Vectors that may be used to transform plant tissue with
nucleic acid constructs of the present invention include both
Agrobacterium vectors and ballistic vectors, as well as vectors
suitable for DNA-mediated transformation. The term `promoter`
refers to a region of a DNA sequence that incorporates the
necessary signals for the efficient expression of a coding
sequence. This may include sequences to which an RNA polymerase
binds, but is not limited to such sequences, and may include
regions to which other regulatory proteins bind along with regions
involved in the control of protein translation. They may also
include coding sequences.
[0088] Promoters employed in carrying out the invention may be
constitutively active promoters. Numerous constitutively active
promoters that are operable in plants are available. A preferred
example is the Cauliflower Mosaic Virus (CaMV) 35S promoter, which
is expressed constitutively in most plant tissues. As an
alternative, the promoter may be a root-specific promoter or root
cortex specific promoter, as explained in greater detail below.
[0089] Antisense sequences have been expressed in transgenic
tobacco plants utilizing 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).
[0090] Use of the CaMV 35S promoter for expression of QPTase 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.
Nat. 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)).
[0091] Other promoters that are active only in root tissues (root
specific promoters) are also particularly suited to the methods of
the present invention. See, e.g., U.S. Pat. No. 5,459,252 to
Conkling et al.; Yamamoto et al., The Plant Cell, 3:371 (1991). The
TobRD2 root-cortex specific promoter may also be utilized. See,
eg., U.S. patent application Ser. No. 08/508,786, now allowed, to
Conkling et al; PCT WO 9705261. All patents cited herein are
intended to be incorporated herein by reference, in their
entirety.
[0092] The QPTase recombinant DNA molecules and vectors used to
produce the transformed tobacco cells and plants of this invention
may further comprise a dominant selectable marker gene. Suitable
dominant selectable markers for use in tobacco include, inter alia,
antibiotic resistance genes encoding neomycin phosphotransferase
(NPTII) and hygromycin phosphotransferase (HPT). Other well-known
selectable markers that are suitable for use in tobacco include a
mutant dihydrofolate reductase gene that encodes
methotrexate-resistant dihydrofolate reductase. DNA vectors
containing suitable antibiotic resistance genes, and the
corresponding antibiotics, are commercially available.
[0093] 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.
Additionally, the positive selection techniques described by
Jefferson (e.g., WO 00055333; WO 09913085; U.S. Pat. Nos.
5,599,670; 5,432,081; and 5,268,463, hereby expressly incorporated
by reference in their entireties) can be used.
[0094] Methods of making recombinant plants of the present
invention, in general, involve first providing a plant cell capable
of regeneration (the plant cell typically residing in a tissue
capable of regeneration). The plant cell is then transformed with a
DNA construct comprising a transcription cassette of the present
invention (as described herein) and a recombinant plant is
regenerated from the transformed plant cell. As explained below,
the transforming step is carried out by techniques as are known in
the art, including but not limited to bombarding the plant cell
with microparticles carrying the transcription cassette, infecting
the cell with an Agrobacterium tumefaciens containing a Ti plasmid
carrying the transcription cassette or any other technique suitable
for the production of a transgenic plant.
[0095] Numerous Agrobacterium vector systems useful in carrying out
the present invention are known. For example, U.S. Pat. No.
4,459,355 discloses a method for transforming susceptible plants,
including dicots, with an Agrobacterium strain containing the Ti
plasmid. The transformation of woody plants with an Agrobacterium
vector is disclosed in U.S. Pat. No. 4,795,855. Further, U.S. Pat.
No. 4,940,838 to Schilperoort et al. discloses a binary
Agrobacterium vector (i.e., one in which the Agrobacterium contains
one plasmid having the vir region of a Ti plasmid but no T region,
and a second plasmid having a T region but no vir region) useful in
carrying out the present invention.
[0096] Microparticles suitable for the ballistic transformation of
a plant cell, carrying a DNA construct of the present invention,
are also useful for making the transformed plants described herein.
The microparticle is propelled into a plant cell to produce a
transformed plant cell and a plant is regenerated from the
transformed plant cell. Any suitable ballistic cell transformation
methodology and apparatus can be used in practicing the present
invention. Exemplary apparatus and procedures are disclosed in
Sanford and Wolf, U.S. Pat. No. 4,945,050, and in Christou et al.,
U.S. Pat. No. 5,015,580. When using ballistic transformation
procedures, the transcription cassette may be incorporated into a
plasmid capable of replicating in or integrating into the cell to
be transformed. Examples of microparticles suitable for use in such
systems include 1 to 5 .mu.m gold spheres. The DNA construct may be
deposited on the microparticle by any suitable technique, such as
by precipitation.
[0097] Plant species may be transformed with the DNA construct of
the present invention by the DNA-mediated transformation of plant
cell protoplasts. Plants may be subsequently regenerated from the
transformed protoplasts in accordance with procedures well known in
the art. Fusion of tobacco protoplasts with DNA-containing
liposomes or with DNA constructs via electroporation is known in
the art. (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)).
[0098] As used herein, transformation refers to the introduction of
exogenous DNA into cells so as to produce transgenic cells stably
transformed with the exogenous DNA. Transformed cells are induced
to regenerate intact 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. The stable presence and the
orientation of the QPTase sequence in transgenic tobacco plants can
be verified by Mendelian inheritance of the QPTase 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.
[0099] For example, to analyze the segregation of the transgene,
regenerated transformed plants (RO) may be grown to maturity,
tested for nicotine and/or TSNA levels, and selfed to produce
R.sub.1 plants. A percentage of R.sub.1 plants carrying the
transgene are homozygous for the transgene. To identify homozygous
R.sub.1 plants, transgenic R.sub.1 plants are grown to maturity and
selfed. Homozygous R.sub.1 plants will produce R.sub.2 progeny
where each progeny plant carries the transgene; progeny of
heterozygous R.sub.1, plants will segregate 3:1.
[0100] Any plant tissue capable of subsequent clonal propagation,
whether by organogenesis or embryogenesis, may be transformed with
a vector of the present invention. The term "organogenesis," as
used herein, means a process by which shoots and roots are
developed sequentially from meristematic centers; the term
"embryogenesis," as used herein, means a process by which shoots
and roots develop together in a concerted fashion (not
sequentially), whether from somatic cells or gametes. The
particular tissue chosen will vary depending on the clonal
propagation systems available for, and best suited to, the
particular species being transformed. Exemplary tissue targets
include leaf disks, pollen, embryos, cotyledons, hypocotyls, callus
tissue, existing meristematic tissue (e.g., apical meristems,
axillary buds, and root meristems) and induced meristem tissue
(e.g., cotyledon meristem and hypocotyl meristem).
[0101] Plants of the present invention may take a variety of forms.
The plants may be chimeras of transformed cells and non-transformed
cells; the plants may be clonal transformants (e.g., all cells
transformed to contain the transcription cassette); the plants may
comprise grafts of transformed and untransformed tissues (e.g., a
transformed root stock grafted to an untransformed scion in citrus
species). The transformed plants may be propagated by a variety of
means, such as by clonal propagation or classical breeding
techniques. For example, first generation (or T.sub.1) transformed
plants may be selfed to give homozygous second generation (or
T.sub.2) transformed plants and the T.sub.2 plants further
propagated through classical breeding techniques. A dominant
selectable marker (such as nptII) can be associated with the
transcription cassette to assist in breeding.
[0102] As used herein, a crop comprises a plurality of plants of
the present invention, and of the same genus, planted together in
an agricultural field. By "agricultural field" is meant a common
plot of soil or a greenhouse. Thus, the present invention provides
a method of producing a crop of plants having lowered QPTase
activity and thus having decreased nicotine and/or TSNA levels, as
compared to a similar crop of non-transformed plants of the same
species and variety. The examples that follow are set forth to
illustrate the present invention, and are not to be construed as
limiting thereof.
EXAMPLE 1
Isolation and Sequencing
[0103] TobRD2 cDNA (Conkling et. al., Plant Phys. 93, 1203 (1990))
was sequenced and is provided herein as SEQ ID NO: 1, and the
deduced amino acid sequence as SEQ ID NO: 2. The deduced amino acid
sequence was predicted to be a cytosolic protein. Although plant
QPTase genes had not yet been reported, comparisons of the NtPT1
amino acid sequence with the GenBank database (FIG. 3) revealed
limited sequence similarity to certain bacterial and other
proteins; quinolate phosphoribosyl transferase (QPTase) activity
has been demonstrated for the S. typhimurium, E. coli. and N.
tabacum genes. The NtQPT1-encoded QPTase has similarity to the
deduced peptide fragment encoded by an Arabidopsis EST (expression
sequence tag) sequence (Genbank Accession number F20096), which may
represent part of an Arabidopsis QPTase gene.
EXAMPLE 2
In-Situ Hybridization
[0104] To determine the spatial distribution of TobRD2 mRNA
transcripts in the various tissues of the root, in situ
hybridizations were performed in untransformed plants. In-situ
hybridizations of the antisense strand of TobRD2 to the TobRD2 mRNA
in root tissue was done using techniques as described in
Meyerowitz, Plant Mol. Biol. Rep. 5: 242 (1987) and Smith et al.,
Plant Mol. Biol. Rep. 5: 237 (1987). Seven day old tobacco
(Nicotania tabacum L.) seedling roots were fixed in
phosphate-buffered glutaraldehyde, embedded in Paraplast Plus
(Monoject Inc., St. Louis, Mo.) and sectioned at 8 micron thickness
to obtain transverse as well as longitudinal sections. Antisense
TobRD2 transcripts, synthesized In vitro in the presence of
.sup.35S-ATP, were used as probes. The labeled RNA was hydrolyzed
by alkaline treatment to yield 100 to 200 base mass average length
prior to use.
[0105] Hybridizations were done in 50% formamide for 16 hours at
42.degree. C., with approximately 5.times.10.sup.6
counts-per-minute (cpm)-labeled RNA per milliliter of hybridization
solution. After exposure, the slides were developed and visualized
under bright and dark field microscopy.
[0106] The hybridization signal was localized to the cortical layer
of cells in the roots. Comparison of both bright and dark field
images of the same sections localized TobRD2 transcripts to the
parenchymatous cells of the root cortex. No hybridization signal
was visible in the epidermis or the stele.
EXAMPLE 3
TobRD2 mRNA Levels in Nicl and Nic2 Tobacco Mutants and Correlation
to Nicotine Levels
[0107] TobRD2 steady-state mRNA levels were examined in Nicl and
Nic2 mutant tobacco plants. Nic1 and Nic2 are known to regulate
quinolate phosphoribosyl. transferase activity and putrescence
methyl-transferase activity, and are co-dominant regulators of
nicotine production. The present results are illustrated in FIGS.
5A and 5B and show that TobRD2 expression is regulated by Nicl and
Nic 2.
[0108] RNA was isolated from the roots of wild-type Burley 21
tobacco plants (Nicl/Nicl Nic2/Nic2), roots of Nicl-Burley 21
(nicl/nicl Nic2/Nic2), roots of Nic2-Burley 21 (Nicl/Nicl
nic2/nic2) and roots of Nic1-Nic2-Burley 21 (nicl/nicl
nic2/nic2).
[0109] Four Burley 21 tobacco lines were grown from seed in soil
for a month and transferred to hydroponic chambers in aerated
nutrient solution in a greenhouse for one month. These lines were
isogenic, except for the two low-nicotine loci, and had genotypes
of Nicl/Nicl Nic2/Nic2; nicl/nicl Nic2/Nic2; Nicl/Nicl nic2/nic2;
nicl/nicl nic2/nic2. Roots were harvested from about 20 plants for
each genotype and pooled for RNA isolation. Total RNA (1 .mu.g)
from each genotype was electrophoresed through a 1% agarose gel
containing 1.1M formaldehyde and transferred to a nylon membrane
according to Sambrook et al. (1989). The membranes were hybridized
with IP-labeled TobRD2 cDNA fragments. Relative intensity of TobRD2
transcripts were measured by densitometry. FIG. 5 (solid bars)
illustrates the relative transcript levels (compared to Nicl/Nicl
Nic2/Nic2) for each of the four genotypes. The relative nicotine
content (compared to Nicl/Nicl Nic2/Nic2) of the four genotypes is
shown by the hatched bars.
[0110] FIG. 5 graphically compares the relative steady state TobRD2
mRNA level, using the level found in wild-type Burley 21 (Nicl/Nicl
Nic2/Nic2) as the reference amount. TobRD2 mRNA levels in nicl/nicl
nic2/nic2 double mutants were approximately 25% that of wild-type
tobacco. FIG. 5B further compares the relative levels of nicotine
in the near isogenic lines of tobacco studied in this example
(solid bars indicate TobRD2 transcript level; hatched bars indicate
nicotine level). There was a close correlation between nicotine
levels and TobRD2 transcript levels.
EXAMPLE 4
Complementation of Bacterial Mutant Lacking QPTase with DNA of SEQ
ID NO: 1
[0111] Escherichia coli strain TH265 is a mutant lacking quinolate
phosphoribosyl transferase (nadC-), and therefore cannot grow on
media lacking nicotinic acids. TH265 cells were transformed with an
expression vector (pWS161) containing DNA of SEQ ID NO: 1, or
transformed with the expression vector (pKK233) only. Growth of the
transformed bacteria was compared to growth of TH265 (pKK233)
transformants, and to growth of the untransformed TH265
nadC-mutant. Growth was compared on ME minimal media (lacking
nicotinic acid) and on ME minimal media with added nicotinic
acid.
[0112] The E. coli strain with the QPTase mutation (nadC), TH265,
was kindly provided by Dr. K. T. Hughes (Hughes et al., J Bact.
175:479 (1993). The cells were maintained on LB media and competent
cells prepared as described in Sambrook et al (1989). An expression
plasmid was constructed in pKK2233 (Brosius, 1984) with the TobRD2
cDNA cloned under the control of the Tac promoter. The resulting
plasmid, pWS161, was transformed into TH265 cells. The transformed
cells were then plated on minimal media (Vogel and Bonner, 1956)
agar plates with or without nicotinic acid (0.0002%) as supplement.
TH265 cells alone and TH265 transformed with pKK2233 were plated on
similar plates for use as controls.
[0113] Results are shown in FIG. 4. Only the TH265 transformed with
DNA of SEQ ID NO: 1 grew in media lacking nicotinic acid. These
results show that expression of DNA of SEQ ID NO: 1 in TH265
bacterial cells conferred the NadC+ phenotype on these cells,
confirming that this sequence encodes QPTase. The TobRD2
nomenclature was thus changed to NtQPT1.
EXAMPLE 5
Transformation of Tobacco Plants
[0114] DNA of SEQ ID NO: 1, in antisense orientation, is operably
linked to a plant promoter (CaMV 35S or TobRD2 root-cortex specific
promoter) to produce two different DNA cassettes: CaMV35S
promoter/antisense SEQ ID NO: 1 and TobRD2 promoter/antisense SEQ
ID NO: 1.
[0115] A wild-type tobacco line and a low-nicotine tobacco line are
selected for transformation, e.g., wild-type Burley 21 tobacco
(Nic1+/Nic2+) and homozygous Nicl-/Nic2-Burley 21. A plurality of
tobacco plant cells from each line are transformed using each of
the DNA cassettes. Transformation is conducted using an
Agrobacterium vector, e.g., an Agrobacterium-binary vector carrying
Ti-border sequences and the nptII gene (conferring resistance to
kanamycin and under the control of the nos promoter (nptII)).
[0116] Transformed cells are selected and regenerated into
transgenic tobacco plants called R.sub.0. The R.sub.0 plants are
grown to maturity and tested for levels of nicotine; a subset of
the transformed tobacco plants exhibit significantly lower levels
of nicotine compared to non-transformed control plants.
[0117] R.sub.0 plants are then selfed and the segregation of the
transgene is analyzed in next generation, the R.sub.1 progeny.
R.sub.1 progeny are grown to maturity and selfed; segregation of
the transgene among R.sub.2 progeny indicates which R.sub.1 plants
are homozygous for the transgene.
EXAMPLE 6
Tobacco Having Reduced Nicotine Levels
[0118] Tobacco of the variety Burley 21 LA was transformed with the
binary Agrobacterium vector pYTY32 to produce a low nicotine
tobacco variety, Vector 21-41. The binary vector pYTY32 carried the
2.0 kb NtQPT1 root-cortex-specific promoter driving antisense
expression of the NtQPT1 cDNA and the nopaline synthase (nos) 3'
termination sequences from Agrobacterium tumefaciens T-DNA. The
selectable marker for this construct was neomycin
phosphotransferase (nptII) from E. coli Tn5, which confers
resistance to kanamycin; the expression of nptII was directed by
the nos promoter from Agrobacterium tumefaciens T-DNA. Transformed
cells, tissues and seedlings were selected by their ability to grow
on Murashige-Skoog (MS) medium containing 300 .mu.g/ml kanamycin.
Burley 21 LA is a variety of Burley 21 with substantially reduced
levels of nicotine as compared with Burley 21 (i.e., Burley 21 LA
has 8% the nicotine levels of Burley 21, see Legg et al., Can J
Genet Cytol, 13:287-91 (1971); Legg et al., J Hered, 60:213-17
(1969))
[0119] One hundred independent pYTY32 transformants of Burley 21 LA
(T.sub.0) were allowed to self. Progeny of the selfed plants
(T.sub.1) were germinated on medium containing kanamycin and the
segregation of kanamycin resistance scored. T.sub.1 progeny
segregating 3:1 resulted from transformation at a single locus and
were subjected to further analysis.
[0120] Nicotine levels of T.sub.1 progeny segregating 3:1 were
measured qualitatively using a micro-assay technique. Approximately
200 mg fresh tobacco leaves were collected and ground in 1 ml
extraction solution (extraction solution: 1 ml Acetic acid in 100
ml H.sub.2O). Homogenate was centrifuged for 5 min at
14,000.times.g and supernatant removed to a clean tube, to which
the following reagents were added: 100 .mu.L NH.sub.4OAC (5 g/100
ml H.sub.2O+50 .mu.L Brij 35); 500 .mu.L Cyanogen Bromide (Sigma
C-6388, 0.5 g/100 ml H.sub.2O+50 .mu.L Brij 35); 400 .mu.L Aniline
(0.3 ml buffered Aniline in 100 ml NH.sub.4OAC+50 .mu.L Brij 35). A
nicotine standard stock solution of 10 mg/ml in extraction solution
was prepared and diluted to create a standard series for
calibration. Absorbance at 460 nm was read and nicotine content of
test samples were determined using the standard calibration
curve.
[0121] T.sub.1 progeny that had less than 10% of the nicotine
levels of the Burley 21 LA parent were allowed to self to produce
T.sub.2 progeny. Homozygous T.sub.2 progeny were identified by
germinating seeds on medium containing kanamycin and selecting
clones in which 100% of the progeny were resistant to kanamycin
(i.e., segregated 4:0; heterozygous progeny would segregate 3:1).
Nicotine levels in homozygous and heterozygous T.sub.2 progeny were
qualitatively determined using the micro-assay and again showed
levels less than 10% of the Burley 21 LA parent. Leaf samples of
homozygous T.sub.2 progeny were sent to the Southern Research and
Testing Laboratory in Wilson, N.C. for quantitative analysis of
nicotine levels using Gas Chromatography/Flame Ionization Detection
(GC/FID). Homozygous T.sub.2 progeny of transformant #41 gave the
lowest nicotine levels (.about.70 ppm), and this transformant was
designated as "Vector 21-41."
[0122] Vector 21-41 plants were allowed to self-cross, producing
T.sub.3 progeny. T.sub.3 progeny were grown and nicotine levels
assayed qualitatively and quantitatively. T.sub.3 progeny were
allowed to self-cross, producing T.sub.4 progeny. Samples of the
bulked seeds of the T.sub.4 progeny were grown and nicotine levels
tested.
[0123] In general, Vector 21-41 is similar to Burley 21 LA in all
assessed characteristics, with the exception of alkaloid content
and total reducing sugars (e.g., nicotine and nor-nicotine). Vector
21-41 may be distinguished from the parent Burley 21 LA by its
substantially reduced content of nicotine, nor-nicotine and total
alkaloids. As shown below, total alkaloid concentrations in Vector
21-41 are significantly reduced to approximately relative to the
levels in the parent Burley 21 LA, and nicotine and nor-nicotine
concentrations show dramatic reductions in Vector 21-41 as compared
with Burley 21 LA. Vector 21-41 also has significantly higher
levels of reducing sugars as compared with Burley 21 LA.
[0124] Field trials of Vector 21-41 T.sub.4 progeny were performed
at the Central Crops Research Station (Clayton, N.C.) and compared
to the Burley 21 LA parent. The design was three treatments (Vector
21-41, a Burley 21 LA transformed line carrying only the NtQPT1
promoter [Promoter-Control], and untransformed Burley 21 LA
[Wild-type]), 15 replicates, 10 plants per replicate. The following
agronomic traits were measured and compared: days from transplant
to flowering; height at flowering; leaf number at flowering; yield;
percent nicotine; percent nor-nicotine; percent total nitrogen; and
percent reducing sugars.
EXAMPLE 7
Low Nicotine and Nitrosamine Blended Tobacco
[0125] The following example describes several ways to create
tobacco products having specific amounts of nicotine and/or TSNAs
through blending. Some blending approaches begin with tobacco
prepared from varieties that have extremely low amounts of nicotine
and/or TSNAs. By blending prepared tobacco from a low nicotine/TSNA
variety (e.g., undetectable levels of nicotine and/or TSNAs ) with
a conventional tobacco (e.g., Burley, which has 30,000 parts per
million (ppm) nicotine and 8,000 parts per billion (ppb) TSNA;
Flue-Cured, which has 20,000 ppm nicotine and 300 ppb TSNA; and
Oriental, which has 10,000 ppm nicotine and 100 ppb TSNA), tobacco
products having virtually any desired amount of nicotine and/or
TSNAs can be manufactured. Tobacco products having various amounts
of nicotine and/or TSNAs can be incorporated into tobacco use
cessation kits and programs to help tobacco users reduce or
eliminate their dependence on nicotine and reduce the carcinogenic
potential.
[0126] For example, a step 1 tobacco product can be comprised of
approximately 25% low nicotine/TSNA tobacco and 75% conventional
tobacco; a step 2 tobacco product can be comprised of approximately
50% low nicotine/TSNA tobacco and 50% conventional tobacco; a step
3 tobacco product can be comprised of approximately 75% low
nicotine/TSNA tobacco and 25% conventional tobacco; and a step 4
tobacco product can be comprised of approximately 100% low
nicotine/TSNA tobacco and 0% conventional tobacco. A tobacco use
cessation kit can comprise an amount of tobacco product from each
of the aforementioned blends to satisfy a consumer for a single
month program. That is, if the consumer is a one pack a day smoker,
for example, a single month kit would provide 7 packs from each
step, a total of 28 packs of cigarettes. Each tobacco use cessation
kit would include a set of instructions that specifically guide the
consumer through the step-by-step process. Of course, tobacco
products having specific amounts of nicotine and/or TSNAs would be
made available in conveniently sized amounts (e.g., boxes of
cigars, packs of cigarettes, tins of snuff, and pouches or twists
of chew) so that consumers could select the amount of nicotine
and/or TSNA they individually desire. There are many ways to obtain
various low nicotine/low TSNA tobacco blends using the teachings
described herein and the following is intended merely to guide one
of skill in the art to one possible approach.
[0127] To obtain a step 1 tobacco product, which is a 25% low
nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA tobacco can be mixed with conventional Burley,
Flue-cured, or Oriental in a 25%/75% ratio respectively to obtain a
Burly tobacco product having 22,500 ppm nicotine and 6,000 ppb
TSNA, a Flue-cured product having 15,000 ppm nicotine and 225 ppb
TSNA, and an Oriental product having 7,500 ppm nicotine and 75 ppb
TSNA. Similarly, to obtain a step 2 product, which is 50% low
nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA tobacco can be mixed with conventional Burley,
Flue-cured, or Oriental in a 50%/50% ratio respectively to obtain a
Burly tobacco product having 15,000 ppm nicotine and 4,000 ppb
TSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb
TSNA, and an Oriental product having 5000 ppm nicotine and 50 ppb
TSNA. Further, a step 3 product, which is a 75%/25% low
nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA tobacco can be mixed with conventional Burley,
Flue-cured, or Oriental in a 75%/25% ratio respectively to obtain a
Burly tobacco product having 7,500 ppm nicotine and 2,000 ppb TSNA,
a Flue-cured product having 5,000 ppm nicotine and 75 ppb TSNA, and
an Oriental product having 2,500 ppm nicotine and 25 ppb TSNA.
[0128] It should be appreciated that tobacco products are often a
blend of many different types of tobaccos, which were grown in many
different parts of the world under various growing conditions. As a
result, the amount of nicotine and TSNAs will differ from crop to
crop. Nevertheless, by using conventional techniques one can easily
determine an average amount of nicotine and TSNA per crop used to
create a desired blend. By adjusting the amount of each type of
tobacco that makes up the blend one of skill can balance the amount
of nicotine and/or TSNA with other considerations such as
appearance and flavor, and smokeability. In this manner, a variety
of types of tobacco products having varying level of nicotine
and/or nitrosamine, as well as varying appearance and flavor and
smokability can be created.
[0129] Although the invention has been described with reference to
embodiments and examples, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims. All references cited herein are hereby expressly
incorporated by reference.
Sequence CWU 1
1
58 1 1399 DNA Nicotiana tabacum 1 caaaaactat tttccacaaa attcatttca
caaccccccc aaaaaaaaac catgtttaga 60 gctattcctt tcactgctac
agtgcatcct tatgcaatta cagctccaag gttggtggtg 120 aaaatgtcag
caatagccac caagaataca agagtggagt cattagaggt gaaaccacca 180
gcacacccaa cttatgattt aaaggaagtt atgaaacttg cactctctga agatgctggg
240 aatttaggag atgtgacttg taaggcgaca attcctcttg atatggaatc
cgatgctcat 300 tttctagcaa aggaagacgg gatcatagca ggaattgcac
ttgctgagat gatattcgcg 360 gaagttgatc cttcattaaa ggtggagtgg
tatgtaaatg atggcgataa agttcataaa 420 ggcttgaaat ttggcaaagt
acaaggaaac gcttacaaca ttgttatagc tgagagggtt 480 gttctcaatt
ttatgcaaag aatgagtgga atagctacac taactaagga aatggcagat 540
gctgcacacc ctgcttacat cttggagact aggaaaactg ctcctggatt acgtttggtg
600 gataaatggg cggtattgat cggtgggggg aagaatcaca gaatgggctt
atttgatatg 660 gtaatgataa aagacaatca catatctgct gctggaggtg
tcggcaaagc tctaaaatct 720 gtggatcagt atttggagca aaataaactt
caaatagggg ttgaggttga aaccaggaca 780 attgaagaag tacgtgaggt
tctagactat gcatctcaaa caaagacttc gttgactagg 840 ataatgctgg
acaatatggt tgttccatta tctaacggag atattgatgt atccatgctt 900
aaggaggctg tagaattgat caatgggagg tttgatacgg aggcttcagg aaatgttacc
960 cttgaaacag tacacaagat tggacaaact ggtgttacct acatttctag
tggtgccctg 1020 acgcattccg tgaaagcact tgacatttcc ctgaagatcg
atacagagct cgcccttgaa 1080 gttggaaggc gtacaaaacg agcatgagcg
ccattacttc tgctataggg ttggagtaaa 1140 agcagctgaa tagctgaaag
gtgcaaataa gaatcatttt actagttgtc aaacaaaaga 1200 tccttcactg
tgtaatcaaa caaaaagatg taaattgctg gaatatctca gatggctctt 1260
ttccaacctt attgcttgag ttggtaattt cattatagct ttgttttcat gtttcatgga
1320 atttgttaca atgaaaatac ttgatttata agtttggtgt atgtaaaatt
ctgtgttact 1380 tcaaatattt tgagatgtt 1399 2 351 PRT Nicotiana
tabacum 2 Met Phe Arg Ala Ile Pro Phe Thr Ala Thr Val His Pro Tyr
Ala Ile 1 5 10 15 Thr Ala Pro Arg Leu Val Val Lys Met Ser Ala Ile
Ala Thr Lys Asn 20 25 30 Thr Arg Val Glu Ser Leu Glu Val Lys Pro
Pro Ala His Pro Thr Tyr 35 40 45 Asp Leu Lys Glu Val Met Lys Leu
Ala Leu Ser Glu Asp Ala Gly Asn 50 55 60 Leu Gly Asp Val Thr Cys
Lys Ala Thr Ile Pro Leu Asp Met Glu Ser 65 70 75 80 Asp Ala His Phe
Leu Ala Lys Glu Asp Gly Ile Ile Ala Gly Ile Ala 85 90 95 Leu Ala
Glu Met Ile Phe Ala Glu Val Asp Pro Ser Leu Lys Val Glu 100 105 110
Trp Tyr Val Asn Asp Gly Asp Lys Val His Lys Gly Leu Lys Phe Gly 115
120 125 Lys Val Gln Gly Asn Ala Tyr Asn Ile Val Ile Ala Glu Arg Val
Val 130 135 140 Leu Asn Phe Met Gln Arg Met Ser Gly Ile Ala Thr Leu
Thr Lys Glu 145 150 155 160 Met Ala Asp Ala Ala His Pro Ala Tyr Ile
Leu Glu Thr Arg Lys Thr 165 170 175 Ala Pro Gly Leu Arg Leu Val Asp
Lys Trp Ala Val Leu Ile Gly Gly 180 185 190 Gly Lys Asn His Arg Met
Gly Leu Phe Asp Met Val Met Ile Lys Asp 195 200 205 Asn His Ile Ser
Ala Ala Gly Gly Val Gly Lys Ala Leu Lys Ser Val 210 215 220 Asp Gln
Tyr Leu Glu Gln Asn Lys Leu Gln Ile Gly Val Glu Val Glu 225 230 235
240 Thr Arg Thr Ile Glu Glu Val Arg Glu Val Leu Asp Tyr Ala Ser Gln
245 250 255 Thr Lys Thr Ser Leu Thr Arg Ile Met Leu Asp Asn Met Val
Val Pro 260 265 270 Leu Ser Asn Gly Asp Ile Asp Val Ser Met Leu Lys
Glu Ala Val Glu 275 280 285 Leu Ile Asn Gly Arg Phe Asp Thr Glu Ala
Ser Gly Asn Val Thr Leu 290 295 300 Glu Thr Val His Lys Ile Gly Gln
Thr Gly Val Thr Tyr Ile Ser Ser 305 310 315 320 Gly Ala Leu Thr His
Ser Val Lys Ala Leu Asp Ile Ser Leu Lys Ile 325 330 335 Asp Thr Glu
Leu Ala Leu Glu Val Gly Arg Arg Thr Lys Arg Ala 340 345 350 3 1053
DNA Nicotiana tabacum 3 atgtttagag ctattccttt cactgctaca gtgcatcctt
atgcaattac agctccaagg 60 ttggtggtga aaatgtcagc aatagccacc
aagaatacaa gagtggagtc attagaggtg 120 aaaccaccag cacacccaac
ttatgattta aaggaagtta tgaaacttgc actctctgaa 180 gatgctggga
atttaggaga tgtgacttgt aaggcgacaa ttcctcttga tatggaatcc 240
gatgctcatt ttctagcaaa ggaagacggg atcatagcag gaattgcact tgctgagatg
300 atattcgcgg aagttgatcc ttcattaaag gtggagtggt atgtaaatga
tggcgataaa 360 gttcataaag gcttgaaatt tggcaaagta caaggaaacg
cttacaacat tgttatagct 420 gagagggttg ttctcaattt tatgcaaaga
atgagtggaa tagctacact aactaaggaa 480 atggcagatg ctgcacaccc
tgcttacatg ttggagacta ggaaaactgc tcctggatta 540 cgtttggtgg
ataaatgggc ggtattgatc ggtgggggga agaatcacag aatgggctta 600
tttgatatgg taatgataaa agacaatcac atatctgctg ctggaggtgt cggcaaagct
660 ctaaaatctg tggatcagta tttggagcaa aataaacttc aaataggggt
tgaggttgaa 720 accaggacaa ttgaagaagt acgtgaggtt ctagactatg
catctcaaac aaagacttcg 780 ttgactagga taatgctgga caatatggtt
gttccattat ctaacggaga tattgatgta 840 tccatgctta aggaggctgt
agaattgatc aatgggaggt ttgatacgga ggcttcagga 900 aatgttaccc
ttgaaacagt acacaagatt ggacaaactg gtgttaccta catttctagt 960
ggtgccctga cgcattccgt gaaagcactt gacatttccc tgaagatcga tacagagctc
1020 gcccttgaag ttggaaggcg tacaaaacga gca 1053 4 50 PRT Nicotiana
tabacum 4 Met Phe Arg Ala Ile Pro Phe Thr Ala Thr Val His Pro Tyr
Ala Ile 1 5 10 15 Thr Ala Pro Arg Leu Val Val Lys Met Ser Ala Ile
Ala Thr Lys Asn 20 25 30 Thr Arg Val Glu Ser Leu Glu Val Lys Pro
Pro Ala His Pro Thr Tyr 35 40 45 Asp Leu 50 5 13 PRT Rhodospirillum
rubrum 5 Arg Pro Asn His Pro Val Ala Ala Leu Ser Phe Ala Ile 1 5 10
6 10 PRT Mycobacterium lepre 6 Leu Ser Asp Cys Glu Phe Asp Ala Ala
Arg 1 5 10 7 22 PRT Salmonella typhimurium 7 Pro Pro Arg Arg Asn
Pro Asp Asp Arg Asp Ala Leu Leu Arg Ile Asn 1 5 10 15 Leu Asp Ile
Ala Ala Val 20 8 22 PRT Escherichia coli 8 Pro Pro Arg Arg Asn Pro
Asp Thr Arg Asp Glu Leu Leu Arg Ile Asn 1 5 10 15 Leu Asp Ile Gly
Ala Val 20 9 25 PRT Homo sapiens 9 Asp Glu Gly Ala Leu Leu Leu Pro
Pro Val Thr Leu Ala Ala Leu Val 1 5 10 15 Asp Ser Trp Leu Arg Glu
Asp Cys Gly 20 25 10 26 PRT Saccharomyces cerevisiae 10 Pro Val Tyr
Glu His Leu Leu Pro Val Asn Gly Ala Trp Arg Gln Asp 1 5 10 15 Val
Thr Asn Trp Leu Ser Glu Asp Val Ser 20 25 11 46 PRT Nicotiana
tabacum 11 Lys Glu Val Met Lys Leu Ala Leu Ser Glu Asp Ala Gly Asn
Leu Gly 1 5 10 15 Asp Val Thr Cys Lys Ala Thr Ile Pro Leu Asp Met
Glu Ser Asp Ala 20 25 30 His Phe Leu Ala Lys Glu Asp Gly Ile Ile
Ala Gly Ile Ala 35 40 45 12 29 PRT Rhodospirillum rubrum 12 Asp Ala
Val Arg Arg Ala Leu Arg Ala Ile Ser Thr Ala Ala Thr Arg 1 5 10 15
Ala His Arg Phe Val Arg Gln Pro Leu Leu Gly Cys Ala 20 25 13 38 PRT
Mycobacterium lepre 13 Asp Thr Ile Arg Arg His Leu Arg Tyr Gly Leu
Ile Thr Gln Val Ala 1 5 10 15 Gly Thr Val Val Thr Gly Ser Met Val
Pro Arg Pro Val Ile Ala Gly 20 25 30 Val Asp Val Ala Leu Leu 35 14
38 PRT Nicotiana tabacum 14 Ala Gln Ala Leu Arg Glu Asp Leu Gly Gly
Glu Val Asp Ala Gly Asn 1 5 10 15 Ile Ala Gln Leu Leu Ala Thr Gln
Ala His Thr Val Ile Thr Arg Asp 20 25 30 Val Phe Cys Gly Lys Arg 35
15 37 PRT Salmonella typhimurium 15 Ala Gln Ala Leu Arg Glu Asp Leu
Gly Gly Thr Val Asp Ala Asn Asn 1 5 10 15 Ile Ala Leu Leu Glu Asn
Ser Arg His Thr Val Ile Thr Arg Asn Val 20 25 30 Phe Cys Gly Lys
Arg 35 16 27 PRT Homo sapiens 16 Leu Asn Tyr Ala Ala Leu Val Ser
Gly Ala Gly Pro Gln Ala Ala Leu 1 5 10 15 Trp Ala Lys Ser Pro Val
Leu Ala Gly Gln Pro 20 25 17 28 PRT Sacharomyces cerevisiae 17 Phe
Asp Phe Gly Gly Tyr Val Val Gly Ser Asp Leu Lys Glu Ala Asn 1 5 10
15 Leu Tyr Cys Lys Gln Asp Met Leu Cys Gly Val Pro 20 25 18 43 PRT
Nicotiana tabacum 18 Leu Ala Glu Met Ile Phe Ala Glu Val Asp Pro
Ser Leu Lys Val Glu 1 5 10 15 Trp Tyr Val Asn Asp Gly Asp Lys Val
His Lys Gly Leu Lys Phe Gly 20 25 30 Lys Val Gln Gly Asn Ala Tyr
Asn Ile Val Ile 35 40 19 34 PRT Rhodospirillum rubrum 19 Arg Ser
Ala Phe Ala Leu Leu Asp Asp Thr Val Thr Phe Thr Thr Pro 1 5 10 15
Leu Glu Ala Glu Ile Ala Ala Gln Thr Val Ala Glu Ala Ala Arg Thr 20
25 30 Leu Ala 20 35 PRT Mycobacterium lepre 20 Val Leu Asp Val Phe
Gly Val Asp Gly Tyr Arg Val Leu Tyr Arg Glu 1 5 10 15 Ala Arg Leu
Gln Ser Gln Pro Leu Leu Thr Val Gln Ala Ala Arg Gly 20 25 30 Leu
Leu Thr 35 21 36 PRT Salmonella typhimurium 21 Trp Val Glu Val Phe
Ile Gln Leu Ala Gly Asp Asp Val Arg Leu Thr 1 5 10 15 His Asp Ala
Ile Ala Asn Gln Thr Val Phe Glu Leu Asn Pro Ala Arg 20 25 30 Val
Leu Leu Thr 35 22 37 PRT Escherichia coli 22 Trp Val Glu Val Phe
Ile Gln Leu Ala Gly Asp Asp Val Thr Ile Ile 1 5 10 15 His Asp Val
Ile Asn Ala Asn Gln Ser Leu Phe Glu Leu Glu Pro Ser 20 25 30 Arg
Val Leu Leu Thr 35 23 36 PRT Homo sapiens 23 Phe Phe Asp Ala Ile
Phe Thr Gln Leu Asn Cys Gln Val Ser Phe Leu 1 5 10 15 Pro Glu Ser
Leu Val Pro Val Ala Arg Val Ala Glu Val Arg Pro His 20 25 30 Asp
Leu Leu Leu 35 24 40 PRT Saccharomyces cerevisiae 24 Phe Ala Trp
Val Phe Asn Gln Cys Glu Leu Gln Val Glu Leu Phe Lys 1 5 10 15 Glu
Ser Phe Leu Glu Pro Ser Lys Asn Asp Ser Gly Lys Ile Val Val 20 25
30 Ala Lys Ile Thr Pro Lys Leu Leu 35 40 25 46 PRT Nicotiana
tabacum 25 Ala Glu Arg Val Val Leu Asn Phe Met Gln Arg Met Ser Gly
Ile Ala 1 5 10 15 Thr Leu Thr Lys Glu Met Ala Asp Ala Ala His Pro
Ala Tyr Ile Leu 20 25 30 Glu Thr Arg Lys Thr Ala Pro Gly Leu Arg
Leu Val Asp Lys 35 40 45 26 24 PRT Rhodospirillum rubrum 26 Thr Ala
Leu Gly His Leu Arg Arg Arg Phe Gly Ala Ile His Thr Arg 1 5 10 15
Arg Leu Thr Cys Thr Gly Leu Glu 20 27 25 PRT Mycobacterium lepre 27
Thr Met Val Cys His Met Val Val Ala Trp Val Ala Val Arg Gly Thr 1 5
10 15 Lys Lys Ile Arg Asp Leu Ala Leu Gln 20 25 28 29 PRT
Salmonella typhimurium 28 Gly Thr Ala Val Thr Leu Val Ala Ser Glu
Val Arg Arg Tyr Val Gly 1 5 10 15 Leu Leu Gly Thr Gln Thr Gln Leu
Asp Leu Thr Ala Leu 20 25 29 31 PRT Escherichia coli 29 Gly Pro Thr
Ala Val Thr Leu Val Ala Ser Lys Val Arg His Tyr Val 1 5 10 15 Glu
Leu Leu Glu Gly Thr Asn Thr Gln Leu Asp Leu Ser Ala Leu 20 25 30 30
31 PRT Homo sapiens 30 Gly Ala Thr Leu Ala Arg Cys Ser Ala Ala Ala
Ala Ala Val Glu Ala 1 5 10 15 Ala Arg Gly Ala Gly Trp Thr Gly His
Val Ala Gly Thr Phe Glu 20 25 30 31 32 PRT Saccharomyces cerevisiae
31 Thr Ala Ile Leu Ser Arg Ser Thr Ala Ser His Lys Ile Ile Ser Leu
1 5 10 15 Ala Arg Ser Thr Gly Tyr Lys Gly Thr Ile Ala Gly Thr Arg
Leu Glu 20 25 30 32 50 PRT Nicotiana tabacum 32 Trp Ala Val Leu Ile
Gly Gly Gly Lys Asn His Arg Met Gly Leu Phe 1 5 10 15 Asp Met Val
Met Ile Lys Asp Asn His Ile Ser Ala Ala Gly Gly Val 20 25 30 Gly
Lys Ala Leu Lys Ser Val Asp Gln Tyr Leu Glu Gln Asn Lys Leu 35 40
45 Gln Ile 50 33 26 PRT Rhodospirillum rubrum 33 Tyr Arg Cys Ser
Phe Asp Ala Leu Ala Val Ala Ser Ala Ser Arg Ala 1 5 10 15 Arg Ala
Gly Val Gly His Met Val Arg Ile 20 25 34 26 PRT Mycobacterium lepre
34 Tyr Arg Val Val Leu Gly Thr Ala Leu Val Ala Val Ser Val Asp Arg
1 5 10 15 Ala Arg Ala Ala Ala Pro Glu Leu Pro Cys 20 25 35 25 PRT
Salmonella typhimurium 35 Tyr Cys Ala Leu Thr Ala Phe Leu Ile Ser
Ser Arg Gln Val Glu Lys 1 5 10 15 Ala Phe Trp His Pro Asp Ala Pro
Val 20 25 36 25 PRT Escherichia coli 36 Tyr Cys Ala Leu Ser Ala Phe
Leu Ile Ser Ser Arg Gln Val Glu Lys 1 5 10 15 Ala Ser Trp His Pro
Asp Ala Pro Val 20 25 37 34 PRT Homo sapiens 37 Tyr Gly Leu Val Ala
Ala Ser Tyr Asp Gly Gly Leu Val Met Leu Asp 1 5 10 15 Val Val Pro
Pro Phe Lys Val Arg Ala Ala Arg Gln Ala Ala Asp Phe 20 25 30 Ala
Leu 38 31 PRT Saccharomyces cerevisiae 38 Tyr Ser Met Val Cys Asp
Thr Tyr Asp Ser Ser Met Leu Asp Trp Thr 1 5 10 15 Ser Ile Thr Asn
Val Asn Ala Arg Ala Val Cys Gly Phe Ala Val 20 25 30 39 50 PRT
Nicotiana tabacum 39 Gly Val Glu Val Glu Thr Arg Thr Ile Glu Glu
Val Arg Glu Val Leu 1 5 10 15 Asp Tyr Ala Ser Gln Thr Lys Thr Ser
Leu Thr Arg Ile Met Leu Asp 20 25 30 Asn Met Val Val Pro Leu Ser
Asn Gly Asp Ile Asp Val Ser Met Leu 35 40 45 Lys Glu 50 40 21 PRT
Rhodospirillum rubrum 40 Glu Ile Leu Gln Leu Ala Ala Val Gly Gly
Ala Glu Val Val Leu Asp 1 5 10 15 Ala Pro Thr Thr Arg 20 41 25 PRT
Mycobacterium lepre 41 Glu Ser Leu Gln Leu Asp Ala Met Ala Glu Glu
Pro Glu Leu Leu Phe 1 5 10 15 Val Trp Gln Thr Gln Val Ala Val Gln
20 25 42 21 PRT Salmonella typhimurium 42 Glu Asn Leu Asp Glu Leu
Asp Asp Ala Lys Gly Ala Asp Ile Phe Asn 1 5 10 15 Thr Asp Gln Met
Arg 20 43 18 PRT Escherichia coli 43 Glu Asn Leu Leu Asp Ala Lys
Gly Ala Asp Ile Phe Glu Thr Glu Gln 1 5 10 15 Met Arg 44 28 PRT
Homo sapiens 44 Lys Cys Ser Ser Leu Gln Val Gln Ala Ala Glu Gly Ala
Asp Leu Val 1 5 10 15 Leu Phe Lys Pro Glu Glu Leu His Pro Thr Ala
Thr 20 25 45 26 PRT Saccharomyces cerevisiae 45 Lys Ile Cys Leu Ser
Glu Asp Ala Thr Ala Ile Glu Gly Ala Asp Val 1 5 10 15 Phe Lys Gly
Asp Gly Leu Lys Cys Ala Gln 20 25 46 46 PRT Nicotiana tabacum 46
Ala Val Glu Leu Ile Asn Gly Arg Phe Asp Thr Glu Ala Ser Gly Asn 1 5
10 15 Val Thr Leu Glu Thr Val His Lys Ile Gly Gln Thr Gly Val Thr
Tyr 20 25 30 Ile Ser Ser Gly Ala Leu Thr His Ser Val Lys Ala Leu
Asp 35 40 45 47 20 PRT Rhodospirillum rubrum 47 Asp Met Val Ala Leu
Val Gly Ser Asp Ile Ala Ala Leu Ala Glu Ser 1 5 10 15 Asp Val Thr
Thr 20 48 29 PRT Mycobacterium lepre 48 Arg Arg Asp Ile Arg Ala Pro
Thr Val Leu Leu Ser Gly Leu Ser Asn 1 5 10 15 Ala Ala Ile Tyr Ala
Gly Asp Tyr Leu Ala Val Arg Ile 20 25 49 21 PRT Salmonella
typhimurium 49 Lys Arg Val Gln Ala Arg Leu Val Ala Glu Leu Arg Glu
Phe Ala Glu 1 5 10 15 Asp Phe Val Gly Arg 20 50 20 PRT Escherichia
coli 50 Lys Arg Thr Lys Ala Leu Leu Val Asp Lys Leu Arg Glu Phe Ala
Glu 1 5 10 15 Asp Phe Val Gln 20 51 33 PRT Homo sapiens 51 Leu Lys
Ala Gln Phe Pro Ser Val Ala Val Glu Ala Gly Ile Thr Asp 1 5 10 15
Asn Leu Pro Gln Phe Cys Gly
Pro His Ile Asp Val Met Met Gln Ala 20 25 30 Pro 52 44 PRT
Saccharomyces cerevisiae 52 Ser Leu Lys Asn Lys Trp Asn Gly Lys Lys
His Phe Leu Leu Glu Cys 1 5 10 15 Gly Leu Asn Asp Asn Leu Glu Glu
Tyr Leu Cys Asp Asp Ile Asp Ile 20 25 30 Tyr Thr Ser Ser Ile His
Gln Gly Thr Pro Val Ile 35 40 53 20 PRT Nicotiana tabacum 53 Ile
Ser Lys Leu Ile Asp Thr Glu Leu Ala Leu Glu Val Gly Arg Arg 1 5 10
15 Thr Lys Arg Ala 20 54 12 PRT Rhodospirillum rubrum 54 Gly Asp
Val Val Ala Pro Pro Lys Ala Glu Arg Ala 1 5 10 55 6 PRT Salmonella
typhimurium 55 Leu Ser Met Arg Phe Cys 1 5 56 6 PRT Escherichia
coli 56 Leu Ser Met Arg Phe Arg 1 5 57 11 PRT Homo sapiens 57 Phe
Leu Phe Lys Val Ala Pro Val Pro Ile His 1 5 10 58 4 PRT
Saccharomyces cerevisiae 58 Phe Leu Ala His 1
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