U.S. patent application number 11/293680 was filed with the patent office on 2006-07-20 for method of reducing the harmful effects of orally or transdermally delivered nicotine.
Invention is credited to Anthony Albino, Mark Conkling, Wendy Jin.
Application Number | 20060157072 11/293680 |
Document ID | / |
Family ID | 37890300 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060157072 |
Kind Code |
A1 |
Albino; Anthony ; et
al. |
July 20, 2006 |
Method of reducing the harmful effects of orally or transdermally
delivered nicotine
Abstract
The present invention generally relates to the reduction of the
harmful effects of orally or transdermally delivered nicotine in
conventional tobacco-use cessation programs. More specifically,
embodiments concern methods of reducing the harmful effects of
nicotine intake, associated with conventional tobacco-use cessation
programs, by providing tobacco products, which contain a reduced
amount of nicotine and/or tobacco specific nitrosamines
(TSNAs).
Inventors: |
Albino; Anthony; (New York,
NY) ; Conkling; Mark; (Chapel Hill, NC) ; Jin;
Wendy; (Chapel Hill, NC) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
37890300 |
Appl. No.: |
11/293680 |
Filed: |
December 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US04/16958 |
May 27, 2004 |
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11293680 |
Dec 2, 2005 |
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PCT/US05/10733 |
Mar 29, 2005 |
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11293680 |
Dec 2, 2005 |
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11077752 |
Mar 10, 2005 |
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11293680 |
Dec 2, 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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60475945 |
Jun 4, 2003 |
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60557929 |
Mar 30, 2004 |
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60297154 |
Jun 8, 2001 |
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60680283 |
May 11, 2005 |
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Current U.S.
Class: |
131/270 ;
131/352 |
Current CPC
Class: |
C12N 15/8243 20130101;
A24B 15/243 20130101; A61K 9/007 20130101; A24B 15/245 20130101;
A24B 15/20 20130101 |
Class at
Publication: |
131/270 ;
131/352 |
International
Class: |
A24F 47/00 20060101
A24F047/00 |
Claims
1. A tobacco use cessation kit comprising: a first tobacco product
that comprises nicotine and delivers a collective content of
N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT),
N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less
than 5.0 .mu.g/g; and a second tobacco product that comprises an
amount of nicotine that is less than the first tobacco product and
delivers a collective content of N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less
than 5.0 .mu.g/g, wherein said first tobacco product and said
second tobacco product are cigarettes.
2. The tobacco use cessation kit of claim 1, further comprising a
third tobacco product that comprises an amount of nicotine that is
less than the amount of nicotine in said second tobacco product and
delivers a collective content of N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) of less
than 5.0 .mu.g/g, wherein said first tobacco product, said second
tobacco product, and said third tobacco product are cigarettes.
3. The tobacco use cessation kit of claim 1, wherein at least one
of said tobacco products comprise a tobacco that comprises a
genetic modification.
4. The tobacco use cessation kit of claim 2, wherein at least one
of said tobacco products comprise a tobacco that comprises a
genetic modification.
5. The tobacco use cessation kit of claim 3, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 25 consecutive nucleotides in
length.
6. The tobacco use cessation kit of claim 3, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 50 consecutive nucleotides in
length.
7. The tobacco use cessation kit of claim 3, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 100 consecutive nucleotides in
length.
8. The tobacco use cessation kit of claim 3, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 200 consecutive nucleotides in
length.
9. The tobacco use cessation kit of claim 4, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 25 consecutive nucleotides in
length.
10. The tobacco use cessation kit of claim 4, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 50 consecutive nucleotides in
length.
11. The tobacco use cessation kit of claim 4, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 100 consecutive nucleotides in
length.
12. The tobacco use cessation kit of claim 4, wherein said genetic
modification comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 200 consecutive nucleotides in
length.
13. The tobacco use cessation kit of claim 5, wherein said gene
involved in nicotine biosynthesis is A622, QPTase, or PMTase.
14. The tobacco use cessation kit of claim 6, wherein said gene
involved in nicotine biosynthesis is A622, QPTase, or PMTase.
15. The tobacco use cessation kit of claim 7, wherein said gene
involved in nicotine biosynthesis is A622, QPTase, or PMTase.
16. The tobacco use cessation kit of claim 8, wherein said gene
involved in nicotine biosynthesis is A622, QPTase, or PMTase.
17. A method of reducing the nicotine consumption of a tobacco user
comprising: identifying a tobacco user for a reduction in nicotine
consumption; and providing said tobacco user with a tobacco-use
cessation kit of claim 1 or claim 2.
18. A method of reducing the nicotine consumption of a tobacco user
comprising: identifying a tobacco user for a reduction in nicotine
consumption; providing said tobacco user a first tobacco product
that comprises nicotine; and providing said tobacco user a second
tobacco product that comprises an amount of nicotine that is less
than the amount of nicotine in said first tobacco product, wherein
said first and said second tobacco products are the same form of
tobacco product.
19. The method of claim 18, further comprising providing said
tobacco user a third tobacco product that comprises an amount of
nicotine that is less than the amount of nicotine in said second
tobacco product and, wherein said third tobacco product is the same
form as said first tobacco product and said second tobacco product
and said first tobacco product.
20. The method of claim 18, wherein said first tobacco product or
said second tobacco product or both comprise genetically modified
tobacco.
21. The method of claim 18 wherein said genetically modified
tobacco comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 25 consecutive nucleotides in
length.
22. The method of claim 18, wherein said genetically modified
tobacco comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 50 consecutive nucleotides in
length.
23. The method of claim 18, wherein said genetically modified
tobacco comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 100 consecutive nucleotides in
length.
24. The method of claim 18, wherein said genetically modified
tobacco comprises an exogenous fragment of a gene involved in
nicotine biosynthesis at least 200 consecutive nucleotides in
length.
25. The method of claim 18, wherein said exogenous fragment of a
gene involved in nicotine biosynthesis is selected from the group
consisting of A622, putrescine N-methyltransferase,
N-methylputrescine oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase, and quinolate phosphoriosyl
transferase.
26. The method of claim 18, wherein said first tobacco product
comprises less than 0.7 mg/g nicotine and said second tobacco
product comprises less than 0.4 mg/g nicotine.
27. The method of claim 2, wherein said first tobacco product
delivers less than 0.7 mg/g nicotine and said second tobacco
product delivers less than 0.4 mg/g nicotine, and said third
tobacco product delivers less than 0.05 mg/g nicotine.
28. The method of claim 1, wherein said tobacco products comprise
about the same amount of tar.
29. The method of claim 2, wherein said tobacco products comprise
about the same amount of tar.
30. The method of claim 3, wherein said tobacco products comprise
about the same amount of tar.
31. The method of claim 4, wherein said tobacco products comprise
about the same amount of tar.
32. The method of claim 1, wherein at least one of said tobacco
products comprises treated tobacco.
33. The method of claim 2, wherein at least one of said tobacco
products comprises treated tobacco.
34. The method of claim 1, wherein at least one of said tobacco
products comprises selectively bred low nicotine tobacco.
35. The method of claim 2, wherein at least one of said tobacco
products comprises selectively bred low nicotine tobacco.
36. The method of claim 1, wherein at least one of said tobacco
products comprises genetically modified tobacco.
37. The method of claim 2, wherein at least one of said tobacco
products comprises genetically modified tobacco.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of, and claims
the benefit of priority to, international patent application number
PCT/US2004/016958, filed May 27, 2004, which designated the United
States of America and was published in English and which claims the
benefit of priority to U.S. provisional patent application No.
60/475,945, filed Jun. 4, 2003; this application is a
continuation-in-part of and claims the benefit of priority to
PCT/US2005/10733, filed Mar. 29, 2005, which designated the United
States of America and was published in English and which claims the
benefit of priority to No. 60/557,929, filed Mar. 30, 2004; this
application is a continuation-in-part of and claims the benefit of
priority to U.S. patent 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. 5, 2003, now U.S. Pat. No. 6,907,887,
which is a continuation of PCT/US2002/18040, filed Jun. 6, 2002,
which designated the United States of America and was published in
English and which claims the benefit of priority to U.S.
provisional application No. 60/297,154, filed Jun. 8, 2001; and
this application also claims the benefit of priority to U.S.
provisional patent application No. 60/680,283, filed May 11, 2005.
All of the aforementioned patent applications and provisional
patent applications are hereby expressly incorporated by reference
in their entireties.
FIELD OF THE INVENTION
[0002] Aspects of the invention concern tobacco-use cessation
programs that employ reduced nicotine tobacco products. More
specifically, embodiments include methods of reducing nicotine
and/or Tobacco Specific Nitrosamine (TSNA) consumption in a tobacco
user by providing said tobacco user with a plurality of tobacco
products, which contain a reduced amount of nicotine and/or TSNAs,
preferably, in a step-wise manner that gradually reduces the
exposure of the tobacco user to nicotine and/or TSNAs.
BACKGROUND OF THE INVENTION
[0003] The addictive properties of tobacco products are largely
attributable to the presence of nicotine and the habitual use of
the delivery system (e.g., the oral fixation associated with the
act of smoking or chewing tobacco, smoke intake, and taste). Many
tobacco-use cessation programs involve the use of nicotine
replacement therapy (NRT) products, wherein various amounts of
nicotine are given to the individual as a replacement for tobacco
use. Several types of tobacco-use cessation products, which involve
NRT, are currently available. For example, nicotine patches, gums,
capsules, inhalers, nasal sprays, and lozenges are conventional
products of NRT. Although these conventional products of NRT may
help tobacco users by suppressing the symptoms of nicotine
withdrawal, they do little to satisfy a tobacco user's cravings for
the habitual use of the delivery system. (Dotinga, Study Bursts
Nicotine Gum's Bubble, Health--Health Scout News, Sep. 20, 2002).
The factors involved with the habitual use of the delivery system
are hereinafter referred to as "secondary factors of addiction."
These secondary factors of addiction are largely psychological and
have only an incidental relationship to the chemical dependence on
nicotine.
[0004] In addition to the fact that conventional NRT does little to
quell the secondary factors of addiction, NRT has had only limited
success in enabling people to quit tobacco use. For example, among
over-the-counter NRT gum users, abstinence rates were 16.1% at 6
weeks and 8.4% at 6 months; whereas, for prescription NRT gum users
abstinence rates were 7.7% at 6 weeks and 7.7% at 6 months.
(Shiffman et al., Addiction 97:505-516, 2002). Users of the of the
NRT patch experienced only slightly better results; over-the
counter patch users were reported to have 19.0% abstinence at 6
weeks and 9.2% at 6 months; whereas, prescription NRT patch users
experienced 16.0% abstinence at 6 weeks and 3.0% abstinence at 6
months. Id. Others report slightly better results in that smoking
cessation with patch or gum show verified abstinence rates at 12
months in the range of 20%. (O'Brien, Lecture given to medical
students at the University of Pennsylvania on Sep. 22, 1995). One
study, however, goes so far as to say that NRT is no longer
effective in increasing long-term successful cessation in
California smokers. (Pierce and Gilpin, Jama, 288:1260-1264
(2002)). Clearly, it appears that tobacco addiction is a complex
web of psychological factors (i.e., the secondary factors) coupled
with nicotine dependence and existing NRT is largely
ineffective.
[0005] By design, conventional NRT relies on tobacco users to
gradually reduce their daily nicotine intake, while they mentally
curb their cravings for the secondary factors of addiction. In
practice, however, many program participants only replace the
addiction for tobacco with a far more expensive addiction to the
NRT product. In some cases, program participants continue using the
NRT product for long periods after the initial program has been
completed and eventually return to tobacco products. There remains
a need for tobacco-use cessation programs that focus on the
secondary factors of addiction while reducing the harmful effects
of nicotine.
SUMMARY OF THE INVENTION
[0006] Aspects of the present invention relate to methods of
reducing nicotine dependence of a tobacco user by gradually
reducing the exposure of said tobacco user to nicotine while
maintaining use of a tobacco product. It should be appreciated that
although TSNAs themselves may have some addictive properties, the
primary addictive component in tobacco is nicotine. Accordingly,
throughout this disclosure it is intended that embodiments directed
to a reduction in nicotine dependence or consumption of a tobacco
user are focused on a reduction of nicotine in tobacco products
provided to said tobacco user while embodiments directed to a
reduction in carcinogenic potential or consumption of TSNAs by a
tobacco user are focused on a reduction in the presence of TSNAs in
tobacco products that are provided to the tobacco user.
[0007] Tobacco products comprising a reduced amount of nicotine
and/or TSNAs (e.g., N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK)) have been
developed. These reduced nicotine and/or TSNA tobacco products have
been provided to tobacco users in a step-wise program that
gradually reduces the exposure of the tobacco user to nicotine
and/or TSNAs. The nicotine and/or TSNA consumption of a tobacco
user that follows the methods described herein is gradually reduced
and, preferably, the consumption of nicotine of program
participants is reduced to a level that allows the tobacco user to
quit tobacco use entirely. Accordingly, aspects of the invention
concern the tobacco products used in the tobacco-use cessation or
nicotine and/or TSNA reduction programs described herein, kits that
contain reduced nicotine and/or TSNA tobacco products, and methods
of making and using these compositions so as to reduce the exposure
of a tobacco user to nicotine and/or TSNAs, while maintaining use
of a tobacco product (e.g., snuff, chew, loose-leaf, or
cigarettes).
[0008] The reduced nicotine and/or TSNA tobacco products described
herein can be made by a variety of methods, which can be used
separately or in combination. In general, these methods fall into
three categories: "treated tobacco," "selectively bred low nicotine
tobacco," or "genetically modified tobacco." By "reduced nicotine
and/or TSNA tobacco or tobacco product" is meant that the tobacco
or tobacco product comprises an amount of nicotine and/or TSNAs
that is less than that of the same variety of tobacco or tobacco
product (e.g., full-flavor, light, or ultra-light) grown or
prepared under similar conditions, wherein the tobacco and/or
tobacco product has not been treated or modified to have a reduced
amount of nicotine and/or TSNAs. In some embodiments, a wild-type
tobacco of the variety that has been modified, which is grown,
harvested, and cured under the about the same conditions, or a
reference tobacco or tobacco product (e.g., 2R4F or IM-16) is used
as the standard by which to determine the presence or absence of a
particular modification that reduces the amount of nicotine and/or
TSNAs. Accordingly, some embodiments described herein are not bound
by the mechanism or approach used to create the reduced nicotine
and/or TSNA tobacco.
[0009] In the United States, tar, nicotine, and carbon monoxide
yields are obtained using the Federal Trade Commission (FTC)
smoking-machine test method, which defines the measurement of tar
as that material captured by a Cambridge pad when a cigarette is
machine smoked, minus nicotine and water (Pillsbury, et al., 1969,
"Tar and nicotine in cigarette smoke". J. Assoc. Off. Analytical
Chem., 52, 458-62). Specifically, the FTC cigarette-testing method
collects smoke samples by simulating puffing volumes of 35 ml of
cigarette smoke for two seconds every 58 seconds, with none of the
filter ventilation holes blocked (if any), until the burn line
reaches the tipping paper plus 2 mm, or a line drawn 23 mm from the
end of a non-filter cigarette. This FTC smoking-machine test method
has been used in the United States since 1967 to determine smoke
cigarette yields for tar and nicotine. The determination of carbon
monoxide yields in cigarette smoke was added to this method in
1980.
[0010] In 1967, when the FTC introduced its testing method, it
issued a news release and explained that the purpose of the testing
"is not to determine the amount of tar and nicotine inhaled by any
human smoker, but rather to determine the amount of tar and
nicotine generated when a cigarette is smoked by a machine in
accordance with the prescribed method." Nevertheless, the method
serves an important role in providing an accurate way to rank and
compare cigarettes according to tar, nicotine and carbon monoxide
yields.
[0011] The International Standards Organization (ISO) developed a
very similar smoking-machine test method for tar, nicotine, and
carbon monoxide yields of cigarettes (ISO, 1991
"Cigarettes--determination of total and nicotine-free dry
particulate matter using a routine analytical smoking machine" ISO:
4387:1991).
[0012] The FTC and ISO smoking methods differ in the following
eight areas. [0013] The FTC method specifies laboratory
environmental conditions of 75.degree. F..+-.1.degree. F.
(23.8.degree. C..+-.1.degree. C.) and a relative humidity of
60%.+-.2% for both the equilibration and testing. The time of
equilibration is a minimum of 24 hours and a maximum of 14 days.
This is compared to the ISO specifications of 22.degree.
C..+-.1.degree. C. and 60%.+-.2% relative humidity for
equilibration, 22.degree. C..+-.2.degree. C. and 60% relative
humidity.+-.5% for testing. The equilibration time is a minimum of
48 hours and a maximum of 10 days. [0014] The FTC defines the
cigarette butt length as a minimum of 23 millimeters or the tipping
paper plus three millimeters whichever is longer. ISO defines butt
length as the longest of 23 millimeters or tipping paper plus three
millimeters or the filter plus eight millimeters. Both methods
specify a 23-millimeter butt length for non-filter cigarettes.
[0015] ISO defines the position of the ashtray at 20-60 millimeters
below the cigarettes in the smoking machine. FTC does not specify a
position. [0016] ISO specifies a two-piece snap together reusable
filter holder. This filter holder contains the Cambridge pad and
uses a synthetic rubber perforated washer to partly obstruct the
butt end of the cigarette. The FTC method defines the use of a
Cambridge filter pad but does not specify a filter pad holder
assembly. [0017] The ISO method specifies airflow across the
cigarettes at the cigarette level. FTC specifies the use of a
monitor cigarette to adjust airflow. [0018] The ISO procedure
defines the process of wiping the excess total particulate matter
(TPM) out of the used filter holder. The inner surfaces of the
filter holder are wiped with two separate quarters of an unused
conditioned filter pad. The FTC method uses the backside (the side
opposite of the trapped TPM) to wipe the inner surface of the
filter holder. [0019] ISO specifies using 20 ml per Cambridge pad
of extraction solution to analyze nicotine and water in TPM. The
FTC procedure defines 10 ml per Cambridge pad. [0020] ISO defines
the internal standards for the gas chromatographic determination of
nicotine and water. The FTC procedure does not specify the internal
standards.
[0021] These differences typically result in slightly lower
measured deliveries for the ISO Method versus the FTC Method. The
measured values between FTC and ISO methods are within the
detection limits of the test or about no greater than 0.4 mg tar
and about 0.04 mg nicotine for cigarettes that yield over about 10
mg.
[0022] Thus, it should also be mentioned that it is understood in
the art (and as used in the present disclosure) that when a tobacco
or a tobacco product is said to "comprise", "have", or "contain" a
particular amount of nicotine and/or TSNA, the tobacco or tobacco
product itself may comprise, have, or contain the recited amount of
nicotine and/or TSNA (e.g., the amount of nicotine and/or TSNA
present in the tobacco leaf or on the tobacco rod) or, in some
contexts, the recited amount of nicotine and/or TSNAs is the amount
of nicotine and/or TSNAs present in the mainstream or sidestream
smoke obtained from the tobacco or tobacco product using the FTC
and/or ISO methodologies.
[0023] In some embodiments, the reduced nicotine and/or TSNA is
provided in a tobacco product as a neat formulation in that only
one variety or type of reduced nicotine and/or TSNA tobacco is
provided; whereas, in other embodiments, the tobacco product
comprises a blend or mixture of tobacco varieties or types of
reduced nicotine and/or TSNA tobaccos. Preferably, the tobacco
products (e.g., a cigarette) that are used in the tobacco-use
cessation or nicotine and/or TSNA reduction kits and the
tobacco-use cessation or nicotine and/or TSNA reduction methods
described herein comprise a tobacco that comprises (e.g., on the
leaf or tobacco rod) or delivers (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods), for example, less than or
equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine,
or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,
NNN, NAT, NAB, or NNK) of less than or equal to 2.0 .mu.g/g, 1.5
.mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g. Some embodiments
are limited to whether the tobacco is treated tobacco, selectively
bred low nicotine tobacco, or genetically modified tobacco,
however, many embodiments are not so limited. Some embodiments also
include an amount of exogenous nicotine.
[0024] In some embodiments, the reduced nicotine and/or TSNA
tobacco products, which can be incorporated into the tobacco-use
cessation or nicotine and/or TSNA reduction kits and methods
described herein, are made from tobacco that has been treated or
otherwise modified to reduce the amount of nicotine and/or TSNAs.
Examples of "treated tobacco" include tobacco that has been treated
by microbial enzymatic degradation, chemical treatment, high
pressure extraction, reconstitution, and/or flash curing methods.
Examples of these methods can be found, for example, in: U.S. Pat.
No. 4,557,280; U.S. Pat. No. 4,561,452; U.S. Pat. No. 4,848,373;
U.S. Pat. No. 4,183,364; U.S. Pat. No. 4,215,706; U.S. Pat. No.
5,803,081; U.S. Pat. No. 6,202,649; U.S. Pat. No. 6,425,401; U.S.
Pat. No. 5,713,376; U.S. Pat. No. 6,338,348; U.S. Pat. No.
6,834,654; U.S. patent application Ser. No. 10/943,346, U.S. patent
application Ser. No. 10/719,295 and WO 05/018307, which designated
the United States and was published in English. The aforementioned
patents and patent applications are hereby expressly incorporated
by reference in their entireties. The reduced nicotine and/or TSNA
tobaccos described above can be blended with conventional tobacco
and/or can include extenders, fillers, tobacco substitutes (e.g.,
tomato or rosemary powder), stems, and scrap tobacco and can
contain exogenous nicotine. The treated tobacco that can be used in
the methods described herein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to 1.0
mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 2.0 .mu.g/g, 1.5 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g. Similarly, tobacco products
that comprise the treated tobacco above can be used in the
tobacco-use cessation or nicotine and/or TSNA reduction kits and
methods described herein. These tobacco products may comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g
nicotine, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g.
[0025] Desirably, the reduced nicotine and/or TSNA tobacco
products, which can be incorporated into the tobacco-use cessation
or nicotine and/or TSNA reduction kits and methods described
herein, are made from "selectively bred low nicotine tobacco." For
example, some embodiments described herein comprise "selectively
bred low nicotine tobacco," such as low nicotine burley varieties
(e.g., Burley 21 LA), low nicotine flue-cured varieties (e.g.,
NC-13 or LA FC53), low nicotine air-cured varieties (e.g., N506) or
low nicotine oriental varieties (e.g., Nevrokop or Melnik
varieties). Tobacco products comprising tobacco that has been
selectively bred or grown to have a reduced amount of nicotine
and/or TSNAs can also be blended with conventional tobacco and/or
can include extenders, fillers, tobacco substitutes (e.g., tomato
or rosemary powder), stems, and scrap tobacco and can contain
exogenous nicotine. The selectively bred low nicotine tobacco that
can be used in the methods described herein can comprise (e.g., on
the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g
nicotine, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 2.0
.mu.g/g, 1.5 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g.
Similarly, tobacco products that comprise the selectively bred low
nicotine tobacco above can be used in the tobacco-use cessation or
nicotine and/or TSNA reduction kits and methods described herein.
These tobacco products may comprise (e.g., on the leaf or tobacco
rod) or deliver (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods), for example, less than or equal to 1.0 mg/g
nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g.
[0026] Preferably, the reduced nicotine and/or TSNA tobacco
products, which can be incorporated into the tobacco-use cessation
or nicotine and/or TSNA reduction kits and methods described
herein, are made from tobacco that has been genetically modified to
comprise a reduced amount of nicotine and/or TSNAs. In some
embodiments, the "genetically modified" tobacco comprises an
exogenous nucleic acid that encodes a protein in the nicotine
biosynthesis pathway (e.g., A622, quinolate phosphoribosyl
transferase (QPTase), putrescene N-methyl transferase (PMTase),
N-methylputrescene oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase) or a fragment thereof. The
exogenous nucleic acid that encodes a protein in the nicotine
biosynthesis pathway or a fragment thereof (e.g., 25, 30, 50, 75,
100, 200, or 500 consecutive nucleotides in length) can be
incorporated into a construct that inhibits the production of
nicotine by many different modalities, depending on the construct
(e.g., antisense, RNA interference, co-suppression, and/or
molecular decoy), when said construct is introduced into a tobacco
plant cell (e.g., by bacterial or biolistic transformation) and a
transgenic plant is regenerated from the transformants. Exemplary
methods for creating "genetically modified tobacco" include the
approaches described in, for example, WO98/56923; U.S. Pat. Nos.
6,586,661; 6,423,520; 6,907,887; and U.S. patent application Ser.
Nos. 09/963,340; 10/356,076; 09/941,042; 10/363,069; 10/729,121;
10/943,346; 11/077,752; WO00/67558, which designated the United
States and was published in English; U.S. Pat. Nos. 5,684,241;
5,369,023; 5,260,205; and 6,700,040, all of which are hereby
expressly incorporated by reference in their entireties. Tobacco
products comprising tobacco that has been genetically modified to
have a reduced amount of nicotine and/or TSNAs can also be blended
with conventional tobacco and/or can include extenders, fillers,
tobacco substitutes (e.g., tomato or rosemary powder), stems, and
scrap tobacco and can include exogenous nicotine. The genetically
modified tobacco that can be used in the methods described herein
can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g
nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a
collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less
than or equal to 2.0 .mu.g/g, 1.5 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g. Similarly, tobacco products that comprise
the genetically modified tobacco above can be used in the
tobacco-use cessation or nicotine and/or TSNA reduction kits and
methods described herein. These tobacco products may comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g
nicotine, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g.
[0027] As described above, some of the tobaccos described herein
contain an amount of exogenous nicotine. More specifically, some of
the tobaccos described herein comprise an amount of exogenous
nicotine (e.g., nicotine prepared by extraction of conventional
tobacco or synthetically prepared nicotine) so as to adjust the
content of the nicotine in the tobacco to a desirable level, which
allows for fine adjustments in the amount of nicotine present in a
tobacco product that comprises this tobacco and, accordingly, fine
adjustments in the amount of nicotine provided to a tobacco user.
Preferably, the tobacco is maintained in a microbe-free environment
after addition of the exogenous nicotine so as to prevent the
accumulation of TSNAs. Accordingly, the tobacco products described
herein can include exogenous nicotine and the tobacco-use cessation
kits and tobacco-use cessation methods described herein can include
these exogenous nicotine-containing tobacco products.
[0028] Some embodiments concern methods of making the tobacco
products described herein. By some approaches, a blended reduced
nicotine tobacco is made by providing a first tobacco, which can be
a conventional tobacco or a modified tobacco as described above
(e.g., a treated tobacco, a selectively bred low nicotine tobacco,
or a genetically modified tobacco); providing a second tobacco,
which can be a conventional tobacco or a modified tobacco as
described above (e.g., a treated tobacco, a low nicotine
selectively bred tobacco, or a genetically modified tobacco); and
blending the first and second tobaccos so as to obtain a reduced
nicotine and/or TSNA blended tobacco. Tobacco products and
tobacco-use cessation or nicotine and/or TSNA reduction kits that
comprise the blended reduced nicotine and/or TSNA tobacco produced
by this method are also provided. The blended reduced nicotine
and/or TSNA tobacco products can comprise treated tobacco,
selectively bred low nicotine tobacco, or genetically modified
tobacco or combinations thereof. The blended tobacco products can
also include exogenous nicotine. The blended tobacco that can be
used in the methods described herein can comprise (e.g., on the
leaf or tobacco rod) or deliver (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods), for example, less than or
equal to 1.0 mg/g nicotine, 0.6 mg/g nicotine, 0.3 mg/g nicotine,
or 0.05 mg/g nicotine and/or a collective content of TSNAs (e.g.,
NNN, NAT, NAB, or NNK) of less than or equal to 2.0 .mu.g/g, 1.5
.mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g. Similarly,
tobacco products that comprise the blended tobacco above can be
used in the tobacco-use cessation or nicotine and/or TSNA reduction
kits and methods described herein. These tobacco products may
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g nicotine, 0.6 mg/g
nicotine, 0.3 mg/g nicotine, or 0.05 mg/g nicotine and/or a
collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less
than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0
.mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g.
[0029] Additional embodiments include tobacco-use cessation or
nicotine and/or TSNA reduction kits that comprise one or more of
the reduced nicotine and/or TSNA tobacco products described herein.
Although some embodiments comprise a tobacco-use cessation or
nicotine and/or TSNA reduction kit that comprises only one tobacco
product that comprises a tobacco that has a reduced amount of
nicotine and/or TSNAs, as compared to a conventional tobacco of the
same variety grown under the same conditions; preferred embodiments
include tobacco-use cessation or nicotine and/or TSNA reduction
kits that comprise a plurality of tobacco products, wherein at
least two of said tobacco products comprise different amounts of
nicotine and/or TSNAs.
[0030] Some tobacco-use cessation or nicotine and/or TSNA reduction
kits comprise, for example, a conventional tobacco product and a
first reduced nicotine and/or TSNA tobacco product, wherein the
first reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the conventional tobacco product. The
first reduced nicotine and/or TSNA tobacco product (e.g., a
cigarette) or a tobacco therein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to 1.0
mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g so long as the amount of
nicotine and/or TSNAs in or delivered by the first tobacco product
is less than the amount of nicotine and/or TSNAs in or delivered by
the conventional tobacco product. The first reduced nicotine and/or
TSNA tobacco product can comprise treated tobacco, selectively bred
low nicotine tobacco, or genetically modified tobacco or
combinations thereof. The first tobacco product can also include
exogenous nicotine.
[0031] Other embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction kits that comprise a conventional tobacco
product, a first reduced nicotine and/or TSNA tobacco product and a
second reduced nicotine and/or TSNA tobacco product, wherein the
first reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the conventional tobacco product and the
second reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the first reduced nicotine and/or TSNA
tobacco product. The first reduced nicotine and/or TSNA tobacco
product (e.g., a cigarette) or tobacco therein can comprise (e.g.,
on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g and
the second reduced nicotine and/or TSNA tobacco product can
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g so long as the amount of nicotine and/or TSNAs in or
delivered by the first tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the conventional tobacco
product and the amount of nicotine and/or TSNAs in or delivered by
the second tobacco product is less than the amount of nicotine
and/or TSNAs in or delivered by the first tobacco product. The
first and/or second reduced nicotine and/or TSNA tobacco products
can comprise treated tobacco, selectively bred low nicotine
tobacco, or genetically modified tobacco or combinations thereof.
These tobacco products can also include exogenous nicotine.
[0032] More embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction kits that comprise a conventional tobacco
product, a first reduced nicotine and/or TSNA tobacco product, a
second reduced nicotine and/or TSNA tobacco product, and a third
reduced nicotine and/or TSNA tobacco product, wherein the first
reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the conventional tobacco product, the
second reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the first reduced nicotine and/or TSNA
tobacco product and the third reduced nicotine and/or TSNA tobacco
product comprises less nicotine and/or TSNAs than the second
reduced nicotine and/or TSNA tobacco product. The first reduced
nicotine and/or TSNA tobacco product (e.g., a cigarette) or a
tobacco therein can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g; the second reduced nicotine and/or TSNA
tobacco product can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g; and the third reduced nicotine and/or TSNA
tobacco product can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g so long as the amount of nicotine and/or
TSNAs in or delivered by the first tobacco product is less than the
amount of nicotine and/or TSNAs in or delivered by the conventional
tobacco product, the amount of nicotine and/or TSNAs in or
delivered by the second tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the first tobacco product,
and the amount of nicotine and/or TSNAs in or delivered by the
third tobacco product is less than the amount of nicotine and/or
TSNAs in or delivered by the second tobacco product. The first,
second, and/or third reduced nicotine and/or TSNA tobacco products
can comprise treated tobacco, selectively bred low nicotine
tobacco, or genetically modified tobacco or combinations thereof.
These tobacco products can also include exogenous nicotine.
[0033] Still more embodiments include tobacco-use cessation or
nicotine and/or TSNA reduction kits that comprise a conventional
tobacco product, a first reduced nicotine and/or TSNA tobacco
product, a second reduced nicotine and/or TSNA tobacco product, a
third reduced nicotine and/or TSNA tobacco product and a fourth
reduced nicotine and/or TSNA tobacco product, wherein the first
reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the conventional tobacco product, the
second reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the first reduced nicotine and/or TSNA
tobacco product, the third reduced nicotine and/or TSNA tobacco
product comprises less nicotine and/or TSNAs than the second
reduced nicotine and/or TSNA tobacco product, and the fourth
reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the third reduced nicotine and/or TSNA
tobacco product. The first reduced nicotine and/or TSNA tobacco
product (e.g., a cigarette) or a tobacco therein can comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the
second reduced nicotine and/or TSNA tobacco product can comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the
third reduced nicotine and/or TSNA tobacco product can comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; and
the fourth reduced nicotine and/or TSNA tobacco product can
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g so long as the amount of nicotine and/or TSNAs in or
delivered by the first tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the conventional tobacco
product, the amount of nicotine and/or TSNAs in or delivered by the
second tobacco product is less than the amount of nicotine and/or
TSNAs in or delivered by the first tobacco product, the amount of
nicotine and/or TSNAs in or delivered by the third tobacco product
is less than the amount of nicotine and/or TSNAs in or delivered by
the second tobacco product, and the amount of nicotine and/or TSNAs
in or delivered by the fourth tobacco product is less than the
amount of nicotine and/or TSNAs in or delivered by the third
tobacco product. The first, second, third, and/or fourth reduced
nicotine and/or TSNA tobacco products can comprise treated tobacco,
selectively bred low nicotine tobacco, or genetically modified
tobacco or combinations thereof. These tobacco products can also
include exogenous nicotine.
[0034] Preferred tobacco-use cessation or nicotine and/or TSNA
reduction kits comprise, however, a first reduced nicotine and/or
TSNA tobacco product, wherein the first reduced nicotine and/or
TSNA tobacco product comprises less nicotine and/or TSNAs than a
conventional tobacco product. That is, in some embodiments, the
tobacco-use cessation or nicotine and/or TSNA reduction kits do not
contain a conventional tobacco product. The first reduced nicotine
and/or TSNA tobacco product (e.g., a cigarette) or a tobacco
therein can comprise (e.g., on the leaf or tobacco rod) or deliver
(e.g., side-stream or main-stream smoke by the FTC and/or ISO
methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g,
0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g. The first reduced nicotine and/or TSNA
tobacco products can comprise treated tobacco, selectively bred low
nicotine tobacco, or genetically modified tobacco or combinations
thereof. The first tobacco product can also include exogenous
nicotine.
[0035] Other embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction kits that comprise a first reduced nicotine
and/or TSNA tobacco product and a second reduced nicotine and/or
TSNA tobacco product, wherein the second reduced nicotine and/or
TSNA tobacco product comprises less nicotine and/or TSNAs than the
first reduced nicotine and/or TSNA tobacco product. The first
reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or
a tobacco therein can comprise (e.g., on the leaf or tobacco rod)
or deliver (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods), for example, less than or equal to 1.0 mg/g,
0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g and the second reduced
nicotine and/or TSNA tobacco product or a tobacco therein can
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g so long as the amount of nicotine and/or TSNAs in or
delivered by the second tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the first tobacco product.
The first and/or second reduced nicotine and/or TSNA tobacco
products can comprise treated tobacco, selectively bred low
nicotine tobacco, or genetically modified tobacco or combinations
thereof. These tobacco products can also include exogenous
nicotine.
[0036] More embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction kits that comprise a first reduced nicotine
and/or TSNA tobacco product, a second reduced nicotine and/or TSNA
tobacco product, and a third reduced nicotine and/or TSNA tobacco
product, wherein the second reduced nicotine and/or TSNA tobacco
product comprises less nicotine and/or TSNAs than the first reduced
nicotine and/or TSNA tobacco product and the third reduced nicotine
and/or TSNA tobacco product comprises less nicotine and/or TSNAs
than the second reduced nicotine and/or TSNA tobacco product. The
first reduced nicotine and/or TSNA tobacco product (e.g., a
cigarette) or tobacco therein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to 1.0
mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the second reduced nicotine
and/or TSNA tobacco product or tobacco therein can comprise (e.g.,
on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; and
the third reduced nicotine and/or TSNA tobacco product or a tobacco
therein can comprise (e.g., on the leaf or tobacco rod) or deliver
(e.g., side-stream or main-stream smoke by the FTC and/or ISO
methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g,
0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g so long as the amount of nicotine and/or
TSNAs in or delivered by the second tobacco product is less than
the amount of nicotine and/or TSNAs in or delivered by the first
tobacco product, and the amount of nicotine and/or TSNAs in or
delivered by the third tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the second tobacco
product. The first, second, and/or third reduced nicotine and/or
TSNA tobacco products can comprise treated tobacco, selectively
bred low nicotine tobacco, or genetically modified tobacco or
combinations thereof. These tobacco products can also include
exogenous nicotine.
[0037] Still more embodiments include tobacco-use cessation or
nicotine and/or TSNA reduction kits that comprise a first reduced
nicotine and/or TSNA tobacco product, a second reduced nicotine
and/or TSNA tobacco product, a third reduced nicotine and/or TSNA
tobacco product and a fourth reduced nicotine and/or TSNA tobacco
product, wherein the second reduced nicotine and/or TSNA tobacco
product comprises less nicotine and/or TSNAs than the first reduced
nicotine and/or TSNA tobacco product, the third reduced nicotine
and/or TSNA tobacco product comprises less nicotine and/or TSNAs
than the second reduced nicotine and/or TSNA tobacco product, and
the fourth reduced nicotine and/or TSNA tobacco product comprises
less nicotine and/or TSNAs than the third reduced nicotine and/or
TSNA tobacco product. The first reduced nicotine and/or TSNA
tobacco product (e.g., a cigarette) or a tobacco therein can
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g; the second reduced nicotine and/or TSNA tobacco product or
a tobacco therein can comprise (e.g., on the leaf or tobacco rod)
or deliver (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods), for example, less than or equal to 1.0 mg/g,
0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the third reduced nicotine
and/or TSNA tobacco product or a tobacco therein can comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; and
the fourth reduced nicotine and/or TSNA tobacco product or a
tobacco therein can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g so long as the amount of nicotine and/or
TSNAs in or delivered by the second tobacco product is less than
the amount of nicotine and/or TSNAs in or delivered by the first
tobacco product, the amount of nicotine and/or TSNAs in or
delivered by the third tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the second tobacco
product, and the amount of nicotine and/or TSNAs in or delivered by
the fourth tobacco product is less than the amount of nicotine
and/or TSNAs in or delivered by the third tobacco product. The
first, second, third, and/or fourth reduced nicotine and/or TSNA
tobacco products can comprise treated tobacco, selectively bred low
nicotine tobacco, or genetically modified tobacco or combinations
thereof. These tobacco products can also include exogenous
nicotine.
[0038] The tobacco-use cessation or nicotine and/or TSNA reduction
kits described herein can, optionally, comprise instructions or
guidance on use of the kit and/or tobacco-use cessation or nicotine
and/or TSNA reduction and said instructions or guidance can refer
the user to counseling programs and literature on the benefits of
reduced exposure to nicotine and/or TSNAs and/or tobacco products,
in general. The instructions or guidance can be provided in said
kits in the form of a paper, CD-ROM, DVD, video, cassette, website
link, or other tangible medium. Additionally, the tobacco products
provided in said tobacco-use cessation or nicotine and/or TSNA
reduction kits can also comprise indicia showing that the product
is a member of a series of tobacco products to be consumed in a
sequential order.
[0039] For example, in some embodiments, the tobacco products
and/or packaging has been labeled with a number or letter or symbol
or other form of visually identifiable marker to indicate whether
the product is a conventional tobacco product, a first tobacco
product, a second tobacco product, a third tobacco product, or a
fourth tobacco product to be used in said kit or otherwise in
conformance with a tobacco-use cessation or nicotine and/or TSNA
reduction method described herein. Preferred indicia that
identifies the tobacco product as a member of a series of tobacco
products used in a tobacco-use cessation or nicotine and/or TSNA
reduction method include visually identifiable rings or bars that
appear on the tobacco product itself and/or the tobacco product
packaging (see e.g., International Publication Number WO/05041151,
which designates the U.S., and was published in English, herein
expressly incorporated by reference in its entirety) and Quest
1.RTM., Quest 2.RTM., and Quest 3.RTM.. The tobacco-use cessation
or nicotine and/or TSNA reduction kits and tobacco products and
packing of such can also comprise indicia from a regulatory agency
(e.g., a governmental body such as the Federal Drug Administration)
indicating that said kit or the tobacco products contained therein
have been approved for use in a tobacco-use cessation program.
[0040] Other embodiments concern methods of reducing the nicotine
and/or TSNA consumption or exposure of a tobacco user by providing
to said tobacco user a tobacco product or tobacco-use cessation or
nicotine and/or TSNA reduction kit, as described herein. In some
embodiments, a tobacco user is identified as one in need of a
reduction in the consumption and/or exposure to nicotine and/or
TSNAs. The identified tobacco user is then provided one or more of
the aforementioned reduced nicotine and/or TSNA tobacco products
and/or tobacco-use cessation kits described herein. In some
methods, the reduction in consumption or exposure to nicotine
and/or TSNAs in said tobacco user is measured. In some methods, the
abstinence from conventional tobacco use is measured.
[0041] Accordingly, by some approaches, a tobacco user, who is,
optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
conventional tobacco product and then said tobacco user is provided
a first reduced nicotine and/or TSNA tobacco product, wherein the
first reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the conventional tobacco product. The
first reduced nicotine and/or TSNA tobacco product (e.g., a
cigarette) or a tobacco therein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to
less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g. The
first reduced nicotine and/or TSNA tobacco products can comprise
treated tobacco, selectively bred low nicotine tobacco, or
genetically modified tobacco or combinations thereof. The first
tobacco product can also include exogenous nicotine. In some
methods, the reduction in consumption or exposure to nicotine
and/or TSNAs in said tobacco user is measured. In some methods, the
abstinence from conventional tobacco use is measured. In some
methods, a marker of nicotine addiction is measured (e.g., regional
cerebral metabolic rate for glucose and/or cerebral blood flow,
which are measurable using positron emission tomography (PET)).
[0042] Other embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction methods, wherein a tobacco user, who is,
optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
conventional tobacco product and then said tobacco user is provided
a conventional tobacco product, a first reduced nicotine and/or
TSNA tobacco product and a second reduced nicotine and/or TSNA
tobacco product, wherein the first reduced nicotine and/or TSNA
tobacco product comprises less nicotine and/or TSNAs than the
conventional tobacco product and the second reduced nicotine and/or
TSNA tobacco product comprises less nicotine and/or TSNAs than the
first reduced nicotine and/or TSNA tobacco product. The first
reduced nicotine and/or TSNA tobacco product (e.g., a cigarette) or
a tobacco therein can comprise (e.g., on the leaf or tobacco rod)
or deliver (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods), for example, less than or equal to 1.0 mg/g,
0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g and the second reduced
nicotine and/or TSNA tobacco product or a tobacco therein can
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g so long as the amount of nicotine and/or TSNAs in or
delivered by the first tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the conventional tobacco
product and the amount of nicotine and/or TSNAs in or delivered by
the second tobacco product is less than the amount of nicotine
and/or TSNAs in or delivered by the first tobacco product. The
first and/or second reduced nicotine and/or TSNA tobacco products
can comprise treated tobacco, selectively bred low nicotine
tobacco, or genetically modified tobacco or combinations thereof.
These tobacco products can also include exogenous nicotine. In some
methods, the reduction in consumption or exposure to nicotine
and/or TSNAs in said tobacco user is measured. In some methods, the
abstinence from conventional tobacco use is measured. In some
methods, a marker of nicotine addiction is measured (e.g., regional
cerebral metabolic rate for glucose and/or cerebral blood flow,
which are measurable using positron emission tomography (PET)).
[0043] More embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction methods, wherein a tobacco user, who is,
optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
conventional tobacco product, a first reduced nicotine and/or TSNA
tobacco product, a second reduced nicotine and/or TSNA tobacco
product, and a third reduced nicotine and/or TSNA tobacco product,
wherein the first reduced nicotine and/or TSNA tobacco product
comprises less nicotine and/or TSNAs than the conventional tobacco
product, the second reduced nicotine and/or TSNA tobacco product
comprises less nicotine and/or TSNAs than the first reduced
nicotine and/or TSNA tobacco product and the third reduced nicotine
and/or TSNA tobacco product comprises less nicotine and/or TSNAs
than the second reduced nicotine and/or TSNA tobacco product. The
first reduced nicotine and/or TSNA tobacco product (e.g., a
cigarette) or tobacco therein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to 1.0
mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the second reduced nicotine
and/or TSNA tobacco product or tobacco therein can comprise (e.g.,
on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; and
the third reduced nicotine and/or TSNA tobacco product or a tobacco
therein can comprise (e.g., on the leaf or tobacco rod) or deliver
(e.g., side-stream or main-stream smoke by the FTC and/or ISO
methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g,
0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g so long as the amount of nicotine and/or
TSNAs in or delivered by the first tobacco product is less than the
amount of nicotine and/or TSNAs in or delivered by the conventional
tobacco product, the amount of nicotine and/or TSNAs in or
delivered by the second tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the first tobacco product,
and the amount of nicotine and/or TSNAs in or delivered by the
third tobacco product is less than the amount of nicotine and/or
TSNAs in or delivered by the second tobacco product. The first,
second, and/or third reduced nicotine and/or TSNA tobacco products
can comprise treated tobacco, selectively bred low nicotine
tobacco, or genetically modified tobacco or combinations thereof.
These tobacco products can also include exogenous nicotine. In some
methods, the reduction in consumption or exposure to nicotine
and/or TSNAs in said tobacco user is measured. In some methods, the
abstinence from conventional tobacco use is measured. In some
methods, a marker of nicotine addiction is measured (e.g., regional
cerebral metabolic rate for glucose and/or cerebral blood flow,
which are measurable using positron emission tomography (PET)).
[0044] Still more embodiments include tobacco-use cessation or
nicotine and/or TSNA reduction methods, wherein a tobacco user, who
is, optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
conventional tobacco product, a first reduced nicotine and/or TSNA
tobacco product, a second reduced nicotine and/or TSNA tobacco
product, a third reduced nicotine and/or TSNA tobacco product and a
fourth reduced nicotine and/or TSNA tobacco product, wherein the
first reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the conventional tobacco product, the
second reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the first reduced nicotine and/or TSNA
tobacco product, the third reduced nicotine and/or TSNA tobacco
product comprises less nicotine and/or TSNAs than the second
reduced nicotine and/or TSNA tobacco product, and the fourth
reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the third reduced nicotine and/or TSNA
tobacco product. The first reduced nicotine and/or TSNA tobacco
product (e.g., a cigarette) or a tobacco therein can comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the
second reduced nicotine and/or TSNA tobacco product or a tobacco
therein can comprise (e.g., on the leaf or tobacco rod) or deliver
(e.g., side-stream or main-stream smoke by the FTC and/or ISO
methods), for example, less than or equal to 1.0 mg/g, 0.6 mg/g,
0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content of
TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g; the third reduced nicotine and/or TSNA
tobacco product or a tobacco therein can comprise (e.g., on the
leaf or tobacco rod) or deliver (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods), for example, less than or
equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or
a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less
than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0
.mu.g/g, 1.0 g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; and the fourth
reduced nicotine and/or TSNA tobacco product or a tobacco therein
can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g so long as the amount of nicotine and/or TSNAs in or
delivered by the first tobacco product is less than the amount of
nicotine and/or TSNAs in or delivered by the conventional tobacco
product, the amount of nicotine and/or TSNAs in or delivered by the
second tobacco product is less than the amount of nicotine and/or
TSNAs in or delivered by the first tobacco product, the amount of
nicotine and/or TSNAs in or delivered by the third tobacco product
is less than the amount of nicotine and/or TSNAs in or delivered by
the second tobacco product, and the amount of nicotine and/or TSNAs
in or delivered by the fourth tobacco product is less than the
amount of nicotine and/or TSNAs in or delivered by the third
tobacco product. The first, second, third and/or fourth reduced
nicotine and/or TSNA tobacco products can comprise treated tobacco,
selectively bred low nicotine tobacco, or genetically modified
tobacco or combinations thereof. These tobacco products can also
include exogenous nicotine. In some methods, the reduction in
consumption or exposure to nicotine and/or TSNAs in said tobacco
user is measured. In some methods, the abstinence from conventional
tobacco use is measured. In some methods, a marker of nicotine
addiction is measured (e.g., regional cerebral metabolic rate for
glucose and/or cerebral blood flow, which are measurable using
positron emission tomography (PET)).
[0045] Preferred tobacco-use cessation or nicotine and/or TSNA
reduction methods, however, include approaches, wherein a tobacco
user, who is, optionally, identified as one in need of a reduction
in the consumption or exposure to nicotine and/or TSNAs, is
provided a first reduced nicotine and/or TSNA tobacco product,
wherein the first reduced nicotine and/or TSNA tobacco product
comprises less nicotine and/or TSNAs than the conventional tobacco
product. That is, said tobacco-use cessation or nicotine and/or
TSNA reduction methods do not contain the step whereby a
conventional tobacco product is provided. The first reduced
nicotine and/or TSNA tobacco product (e.g., a cigarette) or a
tobacco therein can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g. The first reduced nicotine and/or TSNA
tobacco products can comprise treated tobacco, selectively bred low
nicotine tobacco, or genetically modified tobacco or combinations
thereof. The first tobacco product can also include exogenous
nicotine. In some methods, the reduction in consumption or exposure
to nicotine and/or TSNAs in said tobacco user is measured. In some
methods, the abstinence from conventional tobacco use is measured.
In some methods, a marker of nicotine addiction is measured (e.g.,
regional cerebral metabolic rate for glucose and/or cerebral blood
flow, which are measurable using positron emission tomography
(PET)).
[0046] Other embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction methods, wherein a tobacco user, who is,
optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
first reduced nicotine and/or TSNA tobacco product and a second
reduced nicotine and/or TSNA tobacco product, wherein the second
reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the first reduced nicotine and/or TSNA
tobacco product. The first reduced nicotine and/or TSNA tobacco
product (e.g., a cigarette) or a tobacco therein can comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods), for example, less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g
nicotine and/or a collective content of TSNAs (e.g., NNN, NAT, NAB,
or NNK) of less than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0
.mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g and
the second reduced nicotine and/or TSNA tobacco product or a
tobacco therein can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g so long as the amount of nicotine and/or
TSNAs in or delivered by the second tobacco product is less than
the amount of nicotine and/or TSNAs in or delivered by the first
tobacco product. The first and/or second reduced nicotine and/or
TSNA tobacco products can comprise treated tobacco, selectively
bred low nicotine tobacco, or genetically modified tobacco or
combinations thereof. These tobacco products can also include
exogenous nicotine. In some methods, the reduction in consumption
or exposure to nicotine and/or TSNAs in said tobacco user is
measured. In some methods, the abstinence from conventional tobacco
use is measured. In some methods, a marker of nicotine addiction is
measured (e.g., regional cerebral metabolic rate for glucose and/or
cerebral blood flow, which are measurable using positron emission
tomography (PET)).
[0047] More embodiments include tobacco-use cessation or nicotine
and/or TSNA reduction methods, wherein a tobacco user, who is,
optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
first reduced nicotine and/or TSNA tobacco product, a second
reduced nicotine and/or TSNA tobacco product, and a third reduced
nicotine and/or TSNA tobacco product, wherein the second reduced
nicotine and/or TSNA tobacco product comprises less nicotine and/or
TSNAs than the first reduced nicotine and/or TSNA tobacco product
and the third reduced nicotine and/or TSNA tobacco product
comprises less nicotine and/or TSNAs than the second reduced
nicotine and/or TSNA tobacco product. The first reduced nicotine
and/or TSNA tobacco product (e.g., a cigarette) or tobacco therein
can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g; the second reduced nicotine and/or TSNA tobacco product or
tobacco therein can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g; and the third reduced nicotine and/or TSNA
tobacco product or a tobacco therein can comprise (e.g., on the
leaf or tobacco rod) or deliver (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods), for example, less than or
equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or
a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less
than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0
.mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g so long as the
amount of nicotine and/or TSNAs in or delivered by the second
tobacco product is less than the amount of nicotine and/or TSNAs in
or delivered by the first tobacco product, and the amount of
nicotine and/or TSNAs in or delivered by the third tobacco product
is less than the amount of nicotine and/or TSNAs in or delivered by
the second tobacco product. The first, second, and/or third reduced
nicotine and/or TSNA tobacco products can comprise treated tobacco,
selectively bred low nicotine tobacco, or genetically modified
tobacco or combinations thereof. These tobacco products can also
include exogenous nicotine. In some methods, the reduction in
consumption or exposure to nicotine and/or TSNAs in said tobacco
user is measured. In some methods, the abstinence from conventional
tobacco use is measured. In some methods, a marker of nicotine
addiction is measured (e.g., regional cerebral metabolic rate for
glucose and/or cerebral blood flow, which are measurable using
positron emission tomography (PET)).
[0048] Still more embodiments include tobacco-use cessation or
nicotine and/or TSNA reduction methods, wherein a tobacco user, who
is, optionally, identified as one in need of a reduction in the
consumption or exposure to nicotine and/or TSNAs, is provided a
first reduced nicotine and/or TSNA tobacco product, a second
reduced nicotine and/or TSNA tobacco product, a third reduced
nicotine and/or TSNA tobacco product and a fourth reduced nicotine
and/or TSNA tobacco product, wherein the second reduced nicotine
and/or TSNA tobacco product comprises less nicotine and/or TSNAs
than the first reduced nicotine and/or TSNA tobacco product, the
third reduced nicotine and/or TSNA tobacco product comprises less
nicotine and/or TSNAs than the second reduced nicotine and/or TSNA
tobacco product, and the fourth reduced nicotine and/or TSNA
tobacco product comprises less nicotine and/or TSNAs than the third
reduced nicotine and/or TSNA tobacco product. The first reduced
nicotine and/or TSNA tobacco product (e.g., a cigarette) or a
tobacco therein can comprise (e.g., on the leaf or tobacco rod) or
deliver (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods), for example, less than or equal to 1.0 mg/g, 0.6
mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective content
of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0
.mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, or 0.2 .mu.g/g; the second reduced nicotine and/or TSNA
tobacco product or a tobacco therein can comprise (e.g., on the
leaf or tobacco rod) or deliver (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods), for example, less than or
equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or
a collective content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less
than or equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0
.mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g; the third
reduced nicotine and/or TSNA tobacco product or a tobacco therein
can comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or
0.05 mg/g nicotine and/or a collective content of TSNAs (e.g., NNN,
NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g, 4.0
.mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, 0.5 .mu.g/g, or 0.2
.mu.g/g; and the fourth reduced nicotine and/or TSNA tobacco
product or a tobacco therein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to 1.0
mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine and/or a collective
content of TSNAs (e.g., NNN, NAT, NAB, or NNK) of less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g so long as the amount of
nicotine and/or TSNAs in or delivered by the second tobacco product
is less than the amount of nicotine and/or TSNAs in or delivered by
the first tobacco product, the amount of nicotine and/or TSNAs in
or delivered by the third tobacco product is less than the amount
of nicotine and/or TSNAs in or delivered by the second tobacco
product, and the amount of nicotine and/or TSNAs in or delivered by
the fourth tobacco product is less than the amount of nicotine
and/or TSNAs in or delivered by the third tobacco product. The
first, second, third, and/or fourth reduced nicotine and/or TSNA
tobacco products can comprise treated tobacco, selectively bred low
nicotine tobacco, or genetically modified tobacco or combinations
thereof. These tobacco products can also include exogenous
nicotine. In some methods, the reduction in consumption or exposure
to nicotine and/or TSNAs in said tobacco user is measured. In some
methods, the abstinence from conventional tobacco use is measured.
In some methods, a marker of nicotine addiction is measured (e.g.,
regional cerebral metabolic rate for glucose and/or cerebral blood
flow, which are measurable using positron emission tomography
(PET)).
[0049] In some embodiments, the tobacco-use cessation or nicotine
and/or TSNA reduction kits and tobacco use cessation methods can
also comprise a conventional NRT product (e.g., nicotine patches,
nicotine gum, capsules, inhalers, nasal sprays, and lozenges). That
is, aspects of the invention also include tobacco-use cessation or
nicotine and/or TSNA reduction kits that comprise nicotine patches,
nicotine gum, capsules, inhalers, nasal sprays, and lozenges that
can be used in conjunction with a tobacco product as described
herein. It is contemplated that the ability to quit tobacco use can
be increased by providing a conventional NRT product in conjunction
with one or more of the tobacco products described herein or
supplementing one or more of the tobacco-use cessation methods
described herein with a conventional NRT product and a conventional
NRT nicotine-dependence reduction strategy. For example, a
tobacco-use cessation or nicotine and/or TSNA reduction program can
include the steps of providing a tobacco user who has, optionally,
been identified as one in need of a reduction in conventional
tobacco use one or more of the tobacco products described herein
and a nicotine patch. Preferably, said tobacco user is provided a
plurality of tobacco products described herein and a plurality of
nicotine patches, wherein at least two tobacco products and at
least two nicotine patches have different amounts of nicotine. That
is, in some embodiments, a tobacco user is provided a first tobacco
product that comprises a tobacco that has a reduced amount of
nicotine (e.g., comprising on the leaf or tobacco rod or delivering
in the side-stream or main-stream smoke, as determined by the FTC
and/or ISO methods) less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3
mg/g, or 0.05 mg/g) and a nicotine patch comprising an amount of
nicotine (e.g., 21 mg, 14 mg, or 7 mg).
[0050] In some embodiments, a tobacco user is provided at least two
reduced nicotine tobacco products (e.g., a first tobacco product
comprising on the leaf or tobacco rod or delivering in the
side-stream or main-stream smoke, as determined by the FTC and/or
ISO methods) less than or equal to 1.0 mg/g nicotine and a second
tobacco product comprising (e.g., on the leaf or tobacco rod) or
delivering (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods) less than or equal to 0.6 mg/g nicotine and a
nicotine patch (e.g., 21 mg, 14 mg, or 7 mg nicotine); and, in
other embodiments, a tobacco user is provided at least three
reduced nicotine tobacco products described herein, for example, a
first tobacco product comprising (e.g., on the leaf or tobacco rod)
or delivering (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods) less than or equal to 1.0 mg/g nicotine, a
second tobacco product comprising (e.g., on the leaf or tobacco
rod) or delivering (e.g., side-stream or main-stream smoke by the
FTC and/or ISO methods) less than or equal to 0.6 mg/g nicotine,
and a third reduced nicotine tobacco product comprising (e.g., on
the leaf or tobacco rod) or delivering (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods) less than or equal
to 0.3 mg/g nicotine) and a nicotine patch (e.g., 21 mg, 14 mg, or
7 mg nicotine); and, in some embodiments, a tobacco user is
provided at least four tobacco products described herein, for
example, a first tobacco product comprising (e.g., on the leaf or
tobacco rod) or delivering (e.g., side-stream or main-stream smoke
by the FTC and/or ISO methods) less than or equal to 1.0 mg/g
nicotine, a second tobacco product comprising (e.g., on the leaf or
tobacco rod) or delivering (e.g., side-stream or main-stream smoke
by the FTC and/or ISO methods) less than or equal to 0.6 mg/g
nicotine, a third reduced nicotine tobacco product comprising
(e.g., on the leaf or tobacco rod) or delivering (e.g., side-stream
or main-stream smoke by the FTC and/or ISO methods) less than or
equal to 0.3 mg/g nicotine, and a fourth reduced nicotine tobacco
product comprising (e.g., on the leaf or tobacco rod) or delivering
(e.g., side-stream or main-stream smoke by the FTC and/or ISO
methods) less than or equal to 0.05 mg/g nicotine) and a nicotine
patch (e.g., 21 mg, 14 mg, or 7 mg nicotine). Preferably, a tobacco
user is provided a tobacco product that comprises (e.g., on the
leaf or tobacco rod) or delivers (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods) less than or equal to 0.05
mg/g nicotine and a nicotine patch comprising 21 mg, 14 mg, or 7
mg.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1. An illustration of a QPTase inhibition construct
comprising a QPTase inhibition cassette including full-length
QPTase coding sequence and a GUS selection cassette.
[0052] FIG. 2. An illustration of a QPTase inhibition construct
comprising a QPTase inhibition cassette including a 360 bp fragment
of the QPTase gene and a norflurazone resistance selection cassette
including a mutant phytoene desaturase gene (PDSM-1).
[0053] FIG. 3. An illustration of a PMTase inhibition construct
comprising a PMTase inhibition cassette including a 241 bp fragment
of the PMTase gene and a norflurazone resistance selection cassette
including a mutant phytoene desaturase gene (PDSM-1).
[0054] FIG. 4. An illustration of an A622 inhibition construct
comprising an A622 inhibition cassette including a 628 bp fragment
of the A622 gene and a norflurazone resistance selection cassette
including a mutant phytoene desaturase gene (PDSM-1).
[0055] FIG. 5. An illustration of a QPTase/A622 double inhibition
construct comprising a QPTase/A622 inhibition cassette including a
360 bp fragment of the QPTase gene and a 628 bp fragment of the
A622 gene and a norflurazone resistance selection cassette
including a mutant phytoene desaturase gene (PDSM-1).
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention concerns nicotine reduction and/or
tobacco-use cessation programs, which involve the use of modified
tobacco products that contain reduced amounts of nicotine and/or
TSNAs. While most tobacco cessation programs rely heavily on
nicotine replacement therapy (NRT), many of the embodiments
described herein focus less on nicotine replacement and more on
replacing the secondary factors of addiction such as smoke intake,
oral fixation, and taste. An application entitled "Modifying
Nicotine and Nitrosamine Levels in Tobacco" (WO02100199), which was
published in English designating the United States of America and
claiming priority to U.S. Provisional Application No. 60/371,635,
is hereby incorporated by reference in its entirety. Also
incorporated by reference in their entireties are related U.S. Pat.
Nos. 6,586,661 and 6,423,520.
[0057] Some embodiments of the invention concern the use of low
nicotine and/or TSNA tobacco products that have burning and taste
characteristics that are virtually indistinguishable from
conventional tobacco products. In some embodiments, taste
characteristics are maintained by providing a tobacco product
having an amount of tar similar to the amount of tar in standard
tobacco products. That is, in some embodiments, the amount of tar
in the various reduced nicotine and/or TSNA tobacco products (e.g.,
cigarettes comprising (e.g., on the leaf or tobacco rod) or
delivering (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods) less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3
mg/g, or 0.05 mg/g nicotine) is about the same even though the
amount of nicotine in the tobacco products differs. The term
"about" is used in this context and others to indicate that minor
fluctuations in the levels of tar or other compounds are acceptable
(e.g., in the context of tar, the term can signify that less than
or equal to 1 mg, 0.7 mg, 0.5 mg, 0.25 mg, 0.1 mg, or 0.5 mg of tar
higher or lower than a set value is acceptable). Thus, some
embodiments described herein include tobacco products that comprise
(e.g., on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods) different levels
of nicotine (e.g., less than or equal to 1.0 mg/g, 0.6 mg/g, 0.3
mg/g, or 0.05 mg/g nicotine) and amounts of tar that are within 1
mg, 0.7 mg, 0.5 mg, 0.25 mg, 0.1 mg, or 0.05 mg higher or lower
than a set value (e.g., 9 mg/g, 10 mg/g, 11 mg/g, or 12 mg/g) among
different tobacco products. Tobacco use cessation programs that
utilize these tobacco products are also embodiments.
[0058] While there are many ways to create reduced nicotine and/or
TSNA products, the preferred methods use techniques in plant
genetic engineering to reduce or eliminate enzymes involved in
nicotine biosynthesis. Preferably, techniques in plant genetic
engineering are used to selectively reduce the amount of an enzyme
involved in nicotine biosynthesis, (e.g., the enzyme quinolate
phosphoribosyl transferase (QPTase), which is involved in the
production of nicotine at the root cortex). There may be many ways
to reduce levels of QPTase in tobacco plants, given the teachings
described herein and the level of skill in the art, however, the
preferred methods involve the use of antisense, RNAi,
cosuppression, or molecular decoy technology.
[0059] Several approaches to create tobacco and tobacco products
that have a reduced amount of nicotine and/or TSNAs have been
discovered. Interestingly, it was discovered that TSNA content in a
tobacco plant can be lowered by reducing the nicotine content in
the tobacco plant. In some embodiments, antisense technology is
used to lower nicotine and TSNA levels in tobacco plants. (See
PCT/US98/11893, which is hereby expressly incorporated by reference
in its entirety). In other embodiments, molecular decoy technology
is used to lower nicotine and/or TSNA levels in tobacco plants (See
U.S. patent application Ser. No. 09/941,042, which is hereby
expressly incorporated by reference in its entirety). In some
embodiments, nucleic acid constructs encoding interfering RNAs
(RNAi) comprising a first strand having a sequence substantially
similar or identical to the entire coding sequence of a target gene
and/or target gene product involved in nicotine biosynthesis, and a
second strand that is complementary or substantially complementary
to the first strand, are contemplated.
[0060] In some embodiments, the reduced nicotine tobacco products
of the present invention are made utilizing tobacco that is treated
to reduce its nicotine content after the tobacco has been
harvested. Examples of such treatment include microbial enzymatic
degradation, chemical treatment, high pressure extraction,
reconstitution, and flash curing methods. (E.g., U.S. Pat. No.
4,557,280; U.S. Pat. No. 4,561,452; U.S. Pat. No. 4,848,373; U.S.
Pat. No. 4,183,364; U.S. Pat. No. 4,215,706; U.S. Pat. No.
5,803,081; U.S. Pat. No. 6,202,649; U.S. Pat. No. 6,425,401; U.S.
Pat. No. 5,713,376; U.S. Pat. No. 6,338,348; U.S. Pat. No.
6,834,654; U.S. patent application Ser. No. 10/943,346, and WO
05/018307). In other embodiments, the reduced nicotine and/or TSNA
tobacco products are made from tobacco that has been selectively
bred to reduce its nicotine and/or TSNA content. Accordingly, three
general approaches to reducing nicotine and/or TSNAs are described:
genetic modification, treatment (e.g., chemical or microbial), and
selective breeding of low nicotine plants.
[0061] By a preferred approach, for example, a DNA construct
encoding an antisense RNA that complements at least a portion of
the QPTase gene (SEQ. ID. No. 1) is prepared such that
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. By another approach, transcription
factor molecular decoys for the QPTase gene, which are nucleic acid
fragments that correspond to the 5' upstream regulatory elements
(e.g., Nic 1 and Nic 2 transcription factor binding sites) are
inserted into the plant cell. The transcription factors bind to the
decoy fragments rather than the endogenous transcription factor
binding sites and a reduction in the level of transcription of
QPTase is obtained.
[0062] Once the transgenic tobacco plants having reduced nicotine
are made, the tobacco is harvested and cured by conventional
methods and is incorporated into a variety of tobacco products.
Preferably, the transgenic tobacco is blended such that specific
amounts of nicotine and/or TSNA are obtained in specific products.
That is, the blending is conducted so that tobacco products of
varying amounts of nicotine and/or TSNAs are made. In this manner,
a step-wise tobacco-use cessation program can be established,
wherein a program participant begins the program at step 1 with a
tobacco product having only slightly less nicotine than a
conventional tobacco product; at step 2 the program participant
begins using a tobacco product with less nicotine than the products
used in step 1; and so on, for as many steps as desired for a
particular tobacco-use cessation or nicotine and/or TSNA reduction
program. Ultimately, the tobacco product used by the program
participant can have an amount of nicotine that is less than that
which is required to become addicted or maintain an addiction. In
this manner, a nicotine and/or TSNA reduction and/or tobacco-use
cessation program is provided that limits the exposure of a program
participant to nicotine and/or TSNAs yet retains the secondary
factors of addiction, including but not limited to, smoke intake,
oral fixation, and taste. The following section describes tobacco
products that can be used with the tobacco-use cessation programs
described herein.
[0063] Tobacco Products for Use in Nicotine Reduction and/or
Tobacco-Use Cessation Programs
[0064] Wild type tobacco varies significantly in the amount of
TSNAs and nicotine depending on the variety and the manner it is
grown, harvested, and cured. For example, a typical Burley tobacco
leaf can have about 30,000 parts per million (ppm) nicotine and
8,000 parts per billion (ppb) TSNA; a typical Flue-Cured Burley
leaf can have about 20,000 ppm nicotine and 300 ppb TSNA; and a
typical Oriental cured leaf can have about 10,000 ppm nicotine and
100 ppb TSNA. A tobacco plant or portion thereof having a reduced
amount of nicotine and/or TSNA, for use with aspects of the
invention, can have no detectable nicotine and/or TSNA, or may
contain some detectable amounts of one or more TSNA and/or nicotine
so long as the amount of nicotine and/or TSNA is less than that
found in a control plant of the same variety.
[0065] That is, a Burley tobacco leaf embodiment of the invention
having a reduced amount of nicotine can have between about 0 and
about 30,000 ppm nicotine and about 0 and about 8,000 ppb TSNA
desirably, between about 0 and about 20,000 ppm nicotine and about
0 and about 6,000 ppb TSNA more desirably, between about 0 and
about 10,000 ppm nicotine and about 0 and about 5,000 ppb TSNA
preferably, between about 0 and about 5,000 ppm nicotine and about
0 and about 4,000 ppb TSNA more preferably, between about 0 and
about 2,500 ppm nicotine and about 0 and about 2,000 ppb TSNA even
more preferably, and most preferably between about 0 and about
1,000 ppm nicotine and about 0 and about 1,000 ppb TSNA.
Embodiments of Burley leaf prepared by the methods described herein
can also have between about 0 and about 1000 ppm nicotine and about
0 and about 500 ppb TSNA and some embodiments of Burley leaf
prepared by the methods described herein have virtually no
detectable amount of nicotine or TSNA.
[0066] Similarly, a Flue-cured tobacco leaf for use with the
disclosed methods can have a reduced amount of nicotine, which is
between about 0 and about 20,000 ppm nicotine and about 0 and about
300 ppb TSNA desirably between about 0 and about 15,000 ppm
nicotine and about 0 and about 250 ppb TSNA more desirably between
about 0 and about 10,000 ppm nicotine and about 0 and about 200 ppb
TSNA preferably between about 0 and about 5,000 ppm nicotine and
about 0 and about 150 ppb TSNA more preferably between about 0 and
about 2,500 ppm nicotine and about 0 and about 100 ppb TSNA and
most preferably between about 0 and about 1,000 ppm nicotine and
about 0 and about 50 ppb TSNA. Embodiments of flue-cured tobacco
prepared by the methods described herein can also have between
about 0 and about 500 ppm nicotine and about 0 and about 25 ppb
TSNA and some embodiments of flue-cured tobacco prepared by the
methods described herein have virtually no detectable amount of
nicotine or TSNA.
[0067] Further, an Oriental cured tobacco for use with the embodied
methods can have a reduced amount of nicotine having between about
0 and about 10,000 ppm nicotine and about 0 and about 100 ppb TSNA
desirably between about 0 and about 7,000 ppm nicotine and about 0
and about 75 ppb TSNA more desirably between about 0 and about
5,000 ppm nicotine and about 0 and about 50 ppb TSNA preferably
between about 0 and about 3,000 ppm nicotine and about 0 and about
25 ppb TSNA more preferably between about 0 and about 1,500 ppm
nicotine and about 0 and about 10 ppb TSNA and most preferably
between about 0 and about 500 ppm nicotine and essentially no TSNA.
Embodiments of Oriental cured tobacco prepared by the methods
described herein can also have between about 0 and about 250 ppm
nicotine and essentially no TSNA and some embodiments of Oriental
cured tobacco prepared by the methods described herein have
virtually no detectable amount of nicotine or TSNA.
[0068] 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 a reduced amount of TSNA and nicotine
have tremendous utility. 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.
[0069] It should be emphasized that the phrase "a reduced amount"
is intended to refer to an amount of nicotine and/or TSNA in a
tobacco plant, tobacco, or a tobacco product that is less than that
found in a tobacco plant, tobacco, or a tobacco product from the
same variety of tobacco grown and processed in the same manner,
which has not been treated or was not made transgenic for reduced
nicotine and/or TSNA. Thus, in some contexts, wild-type tobacco of
the same variety that has been grown and processed in the same
manner is used as a control by which to measure whether a reduction
in nicotine and/or TSNA has been obtained.
[0070] In some contexts, the phrase reduced amount of nicotine
and/or TSNAs refers to the tobacco plants, tobacco and tobacco
products of the invention that have less nicotine and/or TSNAs by
weight than the same variety of tobacco grown, processed, and cured
in the same way. A typical cigarette has 11 mg of nicotine and 8
.mu.g of TSNAs. Thus, the tobacco plants, tobacco and tobacco
products of the invention can have, in dry weight for example, less
than or equal to 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 or equal to 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.
[0071] Alternatively, a tobacco product, e.g., a cigarette, for use
in the methods described herein can comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods), for example, less than or equal to
0.01 mg, 0.05 mg, 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, 11.0 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 mg, 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, 11.0 mg, 12.0 mg, 13.0 mg, 14.0
mg, 15.0 mg, 16.0 mg, 17.0 mg, 18.0 mg, 19.0 mg, or 20.0 mg
nicotine and less than or equal to 0.1 micrograms, 0.15 micrograms,
0.2 micrograms, 0.25 micrograms, 0.3 micrograms, 0.35 micrograms,
0.4 micrograms, 0.45 micrograms, 0.5 micrograms, 0.55 micrograms,
0.6 micrograms, 0.65 micrograms, 0.7 micrograms, 0.75 micrograms,
0.8 micrograms, 0.85 micrograms, 0.9 micrograms, 0.95 micrograms,
1.0 micrograms, 1.1 micrograms, 1.15 micrograms, 1.2 micrograms,
1.25 micrograms, 1.3 micrograms, 1.35 micrograms, 1.4 micrograms,
1.45 micrograms, 1.5 micrograms, 1.55 micrograms, 1.6 micrograms,
1.65 micrograms, 1.7 micrograms, 1.75 micrograms, 1.8 micrograms,
1.85 micrograms, 1.9 micrograms, 1.95 micrograms, 2.0 micrograms,
2.1 micrograms, 2.15 micrograms, 2.2 micrograms, 2.3 micrograms,
2.4 micrograms, 2.5 micrograms, 3.0 micrograms, 3.5 micrograms, 4.0
micrograms, 4.5 micrograms, or 5.0 micrograms TSNA (NNN, NNK, NAT,
and NAB).
[0072] Moreover, the tobacco used in the claimed methods can
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, less than or equal to 0.01 mg/g, 0.05 mg/g, 0.1 mg/g,
0.15 mg/g, 0.2 mg/g, 0.25 mg/g, 0.3 mg/g, 0.35 mg/g, 0.4 mg/g, 0.45
mg/g, 0.5 mg/g, 0.55 mg/g, 0.6 mg/g, 0.65 mg/g, 0.7 mg/g, 0.75
mg/g, 0.8 mg/g, 0.85 mg/g, 0.9 mg/g, 0.95 mg/g, 1.0 mg/g, 1.1 mg/g,
1.15 mg/g, 1.2 mg/g, 1.25 mg/g, 1.3 mg/g, 1.35 mg/g, 1.4 mg/g, 1.45
mg/g, 1.5 mg/g, 1.55 mg/g, 1.6 mg/g, 1.65 mg/g, 1.7 mg/g, 1.75
mg/g, 1.8 mg/g, 1.85 mg/g, 1.9 mg/g, 1.95 mg/g, 2.0 mg/g, 2.1 mg/g,
2.15 mg/g, 2.2 mg/g, 2.25 mg/g, 2.3 mg/g, 2.35 mg/g, 2.4 mg/g, 2.45
mg/g, 2.5 mg/g, 2.55 mg/g, 2.6 mg/g, 2.65 mg/g, 2.7 mg/g, 2.75
mg/g, 2.8 mg/g, 2.85 mg/g, 2.9 mg/g, 2.95 mg/g, 3.0 mg/g, 3.1 mg/g,
3.15 mg/g, 3.2 mg/g, 3.25 mg/g, 3.3 mg/g, 3.35 mg/g, 3.4 mg/g, 3.45
mg/g, 3.5 mg/g, 3.55 mg/g, 3.6 mg/g, 3.65 mg/g, 3.7 mg/g, 3.75
mg/g, 3.8 mg/g, 3.85 mg/g, 3.9 mg/g, 3.95 mg/g, 4.0 mg/g, 4.1 mg/g,
4.15 mg/g, 4.2 mg/g, 4.25 mg/g, 4.3 mg/g, 4.35 mg/g, 4.4 mg/g, 4.45
mg/g, 4.4 mg/g, 4.45 mg/g, 4.5 mg/g, 4.55 mg/g, 4.6 mg/g, 4.65
mg/g, 4.7 mg/g, 4.75 mg/g, 4.8 mg/g, 4.85 mg/g, 4.9 mg/g, 4.95
mg/g, 5.0 mg/g, 5.5 mg/g, 5.7 mg/g, 6.0 mg/g, 6.5 mg/g, 6.7 mg/g,
7.0 mg/g, 7.5 mg/g, 7.7 mg/g, 8.0 mg/g, 8.5 mg/g, 8.7 mg/g, 9.0
mg/g, 9.5 mg/g, 9.7 mg/g, 10.0 mg/g, 10.5 mg/g, 10.7 mg/g, 11.0
mg/g, 12.0 mg/g, 13.0 mg/g, 14.0 mg/g, 15.0 mg/g, 16.0 mg/g, 17.0
mg/g, 18.0 mg/g, 19.0 mg/g, or 20.0 mg/g nicotine and less than 0.1
micrograms/g, 0.15 micrograms/g, 0.2 micrograms/g, 0.25
micrograms/g, 0.3 micrograms/g, 0.35 micrograms/g, 0.4
micrograms/g, 0.45 micrograms/g, 0.5 micrograms/g, 0.55
micrograms/g, 0.6 micrograms/g, 0.65 micrograms/g, 0.7
micrograms/g, 0.75 micrograms/g, 0.8 micrograms/g, 0.85
micrograms/g, 0.9 micrograms/g, 0.95 micrograms/g, 1.0
micrograms/g, 1.1 micrograms/g, 1.15 micrograms/g, 1.2
micrograms/g, 1.25 micrograms/g, 1.3 micrograms/g, 1.35
micrograms/g, 1.4 micrograms/g, 1.45 micrograms/g, 1.5
micrograms/g, 1.55 micrograms/g, 1.6 micrograms/g, 1.65
micrograms/g, 1.7 micrograms/g, 1.75 micrograms/g, 1.8
micrograms/g, 1.85 micrograms/g, 1.9 micrograms/g, 1.95
micrograms/g, 2.0 micrograms/g, 2.1 micrograms/g, 2.15
micrograms/g, 2.2 micrograms/g, 2.3 micrograms/g, 2.4 micrograms/g,
2.5 micrograms/g, 2.6 micrograms/g, 2.7 micrograms/g, 2.8
micrograms/g, 2.9 micrograms/g, 3.0 micrograms/g, 3.5 micrograms/g,
4.0 micrograms/g, 4.5 micrograms/g, or 5.0 micrograms/g TSNA (NNN,
NNK, NAT, and NAB).
[0073] It should also be appreciated that in some embodiments, the
tobacco products of the present invention comprise an amount of tar
similar to the amount of tar in standard cigarettes. In such
embodiments, the tobacco product (e.g. a cigarette) can have about
0.5 mg to about 30 mg of tar. Such a tobacco product comprises
(e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream
or main-stream smoke by the FTC and/or ISO methods), for example,
less than or equal to 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5
mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg,
8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5
mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 m,
17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21
mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg,
25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg,
29.5 mg, or 30 mg of tar. Accordingly, some embodiments described
herein include tobacco-use cessation kits and tobacco-use cessation
methods that comprise a plurality of tobacco products, wherein at
least two of said tobacco products comprise or deliver different
amounts of nicotine but the same amounts of tar. For example, some
embodiments include tobacco products that comprise (e.g., on the
leaf or tobacco rod) or deliver (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods) less than or equal to 1.0
mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g nicotine, wherein each
product comprises about 10 mg tar.
[0074] Several methods for reducing endogenous levels of nicotine
and TSNAs in a tobacco plant have been discovered. These approaches
can be used to create the tobacco products described herein.
Tobacco plants having a reduced amount of nicotine and/or TSNAs
that retain good smoking characteristics and taste, as manufactured
by the methods described in the following section, can be used in
the embodiments described herein.
[0075] Approaches to Make Tobacco Products Having Reduced Nicotine
and/or TSNA Levels
[0076] There are many methods for reducing the amount of nicotine
and/or TSNAs present in tobacco and these approaches can be divided
into three general categories: genetic modification, selective
breeding to obtain low nicotine plants, and treatment of tobacco
(e.g., microbe or chemical). (See e.g., U.S. Pat. No. 4,557,280;
U.S. Pat. No. 4,561,452; U.S. Pat. No. 4,848,373; U.S. Pat. No.
4,183,364; U.S. Pat. No. 4,215,706; U.S. Pat. No. 5,803,081; U.S.
Pat. No. 6,202,649; U.S. Pat. No. 6,425,401; U.S. Pat. No.
5,713,376; U.S. Pat. No. 6,338,348; U.S. Pat. No. 6,834,654; U.S.
patent application Ser. No. 10/943,346, and WO 05/018307). In some
embodiments, for example, tobacco can be contacted with an
oxidizing agent (e.g., hydrogen peroxide) such that nicotine is
converted to nicotinic acid. Another example is contacting tobacco
with a substrate (e.g., another tobacco) that has been contacted
with a strong acid (e.g., hydrochloric acid) such that the nicotine
is transferred from the tobacco of interest to the substrate. Yet
another example involves treating tobacco with a hot solution of
potassium metabisulfite and, subsequently, boiled in a solution of
potassium sulfate and potassium nitrate. A final example is
contacting an unharvested tobacco plant with auxin, an auxin
analog, or a jasmonate antagonist.
[0077] Nicotine can also be extracted from tobacco utilizing high
pressure extraction. For example, tobacco can be subjected to a
mixture of nitrogen and carbon dioxide gases (with the nitrogen
being 50-80% of the mixture) at a pressure of 250 to 600 bar at
temperatures greater than 50.degree. C. Under these conditions,
nicotine is removed from the tobacco. The extracted nicotine can be
used in some embodiements that comprise exogenous nicotine, as
described herein.
[0078] Nicotine can also be removed using microbial enzymatic
degradation. For example, tobacco can be contacted with a
microorganism (e.g., Cellulomonas sp. grown in a nitrate-containing
medium) that effectively degrades alkaloids (including nicotine),
resulting in a tobacco with a reduced amount of nicotine and
nitrates.
[0079] Flash curing methods can also be used to reduce the amount
of TSNAs present in tobacco. For example, tobacco can be exposed to
microwave radiation as part of the curing process. Another example
is curing tobacco under conditions that prevent anaerobic
conditions around the tobacco. A curing system utilizing a high
airflow of heated air (e.g. 1000-250.degree. F.) is believed to
result in a tobacco having a lower amount of TSNAs.
[0080] In addition, reconstituted or expanded tobacco can be used
to make reduced nicotine tobacco products. Reconstituted tobacco is
made from tobacco dust and other tobacco scrap material. It is
processed into a sheet and then cut into strips to resemble
tobacco. Expanded tobacco is tobacco that has been processed
through expansion of suitable gasses, leaving the tobacco "puffed"
and therefore having a reduced density and greater filling
capacity. Expanded tobacco can be used, for example, to reduce the
weight of a cigarette which can reduced the amount of nicotine
and/or TSNAs per cigarette.
[0081] Nicotine is produced in tobacco plants by the condensation
of nicotinic acid and 4-methylaminobutanal. Two regulatory loci
(Nic1 and Nic2) act as co-dominant regulators of nicotine
production. These two loci are unlinked and the gene action is
semi-dominant and primarily additive (Legg et al. (1969) J. Hered.,
60, 213-217).
[0082] Genetic and enzyme analyses have been used to investigate
the Nic1 and Nic2 genes. Collins et al. ((1974) Crop Sci., 14,
77-80) prepared doubled haploid tobacco breeding lines of these
four alkaloid genotypes. The genotype of standard cultivars is
Nic1/Nic1 Nic2/Nic2 and that of low nicotine lines is nic1/nic1
nic2/nic2. Nic1/Nic1 nic2/nic2 is a high intermediate and nic1/nic1
Nic2/Nic2 is a low intermediate (Legg and Collins (1971) Can. J
Genet. Cytol. 13, 287-291). These lines are similar in
days-to-flower, number of leaves, leaf size, and plant height.
Enzyme analyses of roots of single and double Nic mutants show that
the activities of two enzymes, quinolate phosphoribosyl transferase
(QPTase) and putrescine methyl transferase (PMTase), are directly
proportional to levels of nicotine biosynthesis (Saunders and Bush
(1979) Plant Physiol 64:236). Both Nic1 and Nic2 affect PMTase and
QPTase activities in roots, and thus, regulate nicotine synthesis
(Leete (1983) In: Alkaloids: Chemical and Biological Perspectives,
S. W. Pelletier, ed. John Wiley & Sons, pp. 85-152).
[0083] Enzyme analyses of roots of single and double Nic mutants
show that the activities of QPTase and PMTase are directly
proportional to levels of nicotine biosynthesis. An obligatory step
in nicotine biosynthesis is the formation of nicotinic acid from
quinolinic acid, which step is catalyzed by QPTase. QPTase appears
to be a rate-limiting enzyme in the pathway supplying nicotinic
acid for nicotine synthesis in tobacco (See, e.g., 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
levels of nicotine in the leaves.
[0084] Hibi et al. ((1994) Plant Cell, 6, 723-735) isolated the
cDNA encoding PMTase, PMT, and showed that PMT transcript levels
are regulated by Nic1 and Nic2. The QPTase cDNA and genomic clones
(NtQPT1) have also been isolated and the transcript levels of
NtQPT1 are also regulated by Nic1 and Nic2. Thus, it appears that
the Nic genes regulate nicotine content by regulating the
transcript levels of genes encoding the two rate-limiting enzymes,
PMTase and QPTase. Further, Nic1 and Nic2 have been shown to be
positive regulators of NtQPT1 transcription and that promoter
sequences upstream of the transcription initiation site contain the
cis-acting sequences necessary for Nic gene product activation of
NtQPT1 transcription. Because expression of QPTase and PMTase are
coordinately-regulated by the Nic gene products, it likely that the
Nic gene products also directly regulate transcription of the PMT
gene.
[0085] It has also been discovered that inhibition of A622 reduces
the amount of nicotine in a tobacco plant. Accordingly, A622
encodes a gene product that regulates the production of nicotine.
In some embodiments that employed the A622 inhibition construct
described herein, transgenic tobacco that had conventional levels
of nicotine but significantly reduced levels of nornicotine were
produced. These lines of tobacco are particularly useful because
nornicotine may be the most significant precursor for NNN in
tobacco. Accordingly, reduced risk conventional cigarettes and
other tobacco products (e.g., snuff) comprising the A622 inhibition
construct are embodiments.
[0086] One approach for reducing nicotine involves reducing the
amount of a required enzyme in the biosynthetic pathway leading to
its production. Where the affected enzyme naturally occurs in a
rate-limiting amount (relative to the other enzymes required in the
pathway), any reduction in that enzyme's abundance will decrease
the production of the end product. If the amount of the enzyme is
not normally rate-limiting, its presence in a cell must be reduced
to rate-limiting levels in order to diminish the pathway's output.
Conversely, if the naturally-occurring amount of enzyme is rate
limiting, then any increase in the enzyme's activity will result in
an increase in the biosynthetic pathway's end product.
[0087] In some embodiments, the tobacco that is substantially free
or comprises a reduced amount of nicotine, nornicotine, TSNAs, is
made by exposing at least one tobacco cell of a selected variety
(e.g., Burley, Virginia Flue, or Oriental) to an exogenous nucleic
acid construct encoding an interfering RNA comprising an RNA duplex
that comprises a first strand having a sequence that is
substantially similar or identical to at least a portion of the
coding sequence of a target gene and/or target gene product
involved in nicotine biosynthesis, and a second strand that is
complementary or substantially complementary to the first strand.
In some embodiments, the nucleic acid construct further comprises a
nucleotide sequence encoding the interfering RNA operably linked to
a promoter operable in a plant cell. The tobacco cell is
transformed with the nucleic acid construct, transformed cells are
selected and at least one transgenic tobacco plant is regenerated
from the transformed cells. The transgenic tobacco plants described
herein can contain a reduced amount of anyone of nicotine,
nornicotine, and/or TSNAs, as compared to a control tobacco plant
of the same variety. In some embodiments, nucleic acid constructs
encoding interfering RNAs (RNAi) comprising a first strand having a
sequence substantially similar or identical to the entire coding
sequence of a target gene and/or target gene product involved in
nicotine biosynthesis, and a second strand that is complementary or
substantially complementary to the first strand, are
contemplated.
[0088] Accordingly, some embodiments concern a tobacco that
comprises a genetic modification comprising a reduced amount or a
reduced level of expression of QPTase, PMTase, A622, or another
enzyme in the plant's nicotine biosynthesis pathway, and/or
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods) a
reduced amount of nicotine or total alkaloid and/or a collective
content of TSNA (e.g., NNN, NAT, NAB, or NNK) of less than or equal
to 2.0 .mu.g/g (e.g., 2.0 .mu.g/g, 1.75 .mu.g/g, 1.5 .mu.g/g, 1.0
g/g, 0.5 .mu.g/g, or 0.2 .mu.g/g). Other embodiments concern a
tobacco that comprises a reduced amount or a reduced level of
expression of A622, a normal or conventional amount of nicotine,
for example, comprising (e.g., on the leaf or tobacco rod) or
delivering (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods) equal to, less than, or greater than 0.9 mg/g,
11.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g, 1.6
mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, 2.0 mg/g, 3.0 mg/g, 4.0 mg/g,
5.0 mg/g, 6.0 mg/g, 7.0 mg/g, or 8.0 mg/g), and a reduced amount of
nornicotine (e.g., less than or equal to 0.5 .mu.g/g, 0.4 .mu.g/g,
0.3 .mu.g/g, 0.2 .mu.g/g, or 0.1 .mu.g/g and/or a reduced amount of
NNN (e.g., less than or equal to 1.0 .mu.g/g, 0.8 .mu.g/g, 0.6
.mu.g/g, 0.4 .mu.g/g. 0.2 .mu.g/g or 0.1 .mu.g/g). That is,
particular lines of transgenic tobacco containing the A622
inhibition cassette described herein were unexpectedly found to
have a reduced level of nornicotine but conventional levels of
nicotine. This finding is particularly important since nornicotine
may be a more important precursor for NNN than nicotine. (See
Carmella et al., Carcinogenesis, Vol. 21, No. 4, 839-843, (April
2000), herein expressly incorporated by reference in its entirety).
In other transgenic lines, wherein the A622 gene was inhibited
using one of the constructs described herein, it was found that
both nicotine and nornicotine were effectively reduced (e.g., total
alkaloids were less than or equal to 7,000 ppm, 5000 ppm, 3000 ppm,
1000 ppm, or 500 ppm).
[0089] In some embodiments, the gene product involved in nicotine
biosynthesis is an enzyme. Such enzymes include, but are not
necessarily limited to, putrescene N-methyltransferase (PMTase),
N-methylputrescene oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase and quinolate phosphoribosyl
transferase (QPTase). In preferred embodiments, the gene product
that is inhibited using a construct described herein is QPTase and
PMTase. In some embodiments, the tobacco that is made substantially
free of nicotine and/or TSNAs (e.g., less than or equal to 0.5 mg/g
nicotine and/or less than or equal to 0.5 .mu.g/g collective
content of NNN, NAT, NAB, and NNK) is prepared from a variety of
Burley tobacco (e.g., Burley 21 LA), Oriental tobacco (Djebal 174),
or Virginia flue (Tn90 or K326) tobacco. It should be understood,
however, that most tobacco varieties (e.g., Burley, Flue, and
Oriental) can be made to have reduced amounts of nicotine and/or
TSNAs or can be made substantially free of nicotine and/or TSNAs by
using the embodiments described herein. For example, plant cells of
the variety Burley 21 LA 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 LA variety
that has a reduced amount of nicotine and/or TSNAs.
[0090] Some of the nucleic acid constructs of the present invention
employ interfering RNAs (e.g., siRNAs or dsRNAs) that comprise an
RNA duplex wherein each RNA portion of the duplex is at least,
greater than, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380,
400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640,
660, 680, 700, 750, 1000, 1500, 2000, 2500, or 5000 consecutive
nucleotides complementary or substantially complementary to an mRNA
that encodes a gene product or the entire coding sequence of the
enzyme or complement thereof of an enzyme that regulates nicotine
biosynthesis. In some embodiments, the RNA duplex comprises a first
RNA strand that is complementary to an mRNA that encodes a gene
product involved in nicotine biosynthesis and a second RNA stand
that is complementary to said first strand. Some interfering RNAs
of the present invention can comprise two separate RNA strands
hybridized to each other by hydrogen bonding. Other interfering
RNAs comprise a single RNA strand comprising a first and second
regions of nucleotide sequence that are complementary to each
other. In such embodiments, the first and second regions of
nucleotide sequence are separated by a nucleotide sequence (e.g., a
"linker") that permits or, in the case of the FAD2 intron described
herein, facilitates formation of a hairpin structure upon
hybridization of the first and second regions. This "linker" that
permits formation of a hairpin structure is preferably at least,
greater than, or equal to 30, 40, 50, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380,
400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640,
660, 680, 700, 800, 900, 1000 or more nucleotides in length.
[0091] The modification of nicotine levels in tobacco plants by
antisense regulation of PMTase expression is proposed in U.S. Pat.
Nos. 5,369,023 and 5,260,205 to Nakatani and Malik, all of which
are hereby expressly incorporated by reference in their entireties.
PCT application WO 94/28142 to Wahad and Malik describes DNA
encoding PMT and the use of sense and antisense PMT constructs.
Additionally, PCT Application WO98/56923 to Conkling et al.
describes DNA encoding a plant QPTase enzyme, constructs comprising
such DNA, and methods of altering QPTase expression to increase or
decrease nicotine production in tobacco plants. Still further, U.S.
patent application Ser. No. 09/941,042 to Conkling, which is hereby
expressly incorporated by reference in its entirety, describes the
use of DNA encoding regulatory sequences for the QPTase enzyme and
methods of using these sequences as molecular decoys to sequester
transcription factors at sites distant to the endogenous promoter
for the QPTase gene, thereby decreasing nicotine production in
tobacco plants.
[0092] Most notably, it is presently revealed that there are
several different PMT genes and each may play a role in nicotine
biosynthesis. Inhibition only one PMT gene may create a leaky
system allowing the other genes to compensate for the reduction.
Accordingly, the PMT constructs described herein were designed to
inhibit a plurality of different PMT genes. That is, the PMT
constructs described herein are designed to complement common
regions to all five of the PMT genes so that inhibition of each of
the PMT genes can be accomplished. 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.
[0093] In some embodiments, the reduced nicotine tobacco products
of the present invention are made utilizing tobacco that is treated
to reduce its nicotine content after the tobacco has been
harvested. Examples of such treatment include 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;
4,215,706; and 5,713,376 all of which are hereby expressly
incorporated by reference in their entireties). Tobacco products
made from reconstituted tobacco are also usable in embodiments of
the present invention, as described above.
[0094] Antisense Technology can be Used to Create Tobacco Products
Having a Reduced Level of Nicotine and/or TSNA
[0095] Antisense technology may be used to create tobacco plants
with reduced nicotine levels. The preferred enzyme for antisense
regulation of nicotine levels is the TobRD2 gene (see Conkling et
al., Plant Phys. 93, 1203 (1990)) encoding a Nicotiana tabacum
QPTase (see Example 1) (SEQ. ID. No. 1). In addition to the
description of the technology provided herein, general aspects of
the technology are described in PCT/US98/11893, which is hereby
expressly incorporated by reference in its entirety.
[0096] 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. A preferred approach for reducing
QPTase levels through molecular modification is provided in Example
2 and Example 3.
[0097] In some methods, the antisense product may be complementary
to coding or non-coding (or both) portions of naturally occurring
target RNA. The antisense construct 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. 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 that it is capable of binding to the endogenous sequence
in the cell to be regulated, under stringent conditions as
described below.
[0098] Although the preferred enzyme for antisense regulation is
QPTase, other enzymes that are suitable for antisense regulation
include, for example, A622, putrescine N-methyltransferase,
N-methylputrescine oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase, and any other enzyme linked
to nicotine biosynthesis.
[0099] As an example of the use of antisense technology, tobacco
having a reduced amount of nicotine and TSNA is generated from a
tobacco plant that is created by exposing at least one tobacco cell
of a selected tobacco variety (preferably Burley 21 LA) 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 (e.g., SEQ. ID. No. 1). 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
TSNA present and these values can be compared to the amount of
nicotine and/or TSNA present in a control tobacco plant or portion,
preferably of the same variety.
[0100] 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, 27, 30, 35, 40, 45, 50, 60, 75, 100, 150,
250, 500, 750, 1000, 1500, 2000, 2500, or 5000 bases, or the entire
coding sequence of the enzyme or complement thereof (e.g., SEQ. ID.
No. 1). Accordingly, these DNA constructs have the ability to
inhibit the production of endogenous enzyme in the nicotine
biosynthesis pathway. It is contemplated that antisense, molecular
decoy, RNAi, and cosuppression constructs are effective at reducing
the levels of nicotine and/or TSNAs in tobacco plants.
[0101] Nucleic acid sequences employed in the constructs described
herein include those with sequence similarity to the gene encoding
QPTase, and encoding a protein having quinolate phosphoribosyl
transferase activity, including, for example, allelic variations in
QPTase proteins. Thus, DNA sequences that hybridize to DNA of the
QPTase-encoding gene and code for expression of QPTase,
particularly plant QPTase enzymes, may also be employed in carrying
out the present invention. Multiple forms of 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.
[0102] As used herein, the term `gene` can refer 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. In some
contexts, a gene can include only (2), above, or some combination
of items (1), (3), and (4) with (2). The DNA sequence of the
present invention may comprise or consist essentially of the
sequence encoding the QPTase enzyme, 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.
[0103] By one approach, a novel cDNA sequence encoding a plant
QPTase may be 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.
[0104] 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, as compared to
unmodified or control tobacco cells and plants.
[0105] Promoters to be linked to the antisense constructs of the
present invention may be constitutively active promoters. Numerous
constitutively active promoters which 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. In the alternative, the promoter may be a root-specific
promoter or root cortex specific promoter and others, as explained
in greater detail below.
[0106] Antisense sequences have been expressed in transgenic
tobacco plants utilizing the Cauliflower Mosaic Virus (CaMV)
.sup.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 Mol.
Biol. 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).
[0107] Use of the CaMV 35S promoter for expression of antisense
QPTase genes 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).
[0108] Other promoters, which 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., Plant Cell, 3:371 (1991)). The
TobRD2 root-cortex specific promoter may also be utilized. (See,
e.g., U.S. patent application Ser. No. 08/508,786 and PCT WO
9705261, hereby expressly incorporated by reference n their
entireties.
[0109] Some of the nucleic acids described herein may also be used
in methods of sense co-suppression or RNAi-mediated suppression of
nicotine production. Sense DNAs employed in these methods are
preferably 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, 25, 27, 30, 35, 40, 45, 50, 60, 75, 100, 150,
250, 500, 750, 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.
[0110] In an alternate embodiment, Nicotiana plant cells are
transformed with a DNA construct containing a DNA segment encoding
an enzymatic RNA molecule termed a "ribozyme", which enzymatic RNA
molecule is directed against and cleaves the mRNA transcript of DNA
encoding plant QPTase as described herein. 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 section below describes yet another
method to decrease levels of specific enzymes involved in nicotine
biosynthesis, using decoy nucleic acid fragments.
[0111] Molecular Decoy Technology to Lower Nicotine and/or TSNA
Levels
[0112] The use of nucleic acid-based decoy fragments to reduce gene
expression is referred to as "molecular decoys". In a preferred
example, the "decoy fragment" corresponds to promoter sequences
upstream of the QPTase gene, to reduce QPTase expression. In other
embodiments, the "decoy fragment" corresponds to promoter sequences
upstream of a gene encoding an enzyme involved in the plant's
nicotine biosynthesis pathway (e.g., A622, QPTase, PMTase,
N-methylputrescene oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase), that reduce the expression
of the given gene.
[0113] In some embodiments, an isolated nucleic acid, or a fragment
thereof consisting of at least 20-450 consecutive nucleotides
desirably, at least 30-400 consecutive nucleotides preferably,
50-350 consecutive nucleotides more preferably, and 100-300 or
200-400 consecutive nucleotides most preferably, that is or
contains at least one cis-acting regulatory element, which exists
upstream of the plant QPTase (e.g., SEQ. ID. No. 1) and/or PMTase
coding sequences. Another example is the Nic gene product
responsive element obtained from the sequence disclosed in U.S.
Pat. No. 5,459,252, herein expressly incorporated by reference in
its entirety. In some embodiments, the Nic gene product responsive
element resides between -1000 and -600 or -700 bp of the NtQPT1
promoter. Accordingly, some embodiments involve a 300-400
nucleotide long fragment of the NtQPT1 promoter that corresponds to
the sequence of the NtQPT1 promoter between -1000 and -600 or -700,
as disclosed in U.S. Pat. No. 5,459,252, herein expressly
incorporated by reference in its entirety.
[0114] Thus, in several embodiments, the embodied nucleic acids
have a structure that promotes an interaction with one or more
transcription factors (e.g., Nic1 and Nic2), which are involved in
initiating transcription of QPTase and/or PMTase. Accordingly, said
nucleic acids are said to be or contain at least one transcription
factor (e.g., Nic1 and Nic2) binding sequences, which are also
referred to as "cis-acting regulatory elements." By introducing
multiple copies of these cis-acting regulatory elements (e.g.,
sequences that interact with Nic1 and/or Nic2) into a plant cell,
the ability of the transcription factor to initiate transcription
of the targeted gene (e.g., QPTase and/or PMTase genes) can be
reduced or squelched.
[0115] By one approach, tobacco plants are transformed with an
excess number of DNA sequences (cis-acting elements) from the
promoters of genes encoding, but not limited to, QPTase and PMTase
that are regulated in nicotine biosynthesis. These cis-acting
elements are preferably integrated into the plant genome so as to
allow for transfer to successive generations. Preferred approaches
are provided in Example 4 and Example 5. Typically, the Nic1 and
Nic2 DNA-binding proteins that interact with these cis-acting DNA
sequences are expressed at relatively low levels in the cell, thus
the excess of transgenic cis-acting elements will compete with the
endogenous elements associated with the genes encoding, but not
limited to, QPTase and PMTase for available Nic1 and Nic2
Accordingly, these cis-acting DNA sequences (and those of other
cis-acting elements) are referred to herein as "decoys" or
"molecular decoys". The competition decreases occupancy of
trans-acting DNA-binding proteins on their cognate cis-acting
elements, thereby down-regulating the synthesis of nicotine
biosynthesis enzymes.
[0116] Embodiments of the present invention also provide DNA
molecules of cis-acting elements of QPTase or PMTase, and vectors
comprising those DNA molecules, as well as transgenic plant cells
and plants transformed with those DNA molecules and vectors.
Transgenic tobacco cells and plants of this invention are
characterized by lower nicotine content than untransformed control
tobacco cells and plants.
[0117] Any of a variety of cis-acting elements can be used in
carrying out the molecular decoy methods, depending upon the
particular application. Examples of cis-acting elements (and
corresponding transcription factors) that may be used, alone or in
combination with one another, which may be used in embodiments of
the present invention include, but are not limited to, AS-1 and
ASF-1 (see U.S. Pat. Nos. 4,990,607 and 5,223,419), the AATT repeat
element and PABF (see U.S. Pat. Nos. 5,834,236 and 6,191,258), a
wounding-responsive cis-acting element from potato (Siebert et al.,
Plant Cell 1:961-8 (1989)), an embryo-specific cis-acting element
from bean (Bustos et al, Plant Cell 1:839-853 (1989)), a
root-specific cis-acting element from the tobacco RB7 promoter
(U.S. Pat. No. 5,459,252 and Yamamoto et al., Plant Cell 3:371-382
(1991)), a positive poly(dA-dT) regulatory element and binding
protein and negative CCCAA repeat element and binding protein (Wang
et al., Mol. Cell Biol. 12:3399-3406 (1992)), a root-tip regulatory
element from the tobacco phytochrome A1 promoter of tobacco (Adam
et al., Plant Mol Biol 29:983-993 (1995)), an
anaerobiosis-responsive element from the maize
glyceraldehyde-3-phosphate dehydrogenase 4 gene (Geffers et al.,
Plant Mol Biol 43:11-21 (2000)), and a seed-specific regulatory
region from an Arabidopsis oleosin gene (see U.S. Pat. No.
5,792,922), all of which are hereby expressly incorporated by
reference in their entireties.
[0118] The status of the art is such that large databases list
identified cis-acting regulatory regions (e.g., Plant Cis-acting
Regulatory elements, "PLACE", with about 1,340 entries, and Plant
Cis-acting Regulatory Elements "PlantCARE", which lists about 159
plant promoters. The listed cis-acting regulatory elements in these
databases and the cis-acting regulatory elements that are provided
in Raumbauts et al., Nucleic acids Research 27:295-296 (1999), and
Higo et al., Nucleic acids Research 27:297-300 (1999) can be used
with embodiments of the invention. Accordingly, the databases and
references above are hereby expressly incorporated by reference in
their entireties. The section below describes general methods for
transformation of tobacco plants with modified sequences (RNAi) to
create tobacco plants with low nicotine and/or TSNA levels.
[0119] Inhibition of Gene Expression Using RNAi
[0120] Inhibition of gene expression refers to the absence or
observable reduction in the level of polypeptide and/or mRNA gene
product. Some embodiments of the present invention relate to
inhibiting the expression of one or more genes involved in the
biosynthesis of nicotine and/or nornicotine by genetically
modifying a plant cell, such as a tobacco cell, by providing the
cell with an inhibitory nucleic acid that reduces or eliminates the
production of a gene product involved in nicotine biosynthesis.
Inhibitory nucleic acids include, but are not limited to,
interfering RNAs, antisense nucleic acids and catalytic RNAs. Some
preferred embodiments of the present invention relate to
interfering RNAs (RNAi).
[0121] Target genes that are involved in nicotine and/or
nornicotine biosynthesis are expressed through the transcription a
first gene product, the target mRNA, which is then translated to
produce a second gene product, the target polypeptide. Thus,
reduction or elimination of the expression of one or more target
genes results in the reduction or elimination of one or more target
mRNAs and/or target polypeptides. Target polypeptides involved in
nicotine and nornicotine biosynthesis include, for example, A622,
PMTase, N-methylputrescene oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase, and QPTase. In a preferred
embodiment, the expression of the QPTase, PMTase, and A622 product
is inhibited using an RNAi construct provided herein.
[0122] Reduction of the expression of one or more target genes
and/or target gene products that are involved in nicotine and/or
nornicotine biosynthesis leads to a reduction in the amount of
nicotine and/or TSNAs produced in tobacco. In certain embodiments,
the expression of one or more target gene products involved in
nicotine and/or nornicotine biosynthesis is eliminated. Elimination
of such target gene products can result in the elimination of
nicotine and/or nornicotine biosynthesis, thereby reducing the
amount of nicotine and/or nornicotine present in tobacco to levels
below the detection limit of methods commonly used. Reduction of
the amount of nicotine and/or nornicotine present in tobacco can
lead to a reduction in the amount of TSNAs produced in the tobacco.
In some embodiments, the amount of TSNA present in tobacco is
reduced to levels below the detection limit of methods commonly
used to detect TSNAs.
[0123] The reduction in or elimination of the expression of target
genes or target gene products involved in nicotine and/or
nornicotine biosynthesis is achieved by providing an interfering
RNA specific to one or more such target genes to a tobacco cell,
thereby producing a genetically modified tobacco cell. The
interfering RNA can be provided as a synthetic double-stranded RNA,
or alternatively, as a nucleic acid construct capable of encoding
the interfering RNA. Synthetic double-stranded interfering RNAs are
taken up by the cell directly whereas interfering RNAs encoded by a
nucleic acid construct are expressed from the construct subsequent
to the entry of the construct inside the cell. The reduction in or
elimination of the expression of the target genes and/or the target
gene products is mediated by the presence of the interfering RNA
inside the cell.
[0124] RNA interference and gene silencing are terms that are used
to describe a phenomenon by which the expression of a gene product
is inhibited by an interfering RNA molecule. Interfering RNA
molecules are double-stranded RNAs (dsRNA) that are expressed in or
otherwise introduced into a cell. The dsRNA molecules may by of any
length, however, short dsRNA constructs are commonly used. Such
constructs are known as small interfering RNAs (siRNA), and can be
21-23 bp in length.
[0125] RNA interference is exhibited by nearly every eukaryote and
is thought to function by a highly conserved mechanism (Dillin, A.
PNAS, 100:6289-91). As with antisense inhibition of gene
expression, inhibition mediated by RNA interference is gene
specific. However, in contrast to antisense-mediated inhibition,
inhibition mediated by interfering RNA appears to be inherited
(Dillin, A. PNAS, 100:6289-91). Without being bound by theory, it
is believed that specificity is achieved through nucleotide
sequence interaction between complementary portions of a target
mRNA and the interfering RNA. The target mRNA is selected based on
the specific gene to be silenced. In particular, the target mRNA,
corresponds to the sense strand of the gene to be silenced. An
interfering RNA, such as a dsRNA or an siRNA, comprises an RNA
duplex, which includes a first strand that is substantially similar
or identical to at least a portion of the nucleotide sequence of
the target mRNA, and a second strand having a nucleotide sequence
that is complementary or substantially complementary to the first
strand.
[0126] When used herein with reference to an RNA duplex of the
interfering RNA, it will be appreciated that the terms "first
strand" and "second strand" are used in a relative sense. For
example, the first strand of an RNA duplex can be selected to
comprise either a nucleotide sequence substantially similar or
identical to at least a portion of the nucleotide sequence of the
target mRNA or a nucleotide sequence that is complementary or
substantially complementary to at least a portion of the nucleotide
sequence of the target mRNA. If the first strand is selected to be
substantially similar or identical to at least a portion of the
nucleotide sequence of the target mRNA, then the second strand will
be complementary to at least a portion of the target mRNA because
it is complementary to the first strand. If the first strand is
selected to be complementary or substantially complementary to at
least a portion of the target mRNA, then the second strand will be
substantially similar or identical to at least a portion of the
nucleotide sequence of the target mRNA because it is complementary
to the first strand.
[0127] In general, the interfering RNAs that are produced inside
the cell, whether expressed from a nucleic acid construct or
provided as synthetic double-stranded RNA molecules, include an RNA
duplex having a first and second strand. At least a portion the
first strand of the duplex is substantially similar or identical to
at least a portion of a target mRNA or a target gene involved in
nicotine biosynthesis. Correspondingly, at least a portion of the
second strand of the duplex is complementary or substantially
complementary to the first strand, and thus, at least a portion of
the second strand is complementary or substantially complementary
to at least a portion of the mRNA encoded by the target gene. In
some embodiments, the interfering RNA can comprise a first strand
that is substantially similar or identical to the entire coding
sequence of the target gene or target mRNA involved in nicotine
biosynthesis and a second strand complementary or substantially
complementary to the first strand.
[0128] As used herein with reference to nucleic acids, "portion"
means at least 5 consecutive nucleotides, at least 6 consecutive
nucleotides, at least 7 consecutive nucleotides, at least 8
consecutive nucleotides, at least 9 consecutive nucleotides, at
least 10 consecutive nucleotides, at least 11 consecutive
nucleotides, at least 12 consecutive nucleotides, at least 13
consecutive nucleotides, at least 14 consecutive nucleotides, at
least 15 consecutive nucleotides, at least 16 consecutive
nucleotides, at least 17 consecutive nucleotides, at least 18
consecutive nucleotides, at least 19 consecutive nucleotides, at
least 20 consecutive nucleotides, at least 21 consecutive
nucleotides, at least 22 consecutive nucleotides, at least 23
consecutive nucleotides, at least 24 consecutive nucleotides, at
least 25 consecutive nucleotides, at least 30 consecutive
nucleotides, at least 35 consecutive nucleotides, at least 40
consecutive nucleotides, at least 45 consecutive nucleotides, at
least 50 consecutive nucleotides, at least 60 consecutive
nucleotides, at least 70 consecutive nucleotides, at least 80
consecutive nucleotides, at least 90 consecutive nucleotides, at
least 100 consecutive nucleotides, at least 125 consecutive
nucleotides, at least 150 consecutive nucleotides, at least 175
consecutive nucleotides, at least 200 consecutive nucleotides, at
least 250 consecutive nucleotides, at least 300 consecutive
nucleotides, at least 350 consecutive nucleotides, at least 400
consecutive nucleotides, at least 450 consecutive nucleotides, at
least 500 consecutive nucleotides, at least 600 consecutive
nucleotides, at least 700 consecutive nucleotides, at least 800
consecutive nucleotides, at least 900 consecutive nucleotides, at
least 1000 consecutive nucleotides, at least 1200 consecutive
nucleotides, at least 1400 consecutive nucleotides, at least 1600
consecutive nucleotides, at least 1800 consecutive nucleotides, at
least 2000 consecutive nucleotides, at least 2500 consecutive
nucleotides, at least 3000 consecutive nucleotides, at least 4000
consecutive nucleotides, at least 5000 consecutive nucleotides or
greater than at least 5000 consecutive nucleotides. In some
preferred embodiments, a portion of a nucleotide sequence is
between 20 and 25 consecutive nucleotides. In other preferred
embodiments, a portion of a nucleotide sequence is between 21 and
23 consecutive nucleotides. In some embodiments, a portion of a
nucleotide sequence includes the full-length coding sequence of the
gene or the target mRNA.
[0129] Some preferred interfering RNAs that are described herein
comprise an RNA duplex, which comprises a nucleotide sequence that
is substantially similar or identical to at least a portion of the
coding strand of a gene involved in nicotine biosynthesis. Although
nucleic acid sequences that are substantially similar or identical
to at least a portion of the coding strand of the target gene
involved in nicotine biosynthesis are preferred, it will be
appreciated that nucleotide sequences with insertions, deletions,
and single point mutations relative to the target sequence are also
effective for inhibition of gene expression. Sequence identity may
determined by sequence comparison and alignment algorithms known in
the art (see Gribskov and Devereux, Sequence Analysis Primer,
Stockton Press, 1991, and references cited therein) and calculating
the percent difference between the nucleotide sequences by, for
example, the Smith-Waterman algorithm as implemented in the BESTFIT
software program using default parameters (e.g., University of
Wisconsin Genetic Computing Group). Greater than 90% sequence
identity, or even 100% sequence identity, between the interfering
RNA and a portion of the target gene is preferred. In especially
preferred embodiments, at least about 21 to about 23 contiguous
nucleotides in the target gene are greater than 90% identical to a
sequence present in the interfering RNA.
[0130] In other embodiments, the duplex region of the RNA may be
defined functionally as including a nucleotide sequence that is
capable of hybridizing with a portion of the target gene
transcript. Exemplary hybridization conditions are 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C.
hybridization for 12-16 hours; followed by washing.
[0131] As described above, interfering RNAs disclosed herein
comprise a sequence that is complementary to at least a portion of
the sense strand of a gene encoding a target mRNA, which produces a
polypeptide that is involved in nicotine biosynthesis. Preferred
targets are the products of the A622 gene, quinolate
phosphoribosyltransferase (QPTase) gene and the putrescene
N-methyltransferase (PMTase) gene. However, it will be appreciated
that interfering RNAs specific for other gene products or
combinations of gene products involved in nicotine and/or
nornicotine biosynthesis can be generated using the teachings
herein.
[0132] Additionally, the interfering RNAs described herein can
comprise a plurality nucleotide sequences that are each
complementary to different portions of the sense strand of a gene
involved in nicotine and/or nornicotine biosynthesis.
Alternatively, the interfering RNAs described herein can comprise a
plurality nucleotide sequences that are each complementary to at
least a portion of the sense strands of different genes involved in
nicotine biosynthesis. Still further, a single RNAi construct or
inhibition cassette can be used to inhibit a plurality of genes
involved in the regulation of the production of nicotine and/or
nornicotine. For example, as described below, it was found that the
A622 inhibitory fragment and inhibition cassette (SEQ. ID. Nos. 2
and 3) efficiently reduced production of nicotine and nornicotine
in some lines of tobacco and in other lines of tobacco conventional
levels of nicotine were maintained but the amount of nornicotine in
said tobacco was 0.00 mg/g. Still further, the PMTase inhibitory
sequence and PMTase inhibition cassette (SEQ. ID. Nos. 4 and 5)
were designed to complement common regions of a plurality of PMTase
genes so that the production of multiple gene products can be
inhibited or reduced with a single construct.
[0133] In preferred embodiments, the interfering RNAs described
herein comprise at least one region of double-stranded RNA (duplex
RNA). This duplex RNA can range from about 10 bp in length to about
10,000 bp in length. In some embodiments, the duplex RNA ranges
from about 15 bp in length to about 1500 bp in length. In other
embodiments, the duplex RNA ranges from about 20 bp in length to
about 1200 bp in length. In still other embodiments, the duplex RNA
ranges from about 21 bp in length to about 23 bp in length. In a
preferred embodiment, the duplex RNA has a length of 22 bps. Short
regions of duplex RNA are often designated siRNA, whereas longer
regions of RNA duplex are often termed dsRNA. In some embodiments,
the interfering RNA duplex region is a dsRNA. In other embodiments,
the interfering RNA duplex region is an siRNA. In a preferred
embodiment, the duplex region about the length of the coding
sequence of a target mRNA encoding a polypeptide involved in
nicotine biosynthesis.
[0134] Interfering RNAs described herein can be generated using a
variety of techniques. For example, an interfering RNA can be
generated in a host cell in vivo by providing the cell with one or
more a nucleic acid constructs that comprise the nucleic acids
necessary to encode the strands of a double-stranded RNA. Such
constructs can be included in various types of vectors. Exemplary
vectors contemplated herein include, but are not limited to,
plasmids, viral vectors, viroids, replicable and nonreplicable
linear DNA molecules, replicable and nonreplicable linear RNA
molecules, replicable and nonreplicable circular DNA molecules and
replicable and nonreplicable circular RNA molecules. Preferred
vectors include plasmid vectors, especially vector systems derived
from the Agrobacterium Ti plasmid, such as pCambia vectors and
derivatives thereof.
[0135] In some embodiments, both strands of the double-stranded
region of the interfering RNA can be encoded by a single vector. In
such cases, the vector comprises a first promoter operably linked
to a first nucleic acid which is substantially similar or identical
to at least a portion of the target mRNA. The vector also comprises
a second promoter operably linked to a second nucleic acid, which
is complementary or substantially to the first nucleic acid.
[0136] Another type of single vector construct, which can be used
to generate interfering RNA, encodes a double-stranded RNA hairpin.
In such embodiments, the vector comprises a promoter operably
linked to a nucleic acid that encodes both strands of the duplex
RNA. The first nucleotide sequence, which encodes the strand that
is substantially similar or identical to at least a portion of the
target mRNA, is separated from the second nucleotide sequence,
which encodes a strand complementary or substantially complementary
to the first strand, by a region of nucleotide sequence that does
not substantially hybridize with either of the strands. This
nonhybridizing region permits the RNA sequence transcribed from the
vector promoter to fold back on itself, thereby permitting the
complementary RNA sequences to hybridize so as to produce an RNA
hairpin. Vectors comprising a plurality of nucleic acids, each of
which encode both strands of the duplex RNA are also
contemplated.
[0137] Other embodiments relate to multiple vector systems for the
production of interfering RNA. In one example, a multiple vector
system is used to produce a single interfering RNA that is specific
for a single gene product involved in nicotine biosynthesis. In
such embodiments, at least two vectors are used. The first vector
comprises a promoter operably linked to a first nucleic acid that
encodes a first strand of the RNA duplex that is present in the
interfering RNA. The second vector comprises a promoter operably
linked to a second nucleic acid that encodes the second strand of
the RNA duplex, which is complementary to the first strand.
[0138] Other multiple vector systems are combinations of vectors,
wherein each vector in the system encodes a different interfering
RNA. Each of the interfering RNAs is specific for different gene
products involved in nicotine biosynthesis. In some embodiments,
the vectors in a multiple vector system can encode different
interfering RNAs that are specific to different portions of a
single gene product involved in nicotine biosynthesis.
[0139] It will be appreciated that the promoters used in the
above-described vectors can either be constitutive or regulated.
Constitutive promoters are promoters that are always expressed. The
constitutive promoters selected for use in the above-described
vectors can range from weak promoters to strong promoters depending
on the desired amount of interfering RNA to be produced. Regulated
promoters are promoters for which the desired level of expression
can be controlled. An example of a regulated promoter is an
inducible promoter. Using an inducible promoter in the
above-described vector constructs permits expression of a wide
range of concentrations of interfering RNA inside a cell.
[0140] It will also be appreciated that there is no requirement
that the same or same types of promoters be used in vectors or
multiple vector systems that comprise a plurality of promoters. For
example, in some vectors or vector systems, a first promoter, which
controls the expression of the first interfering RNA strand, can be
an inducible promoter, whereas the second promoter, which controls
the expression of the second RNA strand, can be a constitutive
promoter. This same principal can also be illustrated in a multiple
vector system. For example, a multiple vector system may have three
vectors each of which includes one or more different types of
promoters. Such a system can include, for example, a first vector
having repressible promoter that controls the expression of an
interfering RNA specific for a first gene product involved in
nicotine biosynthesis, a second vector having a constitutive
promoter that controls the expression of an interfering RNA
specific for a second gene product involved in nicotine
biosynthesis and a third vector having an inducible promoter that
controls the expression of an interfering RNA specific for a third
gene product involved in nicotine biosynthesis.
[0141] In other embodiments, interfering RNAs can be produced
synthetically and introduced into a cell by methods known in the
art. Synthetic interfering RNAs can include a variety of RNA
molecules, which include, but are not limited to, nucleic acids
having at least one region of duplex RNA. The duplex RNA in such
molecules can comprise, for example, two antiparallel RNA strands
that form a double-stranded RNA having flush ends, two antiparallel
RNA strands that form a double-stranded RNA having at least one end
that forms a hair pin structure, or two antiparallel RNA strands
that form a double-stranded RNA, wherein both ends form a hair pin
structure. In some embodiments, synthetic interfering RNAs comprise
a plurality of RNA duplexes.
[0142] The regions of RNA duplex in synthetic interfering RNAs can
range from about 10 bp in length to about 10,000 bp in length. In
some embodiments, the duplex RNA ranges from about 15 bp in length
to about 1500 bp in length. In other embodiments, the duplex RNA
ranges from about 20 bp in length to about 1200 bp in length. In
still other embodiments, the duplex RNA ranges from about 21 bp in
length to about 23 bp in length. In a preferred embodiment, the
duplex RNA has a length of 22 bps. In preferred embodiments,
synthetic interfering RNAs are siRNAs. In another preferred
embodiment, the synthetic interfering RNA is an siRNA specific for
the coding sequence of a target mRNA encoding a polypeptide
involved in nicotine biosynthesis.
[0143] Some aspects of the present invention relate to interfering
nucleic acids that are not comprised entirely of RNA. Still other
aspects relate to interfering nucleic acids that do not comprise
any RNA. Such interfering nucleic acids are synthetic interfering
RNA analogs. These analogs substantially mimic the specificity and
activity of interfering RNA from which they are modeled; however,
they typically include additional properties which make their use
desirable. For example, one or both strands of the interfering
nucleic acid may contain one or more nonnatural nucleotide bases
that improve the stability of the molecule, enhance that affinity
of the molecule for the target mRNA and/or enhance cellular uptake
of the molecule. Other modifications are also contemplated. For
example, an interfering nucleic acid can include one or more
nucleic acid strands composed of naturally-occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
non-naturally-occurring nucleobases, sugars and covalent
internucleoside linkages.
[0144] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming nucleic acids, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn the respective ends of this
linear polymeric structure can be further joined to form a circular
structure. Within the nucleic acid structure, the phosphate groups
are commonly referred to as forming the internucleoside backbone of
the oligonucleotide. The normal linkage or backbone of RNA and DNA
is a 3' to 5' phosphodiester linkage.
[0145] Specific examples of interfering nucleic acids useful in
certain embodiments can include one or more nucleic acid strands
containing modified backbones or non-natural internucleoside
linkages. As used herein, nucleic acids having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone.
[0146] In some embodiments, modified nucleic acid backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates and borano-phosphates having normal 3'-5'
linkages, 2'-5' linked analogs of these, and those having inverted
polarity wherein one or more internucleotide linkages is a 3' to
3', 5' to 5' or 2' to 2' linkage. Certain nucleic acids having
inverted polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0147] In some embodiments, modified nucleic acid backbones that do
not include a phosphorus atom therein have backbones that are
formed by short chain alkyl or cycloalkyl internucleoside linkages,
mixed heteroatom and alkyl or cycloalkyl internucleoside linkages,
or one or more short chain heteroatomic or heterocyclic
internucleoside linkages. These include those having morpholino
linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones;
formacetyl and thioformacetyl backbones; methylene formacetyl and
thioformacetyl backbones; riboacetyl backbones; alkene containing
backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts.
[0148] In other embodiments, the interfering nucleic acid can
comprise one or more mimetic regions, wherein both the sugar and
the internucleoside linkage, i.e., the backbone, of the nucleotide
units are replaced with novel groups. In such embodiments, the base
units are maintained for hybridization with an appropriate nucleic
acid target compound. One such compound, a mimetic that has been
shown to have excellent hybridization properties, is referred to as
a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone
of an oligonucleotide is replaced with an amide containing
backbone, in particular an aminoethylglycine backbone. The
nucleobases are retained and are bound directly or indirectly to
aza nitrogen atoms of the amide portion of the backbone.
Representative United States patents that teach the preparation of
PNA compounds include, but are not limited to, U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262, each of which is herein
expressly incorporated by reference in its entirety. Further
teaching of PNA compounds can be found in Nielsen et al., Science,
1991, 254, 1497-1500.
[0149] In still other embodiments, interfering nucleic acids may
include nucleic acid strands having phosphorothioate backbones
and/or heteroatom backbones. Modified interfering nucleic acids may
also contain one or more substituted sugar moieties. In some
embodiments, the interfering nucleic acids comprise one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2 and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. Another modification
includes 2'-methoxyethoxy (2' OCH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78, 486-504).
[0150] Embodiments also include Locked Nucleic Acids (LNAs), which
generate interfering nucleic acids having enhanced affinity and
specificity for the target polynucleotide. LNAs are nucleic acid in
which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom
of the sugar ring thereby forming a bicyclic sugar moiety. The
linkage is preferably a methelyne (--CH.sub.2--).sub.n group
bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1
or 2. LNAs and preparation thereof are described in WO 98/39352 and
WO 99/14226, the disclosures of which are incorporated herein by
reference in their entireties.
[0151] Other modifications include 2'-methoxy (2'-O--CH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Interfering nucleic acids
may also have sugar mimetics such as cyclobutyl moieties in place
of the pentofuranosyl sugar.
[0152] The interfering nucleic acids contemplated herein may also
include nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and
natural nucleobases such as 5-methylcytosine, 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl uracil and cytosine and other alkynyl derivatives of
pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine cytidine
(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine
cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps
such as a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5, 4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC
Press, 1993, the disclosures of which are incorporated herein by
reference in their entireties. Certain of these nucleobases are
particularly useful for increasing the binding affinity of the
interfering nucleic acids described herein. These include
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., eds., Antisense Research and Applications, CRC Press,
Boca Raton, 1993, pp. 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0153] Another modification of the interfering nucleic acids
described herein involves chemically linking to at least one of the
nucleic acid strands one or more moieties or conjugates which
enhance the activity, cellular distribution or cellular uptake of
the of the interfering nucleic acid. The interfering nucleic acids
can include conjugate groups covalently bound to functional groups
such as primary or secondary hydroxyl groups. Conjugate groups
include intercalators, reporter molecules, polyamines, polyamides,
polyethylene glycols, polyethers, groups that enhance the
pharmacodynamic properties of nucleic acids, and groups that
enhance the pharmacokinetic properties of such molecules. Typical
conjugates groups include cholesterols, lipids, phospholipids,
biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,
fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance
the pharmacodynamic properties, in the context of this invention,
include groups that improve interfering nucleic acid uptake,
enhance its resistance to degradation, and/or strengthen
sequence-specific hybridization with target molecules. Groups that
enhance the pharmacokinetic properties, in the context of this
invention, include groups that improve the uptake, distribution,
metabolism or excretion of the interfering nucleic acid. Conjugate
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., dihexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0154] As described above, it is not necessary for all positions in
a given compound to be uniformly modified, and in fact, more than
one of the aforementioned modifications may be incorporated in a
single compound or even at a single nucleoside within an nucleic
acid. The methods described herein also contemplate the use of
interfering nucleic acids which are chimeric compounds. "Chimeric"
interfering nucleic acid compounds or "chimeras," as used herein,
are interfering nucleic acid compounds, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of a nucleic acid compound.
These interfering nucleic acids typically contain at least one
region wherein the nucleic acid is modified so as to confer upon
the interfering nucleic acid increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
nucleic acid may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby contributes further to the inhibition of
gene expression by the interfering nucleic acid.
[0155] The above-described interfering nucleic acids may be
conveniently and routinely made through the well-known technique of
solid phase synthesis. Equipment for such synthesis is sold by
several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art
may additionally or alternatively be employed. It is well known to
use similar techniques to prepare nucleic acids such as the
phosphorothioates and alkylated derivatives. The interfering
nucleic acid compounds for use with the methods described herein
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other compound. Although terms, such as
interfering RNA, dsRNA and siRNA, are used throughout the remainder
of the specification, it will be appreciated that in the context of
synthetically produced interfering nucleic acids, that such terms
are meant to include interfering nucleic acids of all types,
including those which incorporate modifications, such as those
described above.
[0156] The reduction in or elimination of the expression of genes
and/or gene products involved in nicotine and/or nornicotine
biosynthesis can be characterized by comparing the amount of
nicotine and/or nornicotine produced in genetically modified cells,
with the amount of nicotine and/or nornicotine produced in cells
that have not been genetically modified. Alternatively, such
reduction in or elimination of gene expression can be characterized
by genetically analyzing plant cells so as to determine the level
of mRNA present in the genetically modified plant cell as compared
to a non-modified plant cell. Depending on the assay, quantitation
of the amount of gene expression allows one to determine a degree
of reduction in gene expression, which can be greater than 10%,
25%, 33%, 50%, 90%, 95% or 99% as compared to an untreated cell. As
with nicotine and nornicotine, the reduction in or elimination of
TSNA production in tobacco can be characterized by comparing the
amount of TSNAs produced genetically modified cells, with the
amount of TSNAs produced in cells that have not been genetically
modified. The section below provides more description of the
transgenic plants and cells of the invention.
[0157] Transgenic Plant Cells and Plants
[0158] 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. 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, or equivalent nucleotide sequences
representing alleles or polymorphic variants of these genes, or
coding regions thereof. Use of the phrase "substantial sequence
similarity" or "substantially similar" 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.
[0159] As used herein, a "native nucleotide sequence" or "natural
nucleotide sequence" means a nucleotide sequence that can be
isolated from non-transgenic cells or tissue. Native nucleotide
sequences are those which have not been artificially altered, such
as by site-directed mutagenesis. Once native nucleotide sequences
are identified, nucleic acid molecules having native nucleotide
sequences may be chemically synthesized or produced using
recombinant nucleic acid procedures as are known in the art. As
used herein, a "native plant nucleotide sequence" is that which can
be isolated from non-transgenic plant cells or tissue. As used
herein, a "native tobacco nucleotide sequence" is that which can be
isolated from non-transgenic tobacco cells or tissue. Use of the
phrase "isolated" or "substantially pure" in the present
specification and claims as a modifier of nucleic acids,
polypeptides or proteins means that the nucleic acids, polypeptides
or proteins so designated have been separated from their in vivo
cellular environments through the efforts of human beings.
[0160] DNA constructs, or "transcription cassettes," of the present
invention may 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 for polyadenylase.
[0161] The term "operatively associated," as used herein, refers to
nucleotide sequences on a single nucleic acid molecule which are
associated so that the function of one is affected by the other.
Thus, a promoter is operatively associated with a nucleotide
sequence when it is capable of affecting the transcription of that
sequence (i.e., the nucleotide sequence is under the
transcriptional control of the promoter). The promoter is said to
be "upstream" from the transcribed nucleotide sequence, which is in
turn said to be "downstream" from the promoter.
[0162] In embodiments, wherein a termination signal is used, 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 octopine 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. Alternatively, if
nicotine levels are decreased by molecular decoy technology rather
than by antisense or other methods, the molecular decoy fragments,
with or without additional sequences, may be provided to the plant
cell by any means. For example, the molecular decoy fragment may
have an accompanying gene encoding a selectable marker, other
suitable genes, or may be present as part of a plasmid vector. The
molecular decoy fragment may consist of a single or double stranded
DNA or RNA molecule. The molecular decoy may be integrated into the
genome or may exist freely in the cell.
[0163] 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 ColE1, 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; may provide
complementation, by imparting prototrophy to an auxotrophic host;
or may provide a visible phenotype through the production of a
novel compound in the plant.
[0164] 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
exemplified by J. Sambrook et al., Molecular Cloning, A Laboratory
Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).
[0165] Vectors which 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 together with regions
involved in the control of protein translation and may include
coding sequences.
[0166] The recombinant DNA molecules and vectors used to produce
the transformed tobacco cells and plants 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.
[0167] Aspects of the present invention concern transgenic plant
cells comprising one or more interfering RNAs that are capable of
reducing or eliminating the expression of one or more target genes
and/or target gene products involved in nicotine and/or
nornicotine, biosynthesis. As described above, an appropriate
interfering RNA comprises a duplex RNA that comprises a first
strand that is substantially similar or identical to at least a
portion of a target gene or target mRNA, which encodes a gene
product involved in nicotine, and/or nornicotine biosynthesis. The
RNA duplex also comprises a second strand that is complementary or
substantially complementary to the first strand. Examples 6-18
describe the use of several preferred RNAi constructs.
[0168] The interfering RNA or nucleic acid construct comprising the
interfering RNA can be introduced into the plant cell in any
suitable manner. Plant cells possessing stable interfering RNA
activity, for example, by having a nucleic acid construct stably
integrated into a chromosome, can be used to regenerate whole
plants using methods known in the art. As such, some aspects of the
present invention relate to plants, such as tobacco plants,
transformed with one or more nucleic acid constructs and/or vectors
which encode at least one interfering RNA that is capable of
reducing or eliminating the expression of a gene product involved
in nicotine biosynthesis. Transgenic tobacco cells and the plants
described herein are characterized in that they have a reduced
amount of nicotine, nornicotine, and/or TSNA and/or generate a
reduced amount of PAHs upon pyrolysis, as compared to unmodified or
control tobacco cells and plants.
[0169] 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.
[0170] Gene silencing has been employed in several laboratories to
create transgenic plants characterized by lower than normal amounts
of specific gene products. As used herein, "exogenous" or
"heterologous" nucleic acids, including DNAs and/or RNAs, refer to
nucleic acids that have been introduced into a cell (or the cell's
ancestor) through the efforts of humans. The nucleic acid
constructs that are used with the transgenic plants and the methods
for producing the transgenic plants described herein encode one or
more interfering RNA constructs comprising regulatory sequences,
which include, but are not limited to, a transcription initiation
sequence ("promoter") operable in the plant being transformed, and
a polyadenylation/transcription termination sequence. Typically,
the promoter is located upstream of the 5'-end of the nucleotide
sequence to be expressed. The transcription termination sequence is
generally located just downstream of the 3'-end of the nucleotide
sequence to be transcribed.
[0171] In some preferred embodiments, the nucleic acid encoding the
exogenous interfering RNA, which is transformed into a tobacco
cell, comprises a first RNA strand that is identical to the an
endogenous coding sequence of a gene encoding a gene product
involved in nicotine biosynthesis. However, minor variations
between the exogenous and endogenous sequences can be tolerated. It
is preferred, but not necessarily required, that the
exogenously-produced interfering RNA sequence, which is
substantially similar to the endogenous gene coding sequence, be of
sufficient similarity to the endogenous gene coding sequence, such
that the complementary interfering RNA strand is capable of binding
to the endogenous sequence in the cell to be regulated under
stringent conditions as described below.
[0172] In some embodiments, the heterologous sequence utilized in
the methods described herein may be selected so as to produce an
interfering RNA product comprising a first strand that is
substantially similar or identical to the entire mRNA sequence of a
gene involved in nicotine biosynthesis, or to a portion thereof,
and a second strand that is complementary to the mRNA sequence, or
to a portion thereof. The interfering RNA may be complementary to
any contiguous sequence of the natural messenger RNA. For example,
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.
[0173] The nucleotide sequences provided herein, such as
interfering RNAs or nucleic acids encoding interfering RNAs, can be
transformed into a variety of host cells. As used herein,
"transformation" refers to the introduction of exogenous nucleic
acid into cells so as to produce transgenic cells stably
transformed with the exogenous nucleic acid. A variety of suitable
host cells, having desirable growth and handling properties, are
readily available in the art.
[0174] Standard techniques, such as restriction mapping, Southern
blot hybridization, polymerase chain reaction (PCR) and/or
nucleotide sequence analysis can be employed to identify clones
expressing the desired interfering RNA construct. Following the
introduction and verification of the desired interfering RNA or
nucleic acid construct encoding the desired interfering RNA, whole
plants can be regenerated from successfully transformed cells using
conventional techniques.
[0175] "Transcription cassettes" encoding the interfering RNAs that
are used to produce the transgenic cells and plants of the present
invention include, 5' to 3' in the direction of transcription, a
promoter as described herein, a nucleotide sequence as described
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).
[0176] 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.
[0177] Nucleic acid 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)).
[0178] Use of the CaMV 35S promoter for expression of interfering
RNAs 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)). 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. All patents cited herein are intended to be incorporated
herein by reference in their entirety.
[0179] The recombinant interfering nucleic acid 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).
Preferred selectable markers include the norflurazone resistance
genes described in this disclosure. 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.
[0180] 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 5268463, hereby expressly incorporated by
reference in their entireties) can be used.
[0181] Microparticles suitable for the ballistic transformation of
a plant cell, carrying a nucleic acid 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 nucleic acid construct may be deposited on
the microparticle by any suitable technique, such as by
precipitation.
[0182] Plant species may be transformed with the interfering RNA,
nucleic acid construct encoding an interfering RNA, or other DNA
constructs of the present invention by the nucleic acid-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 nucleic acid-containing liposomes or with nucleic
acid 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)).
[0183] These inhibition constructs or RNAi constructs can be
transferred to plant cells by any known method in the art.
Preferably, Agrobacterium-mediated or Biolistic-mediated
transformation are used, according to well-established protocols.
It is also contemplated that Transbacter-mediated transformation
can be used, as described below. (See Broothaerts et al., Nature
433, 629 (2005), herein expressly incorporated by reference in its
entirety).
[0184] 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.
[0185] Numerous Agrobacterium vector systems useful in carrying out
the present invention are known. For example, U.S. Pat. No.
4,459,355, herein expressly incorporated by reference, 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, herein expressly incorporated
by reference. Further, U.S. Pat. No. 4,940,838 to Schilperoort et
al., herein expressly incorporated by reference, 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.
[0186] 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 desired sequence in transgenic tobacco plants
can be verified by Mendelian inheritance of the desired 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.
[0187] By this approach, first bacteria are prepared as follows. YM
plus antibiotic plates (see below) are streaked with bacteria and
the plates are incubated for 2-3 days at 28.degree. C.
Transformation is accomplished by measuring about 20 mL Minimal A
medium for each bacterial strain. Scrapping or washing the Scrape
or wash bacteria from plate with sterile loop and then suspending
said bacteria in 20 mL of Minimal A medium. The cell density is
adjusted to an OD600 0.9-1.0.
[0188] Next, the first healthy fully expanded leaves from 4-5 week
old tissue culture grown tobacco plants are cut into 0.5 cm squares
(or can use a cork borer, which is about 1.0 cm diameter) in deep
petri dish, under sterile RMOP liquid medium. The tissue pieces are
stored in RMOP in a deep petri dish. The leaf pieces (about 20 per
transformation) are then transferred to a deep petri dish
containing bacterial suspension. To ensure that the bacteria have
contacted a cut edge of the leaf, the suspension with leaf cutting
is swirled and is left standing for 5 minutes. The leaf pieces are
then removed from the suspension and blotted dry on filter paper or
on the edge of the container. The leaf pieces are then placed with
adaxial side (upper leaf surface) on solid RMOP at about 10 pieces
per plate. The plates are then incubated in the dark at 28.degree.
C. for: 2-3 days, if A. tumefaciens is used, 5 days if S.
melilotiis used, 5 days M. loti is used, and 5-11 days if Rhizobium
sp. NGR234 is used.
[0189] Over the next week, selection is performed. For the purposes
of this example, hygromycin selction is performed. Accordingly, the
leaf pieces are transferred onto solid RMOP-TCH, with abaxial
surface (lower surface of leaf) in contact with media. The plates
are incubated for 2-3 weeks in the light at 28.degree. C., with 16
hours daylight per day. Subculture occurs every 2 weeks.
[0190] Plantlet formation is accomplished as follows. Once shoots
appear, the plantlet is transferred to MST-TCH pots. The plantlets
are grown with 16 hours daylight for 1-2 weeks. Once roots form the
plants appear, the plants can be transferred to soil in the
greenhouse. TABLE-US-00001 Media and Solutions for Tobacco
Transformation: YM Media (1 L) Mannitol 10 g Yeast extract 0.4 g
K2HPO4 (10% w/v stock) 1 ml KH2PO4 (10% w/v stock) 4 ml NaCl (10%
w/v stock) 1 ml MgSO4.7H2O (10% w/v stock) 2 ml pH 6.8 Agar 15 g/L
Autoclave *When ready to pour add antibiotic selection if required
Keep poured plates for 2 days at room temperature to visualize any
contamination, then store at 4.degree. C.
RMOP+RMOP-TCH Media
[0191] (Svab, Z., et al., 1975. Transgenic tobacco plants by
cocultivation of leaf disks with pPZP Agrobacterium binary vectors.
In "Methods in Plant Molecular Biology-A Laboratory Manual", P.
Maliga, D. Klessig, A. Cashmore, W. Gruissem and J. Varner, eds.
Cold Spring Harbor Press: 55-77), herein expressly incorporated by
reference in its entirety). TABLE-US-00002 1 L Final conc. Sucrose
30 g (3%) Myo-inositol 100 mg (0.1%) MS Macro 10x 100 mL (1x) MS
Micro 1000x 1 mL (1x) Fe2EDTA Iron 100x 10 mL (1x) Thiamine-HCl (10
mg/mL stock) 100 .mu.L (1 mg) NAA (1 mg/mL stock) 100 .mu.L 0.1 mg)
BAP (1 mg/mL stock) 1 mL (1 mg) pH 5.8 Phytagel 2.5 g/L for solid
autoclave *for RMOP-TCH, when ready to pour add: Timentin (200
mg/mL stock) 1 mL, Claforan (250 mg/mL stock) 1 mL, and Hygromycin
(50 mg/mL stock) 1 mL
BAP (1 mg/ml) (6-Benzylaminopurine) Add 1N KOH drop wise to 100 mg
BAP until dissolved. Make up to 100 mL with Milli-Q H2O and store
at 4.degree. C. NAA (1 mg/ml) (Naphthalene Acetic Acid) Dissolve
100 mg NAA in 1 mL absolute ethanol. Add 3 mL 1N KOH. Make up to 80
mL with Milli-Q H.sub.2O. Adjust pH to 6.0 with 1N HCl, make up to
100 mL with Milli-Q H.sub.2O, and store at 4.degree. C.
Cefotaxamine (250 mg/ml) Add 8 ml sterile Milli-Q H.sub.2O to 2 g
Claforan and store at 4.degree. C. in dark Timentin (200 mg/ml) Add
15 ml sterile Milli-Q H2O to 3 g Timentin and store at 4.degree. C.
MST+MST-TCH Media
[0192] (Svab, Z., et al., 1975. Transgenic tobacco plants by
cocultivation of leaf disks with pPZP Agrobacterium binary vectors.
In "Methods in Plant Molecular Biology-A Laboratory Manual", P.
Maliga, D. Klessig, A., Cashmore, W. Gruissem and J. Vamer, eds.
Cold Spring Harbor Press: 55-77), herein expressly incorporated by
reference in its entirety). TABLE-US-00003 1 L Final concentration
Sucrose 30 g (3%) MS Macro 10x 100 mL (1x) MS Micro 1000x 1 mL (1x)
Fe2EDTA Iron 100x 10 mL (1x) pH 5.8 Phytagel 2.5 g/L Autoclave For
MST-TCH, when ready to pour add: Timentin (200 mg/mL stock) (1 mL)
Cefotaxamine (250 mg/mL stock) (1 mL) Hygromycin (50 mg/mL stock)
(1 mL)
[0193] MS Macro 10.times. ((Murashige and Skoog., Phys. Plant. 15:
473-497 (1962), herein expressly incorporated by reference in its
entirety)). TABLE-US-00004 Final concentration 10x (g/L) KNO3 19.0
NH4 N03 16.5 CaCl2.2H2O 4.4 MgS04.7H2O 3.7 KH2PO4 1.7 Store
4.degree. C.
Substituting Chemicals: CaCl2 3.3 g/L MgS04 1.8 g/L MS Micro
1000.times.
[0194] (Murashige and Skoog., Phys. Plant. 15: 473-497 (1962),
herein expressly incorporated by reference in its entirety).
TABLE-US-00005 Final concentration 1000x (g/L) MnS04.4H20 22.3
ZnS04.7H20 8.6 H3BO3 6.2 KI 0.83 Na2MoO4.2H2O 0.25 CuSO4.5H2O 25 mg
CoCl2.6H2O 25 mg Store 4.degree. C.
Substituting Chemicals:
[0195] MnS04.H20 16.9/L TABLE-US-00006 FeSO4EDTA Iron 100x (g/1 L)
FeS04.7H20 2.78 Na2EDTA 3.72 Store 4.degree. C. in dark bottle
[0196] Once the transformed cells are selected, by any of the
approaches described above, they 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 of an interfering RNA or a
nucleic acid encoding an interfering RNA in transgenic tobacco
plants can be verified by Mendelian inheritance of the interfering
RNA or a nucleic acid encoding an interfering RNA sequence, as
revealed by standard methods of nucleic acid analysis applied to
progeny resulting from controlled crosses. After regeneration of
transgenic tobacco plants from transformed cells, the introduced
nucleic acid sequence can be readily transferred to other tobacco
varieties through conventional plant breeding practices and without
undue experimentation.
[0197] For example, to analyze the segregation of the transgene,
regenerated transformed plants (TO) may be grown to maturity,
tested for nicotine and/or TSNA levels, and selfed to produce
T.sub.1 plants. A percentage of T.sub.1 plants carrying the
transgene are homozygous for the transgene. To identify homozygous
T.sub.1 plants, transgenic T.sub.1 plants are grown to maturity and
selfed. Homozygous T.sub.1 plants will produce T.sub.2 progeny
where each progeny plant carries the transgene; progeny of
heterozygous T.sub.1, plants will segregate 3:1.
[0198] Any plant tissue capable of subsequent clonal propagation,
whether by organogenesis or embryogenesis, may be transformed with
a nucleic acid embodiment of the present invention. Preferred
plants for introduction of a nucleic acid embodiment, described
herein, include Nicotiana. Preferred varieties of Nicotana for
introduction of a nucleic acid embodiment as described herein
include the Nicotiana tabacum varieties provided in Table 1.
TABLE-US-00007 TABLE 1 Burley Dark One Newest Varieties Varieties
Flu Cured Other Virginia Hybrid Sucker Varieties Oriental KT 200
BLACK K 149 CU 748 BROWN NBH 98 OS400 GL 350 D174 LC MAMMOTH LEAF
MS LIZARD KT 204 DF 485 K 326 GL 737 TAIL 21 .times. KY KY LC
ORNOCO 10 160 LIZARD KY DF 911 K 346 OX 207 TAIL MS TURTLE 14
.times. KY FOOT L 8 KY 10 DT 508 K 394 PVH 03 M and N TN 97 KY 14
DT 518 K 730 PVH 09 SHIREY KT 200 Coker 371 PVH WALKER KY 17 DT 592
Gold 2040 BROADLEAF KY 907 GREEN CU 748 RG 17 WOOD KY 907 IMPROVED
GL 737 RG 81 LC MADOLE KY 908 KT-D4 LC GL 939 RGH 4 KY 908 KY 160
GL 973 RGH 51 KY 910 KY 171 K 358 RS 1410 MS KY 171 K 399 Speight
Burley 21 .times. KY 10 168 MS KY14 .times. L8 LITTLE NC 102
Speight CRITTENDEN 179 N 126 LITTLE NC 291 Speight WOOD 190 NARROW
N 777 LEAF NC 297 Speight MADOLE 196 N 88 NEWTON'S NC 55 Speight VH
MADOLE 200A NBH 98 NL MADOLE NC 606 Speight 210 TN 86 TN D94 NC 71
Speight 218 TN 86 LC TN D950 NC 72 Speight 220 TN 90 TR MADOLE NC
810 Speight H-20 TN 90 LC VA 309 RGH 4 Speight H-6 TN 97 LC VA 312
RGH 51 Speight NF-3 VA 509 VA 355 VA 119 LA21 VA 359 NC 37 NF Ox
414 NF Sp. G- 172
[0199] 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).
[0200] 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.
[0201] 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, aspects of the present
invention provide a method of producing a crop of plants having
reduced amounts of nicotine and/or nornicotine, as compared to a
similar crop of non-transformed plants of the same species and
variety.
[0202] The modified 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). The harvested
tobacco leaves and stems are suitable for conventional methods of
processing such as curing and blending. The modified tobacco is
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.
[0203] Some embodiments concern the production and identification
of particular lines of a transgenic Burley variety (Vector 21-41),
which have very low levels of nicotine and TSNAs. The constructs
used to create these particular lines of transgenic Burley tobacco
are provided in Conkling et al., WO98/56923; U.S. Pat. Nos.
6,586,661; 6,423,520; and U.S. patent application Ser. Nos.
09/963,340; 10/356,076; 09/941,042; 10/363,069; 10/729,121;
10/943,346, all of which are hereby expressly incorporated by
reference in their entireties. After the creation and analysis of
nearly 2,000 lines of transgenic Burley tobacco, these particular
lines of reduced nicotine and TSNA transgenic tobacco were
identified. Tobacco harvested from these lines were incorporated
into tobacco products (Quest 1.RTM., Quest 2.RTM., and Quest
3.RTM.) and were analyzed for their ability to reduce the potential
to contribute to a tobacco-related disease, as described in the
sections above. It was found that tobacco products comprising these
lines of transgenic Burley tobacco, had a reduced potential to
contribute to a tobacco-related disease (i.e., that these tobacco
products are reduced risk tobacco products).
[0204] Several embodiments concern isolated nucleic acids that
comprise, consist, or consist essentially of the nucleic acids
described in the sequence listing (SEQ. ID. NOs.: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, or 26) and fragments thereof at least 30 consecutive
nucleotides in length. That is, embodiments of the invention
include an isolated nucleic acid comprising, consisting of,
consisting essentially of, any one or more of the sequences of SEQ.
ID. NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, or a fragment thereof
(e.g., a fragment that is at least, less than or equal to or
greater than 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,
200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440,
460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700,
800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,
1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,
3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000,
4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100,
5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200,
6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300,
7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400,
8500, 8600, 8700, 8800, 8900, or 9000 consecutive nucleotides of
SEQ. ID. NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.
[0205] In preferred embodiments, the target gene or target mRNA
encodes QPTase, PMTase, or the A622 gene product. In preferred
embodiments, an interfering RNA comprises, consists, or consists
essentially of an RNA strand that is complementary to at least a
portion (e.g., less than, greater than or equal to 30, 35, 40, 45,
50, 60, 75, 100, 150, 250, 500, 750, or 1000 consecutive
nucleotides) of SEQ ID NOS: 1, 6, 4, or 2, and inhibits the
production of QPTase, PMTase, A622, nicotine, nornicotine, NNN,
NNK, NAT, or NAB in a tobacco. In related embodiments, the
interfering RNA comprises, consists, or consists essentially of an
RNA strand that is complementary to each least a portion (e.g.,
less than, greater than or equal to 30, 35, 40, 45, 50, 60, 75,
100, 150, 250, 500, 750, or 1000 consecutive nucleotides) of SEQ ID
NO: 2, and inhibits production of nornicotine but not nicotine in a
tobacco.
[0206] Some of these nucleic acid embodiments comprise, consist, or
consist essentially of fragments of the QPTase, PMTase, and A622
genes that were found to inhibit gene expression unexpectedly well
in the RNAi constructs described herein, producing reduced alkaloid
tobacco (below 7,000 ppm, 1,000 ppm, or 500 ppm).
[0207] Still more of the nucleic acid embodiments concern several
phytoene desaturase (PDS) mutants (e.g., PDSM-1, PDSM-2, and
PDSM-3, SEQ. ID. NOs.: 7, 8, or 9) that were developed to confer
resistance to norflurazone, which allows both tissue-culture
selection of cells transformed with the construct, as well as,
field-based selection, wherein weeds and tobacco, which do not
contain an herbicide resistance gene, are removed from the field or
crop by spraying the herbicide norflurazone or an herbicide of the
same class or activity (e.g., herbicides that contain
C.sub.12H.sub.9ClF.sub.3N.sub.3O (see U.S. Pat. No. 3,644,355,
herein expressly incorporated by reference in its entirety), but
plants expressing PDSM-1, PDSM-2, or PDSM-3 survive the herbicide
contact). That is, some embodiments include isolated nucleic acids
that comprise, consist, or consist essentially of the PDS mutant
sequences provided by SEQ. ID. NOs.:7, 8, or 9 and fragments
thereof at least 30 nucleotides in length (e.g., less than, greater
than or equal to 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500,
750, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1729
consecutive nucleotides) that include a mutation (e.g., T1478G,
which encodes Val493Gly; G863C, which encodes Arg288Pro; and
T1226C, which encodes Leu409Pro) that confers resistance to
norflurazone). Preferably, the fragments of the PDS mutants
described herein confer resistance to norflurazone, although
fragments that do not confer resistance to the herbicide are also
useful in the field in assays designed to follow the retention of
constructs described herein in successive generations of transgenic
plants. Approaches to develop more norflurazone-resistance genes
are also provided herein.
[0208] Additional embodiments include isolated nucleic acids that
comprise, consist, or consist essentially of root-specific
promoters, constitutive promoters, and developmentally regulated
promoters, which can be used interchangeably with the nucleic acid
sequences described herein. Some embodiments, for example, include
a root-specific promoter such as the truncated RD2 promoter (SEQ.
ID NO. 10) or the Putrescene methyl transferase promoter (PMT-1)
(SEQ. ID NO. 11). Constitutive promoters that can be used with
embodiments described herein include the GapC promoter (SEQ. ID.
NO.: 12), Actin 2 promoter (Act2P) (SEQ. ID NO. 13), the tobacco
alcohol dehydrogenase promoter (ADP) (SEQ. ID NO. 14), and the
Arabidopsis ribosomal protein L2 promoter (RPL2P) (SEQ. ID NO. 15).
Developmentally regulated promoters that can be used with the
nucleic acid sequences described herein include the cinnamyl
alcohol dehydrogenase promoter (SEQ. ID NO. 16) and the
metallothionein I promoter (SEQ. ID NO. 17). Additional embodiments
also include isolated nucleic acids that comprise, consist, or
consist essentially of the GAD2 terminator (SEQ. ID NO. 18), a FAD2
intron (provided by (SEQ. ID NO. 19), which was used as a spacer in
several of the RNAi constructs, and the PAP1 intron (provided by
nucleotides 6446-7625 of (SEQ. ID NO. 20). Because of the unique
properties of the FAD2 intron, in particular the hair-pin secondary
structure afforded by the interaction of splice sites in the
sequence, it was found, unexpectedly, that transgenic tobacco could
be made with various inhibitory sequences with nearly equivalent
success (e.g., approximately 50% of the reduced nicotine lines
created by multiple constructs were found to have less than 1,000
ppm total alkaloid). Accordingly, significantly improved RNAi
constructs were generated using this spacer. That is, aspects of
the invention concern the use of an intronic sequence comprising
splicing recognition sequences (preferably FAD2 or PAP1 intron) to
link or join a first RNA sequence to a second RNA sequence that is
complementary to said first RNA sequence, wherein said first or
second RNA sequence is complementary to a target RNA, which,
preferably, regulates the production of a harmful compound in
tobacco (e.g., nicotine or nornicotine).
[0209] Aspects of the invention also concern isolated nucleic acids
that comprise, consist, or consist essentially of the inhibition
and selection cassettes identified as SEQ. ID. Nos. 21, 22, 5, 3,
23, 24, or 25 and fragments thereof (e.g., a fragment that is at
least, less than or equal to or greater than 30, 40, 50, 60, 70,
80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320,
340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580,
600, 620, 640, 660, 680, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,
2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500,
3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600,
4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700,
5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800,
6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900,
8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, or 9000
consecutive nucleotides) of SEQ. ID. Nos. 21, 22, 5, 3, 23, 24, or
25).
[0210] Aspects of the invention also concern isolated nucleic acids
that comprise, consist, or consist essentially of a plurality of
the nucleic acid sequences described herein. For example, a double
knock-out construct comprising a portion of the A622 gene and a
portion of the QPTase gene has been made and it is expected that
this construct will efficiently reduce expression of at least two
genes involved in the synthesis or regulation of the production of
nicotine (SEQ. ID. No. 23). Accordingly, aspects of the invention
concern an isolated nucleic acid construct that inhibits the
expression of a plurality of genes that regulate the production of
nicotine. In some aspects of these embodiments, said isolated
nucleic acid construct inhibits the expression of at least two
nicotine biosynthesis genes.
[0211] It should also be understood that aspects of the invention
concern tobacco generated by crossing the transgenic tobaccos
described herein. For example, some embodiments concern progeny of
a cross between a transgenic tobacco having a reduced amount of
nicotine created by different methods. Crossings of the transgenic
tobacco described herein and wild-type tobacco are also aspects of
the invention.
[0212] The interfering RNAs used with the embodied nucleic acids
can be expressed from nucleic acid construct that encodes one or
more strands of the RNA duplex of the interfering RNA. In some
embodiments, the nucleic acid construct is present on a vector. The
vectors may be viral vectors, plasmids, or any other vehicles for
nucleic acid delivery. In other embodiments, the interfering RNAs
described herein can be generated synthetically by methods, such as
direct synthesis or in vitro transcription. In some embodiments,
synthetic interfering nucleic acids comprising modified nucleic
acids are contemplated. Other embodiments of the present invention
include multiple vector systems for producing an interfering RNA
wherein a first vector encodes the first strand of the interfering
RNA and a second vector encodes the second strand of the
interfering RNA.
[0213] Still other embodiments relate to tobacco cells comprising
one or more of the nucleic acid constructs described herein, which
encode an interfering RNA that is specific for a gene product
involved in nicotine biosynthesis. In such embodiments, the
interfering RNA reduces or eliminates the expression of such gene
product. Additional embodiments relate to tobacco cells comprising
one or more interfering RNAs that are specific for a gene product
involved in nicotine biosynthesis. In certain embodiments, the
interfering RNAs are synthetic interfering RNAs.
[0214] Some embodiments relate to tobacco plants and cured tobacco
products having a reduced amount or nicotine, nornicotine, and/or
TSNAs. In such embodiments, reduction in nicotine, nornicotine,
and/or TSNA amounts in the tobacco plants and cured tobacco
products is mediated by an interfering RNA comprising an RNA duplex
wherein at least 30 consecutive nucleotides (e.g., at least or
equal to 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,
220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460,
480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 800,
900, 1000 consecutive nucleotides) of the RNA duplex are
complementary or substantially complementary to a target mRNA that
encodes a gene product involved in nicotine biosynthesis. Further
aspects relate to a field or crop of tobacco plants comprising one
or more of the constructs described herein. Still other aspects
relate to a tobacco seed produced from one or more of the tobacco
plants of the present invention.
[0215] Transgenic tobacco plants produced by the methods described
herein can be cured by any of the tobacco curing techniques that
are known in the art. As such, some embodiments relate to cured
tobacco and cure tobacco products made from the transgenic plants
described herein. In some embodiments, the cured tobacco product is
a blended tobacco product. In some embodiments, the cured tobacco
product is processed in a microbe-free environment. In other
embodiments, the cured tobacco is contacted with sterilizing vapor,
heat, or radiation so as to prevent the conversion of alkaloid to
TSNAs.
[0216] Some aspects of the present invention relate to methods of
preparing a tobacco cell having a reduced nicotine content, wherein
the method comprises providing a tobacco cell with one or more
interfering RNAs or one or more nucleic acid constructs encoding an
interfering RNA comprising an RNA duplex, which comprises a first
strand having a sequence substantially similar or identical to at
least a portion of the coding sequence of a target gene and/or
target gene product involved in nicotine and/or nornicotine
biosynthesis, and a second strand that is complementary or
substantially complementary to the first strand. In a preferred
embodiment, the target gene product involved in nicotine
biosynthesis is QPTase, PMTase, or A622.
[0217] Other aspects of the present invention relate to methods of
preparing a tobacco plant having a reduced nicotine content
comprising obtaining a tobacco cell in culture; providing to the
tobacco cell one or more interfering RNAs or one or more nucleic
acid constructs encoding an interfering RNA comprising an RNA
duplex, which comprises a first strand having a sequence
substantially similar or identical to at least a portion of the
coding sequence of a target gene and/or target gene product
involved in nicotine biosynthesis, and a second strand that is
complementary or substantially complementary to the first strand;
allowing expression of the interfering RNA, thereby reducing
cellular nicotine content; and regenerating a tobacco plant from
the tobacco cell. In some embodiments, the tobacco plants prepared
by such method also have a reduced TSNA content, as compared to a
conventional tobacco product of he same class, a reference tobacco
product (e.g., IM16), or the same strain of tobacco prior to
genetic modification.
[0218] Preparation of Preferred Transgenic Tobaccos
[0219] A first generation of transgenic Burley tobacco was created
using a full-length antisense QPTase construct. 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 (SEQ. ID. NO. 1) 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, and the expression 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)).
[0220] 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.
[0221] Nicotine levels of T.sub.1 progeny segregating 3:1 were
measured qualitatively using a micro-assay technique. Approximately
.about.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 H2O+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.
[0222] 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."
[0223] 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.
[0224] 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.
[0225] 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.
[0226] Vector 21-41 was also grown on approximately 5000 acres by
greater than 600 farmers in five states (Pennsylvania, Mississippi,
Louisiana, Iowa, and Illinois). The US Department of Agriculture,
Agriculture Marketing Service (USDA-AMS) quantified nicotine levels
(expressed as percent nicotine per dry weight) using the FTC method
of 2,701 samples taken from these farms. Nicotine levels ranged
from 0.01% to 0.57%. The average percent nicotine level for all
these samples was 0.09%, with the median of 0.07%. Burley tobacco
cultivars typically have nicotine levels between 2% and 4% dry
weight (Tso, T. C., 1972, Physiology and Biochemistry of Tobacco
Plants. Dowden, Hutchinson, and Ross, Inc. Stroudsbury).
[0227] A transgenic Flue-cured tobacco with a reduced amount of
nicotine and TSNAs was created using an RNAi approach. FIG. 1
illustrates an RNAi construct that was used to create a reduced
nicotine tobacco, wherein the root-specific promoter RD2 (Bp
1-2010) was used to drive expression of an RNAi cassette comprising
an antisense full-length QPTase cDNA (Bp 2011-3409) linked to a 382
bp fragment of the cucumber aquaporin gene (Bp 3410-3792), which is
linked to a sense full-length QPTase cDNA (Bp 3793-5191) and the
GapC terminator (Bp5192-5688) (see SEQ. ID. No. 21). This first
RNAi construct also comprises a GUS-selection cassette comprising
the GapC promoter (Bp 1-1291), which drives expression of the GUS
gene (Bp 1292-3103), linked to the GapC terminator (Bp 3104-3600)
(see SEQ. ID. No. 24). This first RNAi construct was ligated into a
binary vector, pBin19 which was then introduced into Agrobacterium
tumefaciens. Leaf disks from flue-cured variety K326 were then
transformed with Agrobacterium that contained the RNAi construct
comprising the RNAi cassette and the GUS selection cassette.
GUS-based selection was then employed to select positively
transformed plantlets (buds), which were then regenerated to
plants. Leaf samples were then harvested and the alkaloid content
was then determined. The alkaloid content of samples obtained from
some of the transgenic lines created with this first RNAi construct
was 6000 ppm. Since the total alkaloid content in tobacco is about
90% nicotine, it is understood by those skilled in the art that the
transgenic Flue-cured tobacco created using the construct shown in
FIG. 1 has reduced levels of nicotine, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0228] FIG. 2 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a QPTase inhibition cassette (SEQ. ID. No. 22)
and a norflurazone selection cassette (SEQ. ID. No. 25). Starting
from the right border (RB), the QPTase inhibition cassette
comprises an RD2 promoter (Bp 1-2010) operably linked to an
antisense fragment (360 bp) (Bp 2011-2370) of the QPTase gene,
joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense
fragment of the QPTase gene (360 bp) (Bp 3502-3861), which is
joined to the GAD2 terminator (Bp 3862-4134). The selection
cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked
to a mutant phytoene desaturase gene (PDSM 1) (Bp 1162-2890) joined
to the GapC terminator (Bp 2891-3387) at the left border (LB).
[0229] Flue-cured tobacco was transformed with the construct shown
in FIG. 2 using Agrobacterium-mediated transformation and 1,140
independent lines were selected, regenerated, and transplanted in
the greenhouse. Of the 1,140 independent lines, 1,097 plants were
harvested and tested for alkaloid content. A total of 608 lines
were identified as having less than 1,000 ppm total alkaloid and
139 lines were identified as having less than 500 ppm total
alkaloid. Accordingly, the transgenic Flue-cured tobacco created
using the construct shown in FIG. 2 has significantly reduced
levels of nicotine and TSNA, as compared to a conventional tobacco,
a reference tobacco, or the parental strain of tobacco prior to
genetic modification.
[0230] Burley tobacco was also transformed with the construct shown
in FIG. 2 using Agrobacterium-mediated transformation and 385
independent lines were selected, regenerated, and transplanted in
the greenhouse. Of the 385 independent lines, 350 lines of plants
were harvested and tested for alkaloid content. A total of 142
lines were identified as having less than 1,000 ppm total alkaloid
and 10 lines were identified as having less than 500 ppm total
alkaloid. Accordingly, it is understood by those skilled in the art
that the transgenic Burley tobacco created using the construct
shown in FIG. 2 also has significantly reduced levels of nicotine
and TSNA, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
[0231] Oriental tobacco was transformed with the construct shown in
FIG. 2 using Agrobacterium-mediated, Transbacter-mediated or
biolistic transformation and 61 independent lines were selected,
regenerated, and transplanted in the greenhouse. All 61 lines were
tested for alkaloid content and a total of 10 lines were identified
as having less than 1,500 ppm total alkaloids and a total of 3
lines were identified as having less than 1,000 ppm total alkaloid.
Accordingly, it is understood by those skilled in the art that the
transgenic Oriental tobacco created using the construct shown in
FIG. 2 also has significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0232] FIG. 3 illustrates another RNAi construct that can be used
to create a reduced nicotine and TSNA transgenic tobacco. This RNAi
construct has a PMTase inhibition cassette (SEQ. ID. No. 5) and a
norflurazone selection cassette (SEQ. ID. No. 25). Starting from
the right border (RB), the PMTase inhibition cassette comprises an
RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic
acid (241 bp) (Bp 2011-2251) of a PMTase gene, joined to a FAD2
intron (Bp 2252-3382), which is joined to a sense nucleic acid of
the PMTase gene (241 bp) (Bp 3383-3623), which is joined to the
GAD2 terminator (Bp 3624-3896). The selection cassette comprises
the Actin 2 promoter (Bp 1-1161) operably linked to a mutant
phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC
terminator (Bp 2891-3387) at the left border (LB).
[0233] Flue-cured tobacco will be transformed with the construct
shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Flue-cured tobacco that will be created using the construct shown
in FIG. 3 will have significantly reduced levels of nicotine and
TSNA, as compared to a conventional tobacco, a reference tobacco,
or the parental strain of tobacco prior to genetic
modification.
[0234] Burley tobacco will be transformed with the construct shown
in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated (see
e.g., Broothaerts et al., Nature 433:629 (2005), herein expressly
incorporated by reference in its entirety) or biolistic
transformation and independent lines will be selected, regenerated,
and transplanted in the greenhouse. Most of the independent lines
grown in the greenhouse will be harvested and tested for alkaloid
content. It is expected that approximately 50% of the lines tested
will have less than 1,000 ppm total alkaloid and approximately 10%
of the lines tested will have less than 500 ppm total alkaloid.
Accordingly, it is expected that the transgenic Burley tobacco that
will be created using the construct shown in FIG. 3 will have
significantly reduced levels of nicotine and TSNA, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0235] Oriental tobacco will also be transformed with the construct
shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Oriental tobacco that will be created using the construct shown in
FIG. 3 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0236] FIG. 4 illustrates another RNAi construct that was used to
create a reduced nicotine and TSNA transgenic tobacco. This RNAi
construct has a A622 inhibition cassette (SEQ. ID. No. 3) and a
norflurazone selection cassette (SEQ. ID. No. 25). Starting from
the right border (RB), the A622 inhibition cassette comprises an
RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic
acid (628 bp) (Bp 2011-2638) of the A622 gene, joined to a FAD2
intron (Bp 2639-3769), which is joined to a sense nucleic acid of
the A622 gene (628 bp) (Bp 3770-4397), which is joined to the GAD2
terminator (Bp 4398-4670). The selection cassette comprises the
Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene
desaturase gene (PDSM 1) (Bp 1162-2890) joined to the GapC
terminator (Bp 2891-3387) at the left border (LB).
[0237] Flue-cured tobacco was transformed with the construct shown
in FIG. 4 using Agrobacterium-mediated transformation and 270
independent lines were selected, regenerated, and transplanted in
the greenhouse. Of the 270 independent lines, 259 plants were
harvested and tested for alkaloid content. A total of 131 lines
were identified as having less than 1,000 ppm total alkaloid and 45
lines were identified as having less than 500 ppm total alkaloid.
Accordingly, it is understood by those skilled in the art that the
transgenic Flue-cured tobacco created using the construct shown in
FIG. 4 also has significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0238] Several lines that were transformed with this construct were
unexpectedly found to have conventional levels of nicotine but a
significantly reduced amount of nornicotine. That is, 9 lines were
found to have nicotine levels ranging from 2.17 mg/g to 3.99 mg/g
and nornicotine levels less than or equal to 0.00 to 0.06 mg/g (see
Table 2). TABLE-US-00008 TABLE 2 Transgenic tobacco having reduced
nornicotine and conventional amounts of nicotine Alkaloid
Nornicotine Nicotine new I.D (ppm) (mg/g) (mg/g) VOG 0 20 2486.53
##STR1## 2.30 VDG 0 32 4683.01 ##STR2## 3.48 VDG 0 45 4490.79
##STR3## 3.94 VDG 0 52 2855.58 ##STR4## 2.61 VDG 0 54 2291.89
##STR5## 2.17 VDG 0 77 4857.86 ##STR6## 3.99 VDG 0 97 3072.40
##STR7## 2.58 VDG 107 4921.31 ##STR8## 3.59 Control- 8 5005.22 0.28
4.02 Control- 20 5711.97 0.34 5.35 Control- 28 5196.25 0.24 4.52
*Highlighted entries show transgenic tobacco lines having a reduced
amount of nornicotine and conventional amounts of nicotine.
[0239] Tobacco products containing the selectively reduced
nornicotine transgenic tobacco described above are also embodiments
of the invention. That is, tobacco products comprising a transgenic
tobacco that comprises (e.g., on the leaf or tobacco rod) or
delivers (e.g., side-stream or main-stream smoke by the FTC and/or
ISO methods) a conventional amount of nicotine (e.g., at least,
less than, greater than, or equal to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/g nicotine) and a
reduced amount of nornicotine (e.g., 0.00, 0.01, 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15,
0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared to a conventional
tobacco, a reference tobacco, or the parental strain of tobacco
prior to genetic modification, are embodiments of the invention.
Particularly preferred are transgenic tobacco and tobacco products
made therefrom, which comprise (e.g., on the leaf or tobacco rod)
or deliver (e.g., side-stream or main-stream smoke by the FTC
and/or ISO methods) a conventional amount of nicotine (e.g., at
least, less than, greater than, or equal to 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,
4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 mg/g
nicotine) and a reduced amount of nornicotine (e.g., 0.00, 0.01,
0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12,
0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mg/g), as compared
to a conventional tobacco, a reference tobacco, or the parental
strain of tobacco prior to genetic modification, and an isolated
fragment of the A622 gene, in particular, comprising, consisting
of, or consisting essentially of an isolated nucleic acid of SEQ.
ID. No. 2, or the cassette of SEQ. ID. No. 3.
[0240] Burley tobacco will be transformed with the construct shown
in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated or
biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Burley tobacco that will be created using the construct shown in
FIG. 4 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification. It is
also expected that some lines of tobacco created with the
afore-mentioned nucleic acid construct will retain conventional
amounts of nicotine but will comprise a reduced amount of
nornicotine, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
[0241] Oriental tobacco will also be transformed with the construct
shown in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated,
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Oriental tobacco that will be created using the construct shown in
FIG. 4 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification. It is
also expected that some lines of tobacco created with the
afore-mentioned nucleic acid construct will retain conventional
amounts of nicotine but will comprise a reduced amount of
nornicotine, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
[0242] FIG. 5 illustrates a double-knock-out RNAi construct, which
has been created to develop a reduced nicotine and TSNA transgenic
tobacco. This double-knock-out RNAi construct has a QPTase/A622
inhibition cassette (SEQ. ID. No. 23) and a norflurazone selection
cassette (SEQ. ID. No. 25). Starting from the right border (RB),
the QPTase/A622 inhibition cassette comprises an RD2 promoter (Bp
1-2010) operably linked to a QPTase antisense nucleic acid (360 bp)
(Bp 2011-2370) of a QPTase gene, which is joined to a A622
antisense nucleic acid (628 bp) (Bp 2371-2998) of a A622 gene,
which is joined to a FAD2 intron (Bp 2999-4129), which is joined to
a sense nucleic acid of the A622 gene (628 bp) (Bp 4130-4757),
which is joined to a sense nucleic acid of the QPTase gene (360 bp)
(Bp 4758-5117), which is joined to the GAD2 terminator (Bp
5118-5390). The selection cassette comprises the Actin 2 promoter
(Bp 1-1161) operably linked to a mutant phytoene desaturase gene
(PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387)
at the left border (LB).
[0243] Flue-cured tobacco was transformed with the construct shown
in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or
biolistic transformation and 444 independent lines were selected,
regenerated, and transplanted in the greenhouse. Each of these
lines were analyzed for alkaloid content and 240 lines were found
to have less than 1,000 ppm total alkaloid and 18 lines were found
to have less than 500 ppm total alkaloid. Accordingly, it is
understood by those skilled in the art that the transgenic
Flue-cured tobacco created using the construct shown in FIG. 5 also
has significantly reduced levels of nicotine and TSNA, as compared
to a conventional tobacco, a reference tobacco, or the parental
strain of tobacco prior to genetic modification.
[0244] Significantly, for the first time, a plurality of different
genes involved in nicotine biosynthesis have been inhibited using a
single construct. This is particularly useful as it has been shown
that genetic modification of genes in nicotine biosynthesis is
leaky. That is, since there are several genes involved in nicotine
biosynthesis, it is contemplated that the inhibition of one gene
may be compensated for by other genes involved in the synthesis of
nicotine. Accordingly, the inhibition of a plurality of genes in
nicotine biosynthesis, as indicated by the present disclosure
allows for a stronger control over the production of nicotine.
[0245] Burley tobacco will be transformed with the construct shown
in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated or
biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Burley tobacco that will be created using the construct shown in
FIG. 5 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0246] Oriental tobacco will also be transformed with the construct
shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated,
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Oriental tobacco that will be created using the construct shown in
FIG. 5 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0247] It should be emphasized that other promoters and terminators
can be used with the nucleic acids of the invention
interchangeably. Although RD2 (SEQ. ID. No. 10) is a preferred
root-specific promoter, there are other root-specific promoters
that can be used, as well. For example, the putrescene methyl
transferase 1 promoter (PMT-1) (SEQ. ID. No. 11) is a root-specific
promoter that can be used in place of the RD2 promoter in any of
the constructs described above. Similarly, although the actin2
promoter (SEQ. ID. No. 13) is preferred for driving expression of a
norflurazone resistance gene, other constitutive promoters such as
the GapC promoter (SEQ. ID. No. 12), the tobacco alcohol
dehydrogenase (ADP) (SEQ. ID. No. 14) and the Arabidopsis ribosomal
protein L2 (RPL2P) (SEQ. ID. No. 15) can be used to drive
expression of the norflurazone resistance gene. Additionally,
developmentally regulated promoters such as, cinnamyl alcohol
dehydrogenase (SEQ. ID. No. 16) and metallothionein I promoter
(SEQ. ID. No. 17) can be used interchangeable with the cassettes
described herein.
[0248] Further, in some embodiments, a plurality of constitutive
promoters, in tandem, can be used to drive expression of the
norflurazone resistance gene. Additionally, a plurality of
root-specific promoters can be used to drive expression one or more
of the inhibition cassettes described above (e.g., the QPTase
inhibition cassette, the PMTase inhibition cassette, the A622
inhibition cassette, or a double-knockout inhibition cassette).
Developmentally regulated promoters, a plurality of developmentally
regulated promoters, constitutive promoters, or a plurality of
constitutive promoters can also be used to drive expression of one
or more of the inhibition or selection cassettes described above.
Accordingly, any promoter operable in tobacco can be used to drive
expression of any of the inhibition cassettes or the selection
cassette described herein (e.g., nos, 35S, or CAMV). Terminators,
such as GAD2 terminator (SEQ. ID. No. 18) and the FAD 2 (SEQ. ID.
No. 19) or PAP1 introns can be used interchangeably, as well.
[0249] Other aspects of the invention concern the discovery of
several mutants of the phytoene desaturase gene that confer
resistance to the herbicide norflurazone (e.g., SEQ. ID. Nos.7, 8,
and 9). These herbicide resistance genes were used as selectable
markers in the transformations above. Typically, the selection was
accomplished by introducing the transformed plant tissue to the
norflurazone (e.g., 0.005 .mu.M-0.1 .mu.Mconc.). That is, the
concentration of norflurazone that can be used to select positive
transformants containing a norflurazone resistance gene, as
described herein can be at least, less than, greater than, or equal
to 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05,
0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, or 1.0 .mu.M. Preferably, less than or equal to 0.05 .mu.M
concentration of norflurazone is used when selecting transformants
with Flue-cured tobacco and less than or equal to 0.0125 .mu.M
concentration norflurazone is used when selecting transformants
with Burley tobacco. As the plantlet develops, selection was
accomplished by differentiating the green shoots (positive
transformants) from the yellow or white shoots (negative
transformants). Once selection was made, the herbicide was removed
and the plantlet was allowed to develop in the greenhouse.
[0250] The norflurazone resistant phytoene desaturase mutants
(PDSM-1, PDSM-2, and PDSM-3) were generated by site-directed
mutagenesis of particular regions of the gene believed to be
involved in binding of the herbicide. Constructs carrying the
various PDSM genes were then transferred to tobacco leaf disks by
conventional Agrobacterium transformation and the resistance to
norflurazone was analyzed at various concentrations. After several
iterations, the mutants described as SEQ. ID. Nos. 7, 8, and 9,
were identified as sequences that confer resistance to
norflurazone. Accordingly, aspects of the invention concern the
PDSM genes described herein, their use in plants as selectable
markers to identify plant cells that contain a transformed gene,
whether in tissue culture or in the field, and methods of
identifying new PDSM genes that confer norflurazone resistance.
[0251] In a first selection construct, the Arabidopsis phytoene
desaturase gene (PDS) (SEQ. ID. No. 26) was mutated using
site-directed mutagenesis, such that a T to G mutation at position
1478, resulting in a Valine to Glycine change at amino acid residue
493 was created. To generate the norflurazone resistance gene, the
open reading frame of the Arabidopsis phytoene desaturase gene was
amplified and cloned into the TOPO vector (Invitrogen). A single
base pair change from T-G at nucleotide position 1478, leading to a
Valine to Glycine change at amino acid residue 493, was introduced
using QuickChange Site-directed Mutagenisis Kit (Stratgene). The
point mutation was verified by sequencing and the resultant mutant
was named PDSM-1 (SEQ. ID. No. 7). The 1.729 Kb PDSM1 sequence was
then amplified and ligated into the binary vector pWJ001, a pCambia
derivative that contained the RNAi cassettes above, which was then
introduced into Agrobacterium tumefaciens. A similar approach was
used to generate the PDSM-2 and PDSM-3 mutants described in the
sequence listing as SEQ. ID. NOs.8 and 9.
[0252] That is, in a second selection construct, the Arabidopsis
phytoene desaturase gene (PDS) (SEQ. ID. No. 26) was mutated using
site-directed mutagenesis, such that a G to C mutation at position
863, resulting in a Arginine to Proline change at amino acid
residue 288 was created. To generate the norflurazone resistance
gene, the open reading frame of the Arabidopsis phytoene desaturase
gene was amplified and cloned into the TOPO vector (Invitrogen). A
single base pair change was introduced using QuickChange
Site-directed Mutagenisis Kit (Stratgene). The point mutation was
verified by sequencing and the resultant mutant was named PDSM-2.
The 1.729 Kb PDSM-2 sequence was then amplified and ligated into
the binary vector pWJ001, a pCambia derivative that contained the
RNAi cassettes above, which was then introduced into Agrobacterium
tumefaciens.
[0253] Further, in a third selection construct, the Arabidopsis
phytoene desaturase gene (PDS) (SEQ. ID. No. 26) was mutated using
site-directed mutagenesis, such that a T to C mutation at position
1226, resulting in a Leucine to Proline change at amino acid
residue 409 was created. To generate the norflurazone resistance
gene, the open reading frame of the Arabidopsis phytoene desaturase
gene was amplified and cloned into the TOPO vector (Invitrogen). A
single base pair change was introduced using QuickChange
Site-directed Mutagenisis Kit (Stratgene). The point mutation was
verified by sequencing and the resultant mutant was named PDSM-3.
The 1.729 Kb PDSM-2 sequence was then amplified and ligated into
the binary vector pWJ001, a pCambia derivative that contained the
RNAi cassettes above, which was then introduced into Agrobacterium
tumefaciens.
[0254] Accordingly, aspects of the invention concern methods of
identifying a mutation on a phytoene desaturase gene that confers
resistance to an herbicide, preferably norflurazone. By one
approach, a phytoene desaturase gene is provided, preferably SEQ.
ID. No. 26, a nucleotide in said gene is mutated so as to generate
a mutant phytoene desaturase gene, said mutant phytoene desaturase
gene is transformed to a plant cell so as to generate a plant cell
comprising said mutant phytoene desaturase gene, said plant cell
comprising said mutant phytoene desaturase gene is then contacted
with an herbicide, preferably norflurazone, and the presence or
absence of a resistance to said herbicide is identified, whereby
the presence of a resistance to said herbicide identifies said
mutation as one that confers resistance to said herbicide. By one
approach, the entire sequence of a phytoene desaturase gene (e.g.,
SEQ. ID. NO. 26) is mutated one residue at a time and each mutant
is screened for resistance to the herbicide. Accordingly, aspects
of the invention include compositions (e.g., nucleic acid
constructs or cassettes, plant cells, plants, tobacco, or tobacco
products) that comprise, consist, consist essentially of a mutant
phytoene desaturase nucleic acid of SEQ. ID. NO. 7, 8, or 9 or
fragment thereof at least or equal to 30, 50, 100, 200, 400, 500,
700, 900, 1000, 1200, 1400, 1600, or 1700 consecutive nucleotides
of in length that confers resistance to an herbicide, in particular
norflurazone. Aspects of the invention also include compositions
(e.g., nucleic acid constructs or cassettes, plant cells, plants,
tobacco, or tobacco products) comprising the mutant phytoene
desaturase protein or fragments thereof (e.g., at least 15, 25, 50,
100, 200, 300, 400, 500 consecutive amino acids of a protein
encoded by SEQ. ID. Nos. 7, 8, or 9) that confer resistance to an
herbicide, in particular norflurazone.
[0255] The nucleic acid sequences, cassettes, and constructs
described herein can also be altered by mutation such as
substitutions, additions, or deletions that provide for sequences
encoding functionally equivalent molecules. Due to the degeneracy
of nucleotide coding sequences, other DNA sequences that encode
substantially the same amino acid sequence can be used in some
embodiments of the present invention. These include, but are not
limited to, nucleic acid sequences comprising all or portions of
the nucleic acid embodiments described herein that complement said
sequences and have been altered by the substitution of different
codons that encode a functionally equivalent amino acid residue
within the sequence, thus producing a silent change. In some
contexts, 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.
[0256] Additional nucleic acid embodiments include sequences that
are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to
the nucleic acids, nucleic acid constructs, and nucleic acid
cassettes provided herein. Preferably these sequences also perform
the functions of the particular nucleic acid embodiment (e.g.,
inhibition of nicotine or nornicotine production or confer
resistance to norflurazone). 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.
[0257] Additional nucleic acid embodiments also include nucleic
acids that hybridize to the nucleic acid sequences disclosed herein
under low, medium, and high stringency, wherein said additional
nucleic acid embodiments also perform the function of the
particular embodiment (e.g., inhibit nicotine or nornicotine
production or confer resistance to norflurazone). Identification of
nucleic acids that hybridize to the embodiments described herein
can be determined in a routine manner. (See J. Sambrook et al.,
Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring
Harbor Laboratory)). For example, hybridization of such sequences
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
degrees C., or even 70 degrees C.). Preferably these sequences also
perform the functions of the particular nucleic acid embodiment
(e.g., inhibition of nicotine or nornicotine production or confer
resistance to norflurazone).
[0258] The examples described herein demonstrate that several
different RNAi constructs can be used to effectively reduce the
levels of nicotine, and/or nornicotine in tobacco. Additionally,
these examples demonstrate that several mutant phytoene desaturase
genes, which confer resistance to the herbicide norflurazone, have
been created and that selection cassettes comprising these
herbicide resistant nucleic acids can be used to determine the
presence of a linked gene in transformed tobacco cells. The section
below describes typical curing methods which may be used to prepare
the tobacco once it is harvested.
[0259] Curing
[0260] The curing process, which typically lasts about 1 week,
brings out the flavor and aroma of tobacco. Several methods for
curing tobacco may be used, and indeed many methods have been
previously disclosed. For example, U.S. Pat. Nos. 4,499,911 to
Johnson; 5,685,710 to Martinez Sagrera; 3,905,123 to Fowler;
3,840,025 to Fowler; and 4,192,323 to Horne, (each hereby expressly
incorporated by reference in its entirety) describe aspects of the
tobacco curing process which may be used for some embodiments of
the present invention. Conventionally, "sticks" that are loaded
with tobacco are placed into bulk containers and placed into closed
buildings having a heat source known as a curing barn. A flue is
often used to control the smoke (thus earning the term
"flue-cured"). The method of curing will depend, in some cases, on
the type of tobacco-use cessation product desired, (i.e., snuff,
cigarettes, or pipe tobacco may preferably utilize different curing
methods) and preferred methods may vary from region to region and
in different countries. In some approaches, the stems and midveins
of the leaf are removed from the leaves prior to curing to yield a
high quality, low nitrosamine tobacco product.
[0261] "Flue curing" is a popular method for curing tobacco in
Virginia, North Carolina, and the Coastal Plains regions of the
United States. This method is used mainly in the manufacture of
cigarettes. Flue curing requires a closed building equipped with a
system of ventilation and a source of heat. The heating can be
direct or indirect (e.g., radiant heat). When heat and humidity are
controlled, leaf color changes, moisture is quickly removed, and
the leaf and stems dry. Careful monitoring of the heating and
humidity can reduce the accumulation of nitrosamines.
[0262] Another curing method is termed "air curing". In this
method, an open framework is prepared in which sticks of leaves (or
whole plants) are hung so as to be protected from both wind and
sun. Leaf color changes from green to yellow, as leaves and stems
dry slowly.
[0263] "Fire curing" employs an enclosed barn similar to that used
for flue curing. The tobacco is hung over low temperature fire so
that the leaves cure in a smoke-laden atmosphere. This process uses
lower temperatures so the process may take up to a month, in
contrast to flue curing, which takes about 6 to 8 days.
[0264] A further curing method, termed "sun curing" is the drying
of uncovered sticks or strings of tobacco leaves in the sun. The
best known sun-cured tobaccos are the so-called oriental tobaccos
of Turkey, Greece, Yugoslavia, and nearby countries.
[0265] The curing process, and most particularly the flue-curing
process, is generally divided into the following four stages:
A) Firing Up: During this step, the tobacco leaves turn bright
lemon-orange in color. This is achieved by a gradual increase in
temperature.
B) Leaf Yellowing: In this step any moisture is removed. This
creates the "yellowing" of the tobacco. It also prepares the
tobacco for drying in the next step.
C) Leaf Drying: Leaf drying, an important step in the curing
process, requires much time for the tobacco to dry properly.
Additionally, air flow is increased in this step to facilitate the
drying process.
D) Stem Drying: The drying process continues, as the stem of the
tobacco leaf becomes dried.
[0266] The cured tobacco may then be blended with other tobaccos or
other materials to create the product to be used for the
tobacco-use cessation method. The section below describes typical
methods of blending and preparing the tobacco product.
[0267] Making Tobacco Cessation Products by Tobacco Blending and/or
Addition of Exogenous Nicotine
[0268] In some embodiments, a tobacco comprising a reduced amount
of alkaloid (e.g., a reduced amount of nicotine, nornicotine,
and/or TSNAs) is contacted with an exogenous nicotine so as to
raise the level of nicotine in the contacted tobacco in a
controlled fashion. By this approach, nicotine levels in tobacco
that comprises a reduced amount of endogenous nicotine (i.e.,
nicotine that is produced by the plant from which the tobacco is
obtained) can be selectively raised to levels that are commensurate
with conventional full-flavor cigarettes, light cigarettes, or
ultra-light cigarettes. (See e.g., WO 2005/018307, which designates
the United States and was published in English, herein expressly
incorporated by reference in its entirety). For example, tobacco
comprising a reduced amount of endogenous nicotine and/or TSNAs can
be contacted with an amount of exogenous nicotine that is at least,
equal to, or more than 0.3 mg/g-20.0 mg/g (nicotine/gram of
tobacco). That is, tobacco comprising a reduced amount of
endogenous nicotine and/or TSNAs can be contacted with an amount of
exogenous nicotine that is at least, equal to, or more than 0.3
mg/g, 0.4 mg/g, 0.5 mg/g, 0.6 mg/g, 0.7 mg/g, 0.8 mg/g, 0.9 mg/g,
11.0 mg/g, 1.1 mg/g, 1.2 mg/g, 1.3 mg/g, 1.4 mg/g, 1.5 mg/g, 1.6
mg/g, 1.7 mg/g, 1.8 mg/g, 1.9 mg/g, 2.0 mg/g, 2.1 mg/g, 2.2 mg/g,
2.3 mg/g, 2.4 mg/g, 2.5 mg/g, 2.6 mg/g, 2.7 mg/g, 2.8 mg/g, 2.9
mg/g, 3.0 mg/g, 3.1 mg/g, 3.2 mg/g, 3.3 mg/g, 3.4 mg/g, 3.5 mg/g,
3.6 mg/g, 3.7 mg/g, 3.8 mg/g, 3.9 mg/g, 4.0 mg/g, 4.1 mg/g, 4.2
mg/g, 4.3 mg/g, 4.4 mg/g, 4.5 mg/g, 4.6 mg/g, 4.7 mg/g, 4.8 mg/g,
4.9 mg/g, 5.0 mg/g, 5.1 mg/g, 5.2 mg/g, 5.3 mg/g, 5.4 mg/g, 5.5
mg/g, 5.6 mg/g, 5.7 mg/g, 5.8 mg/g, 5.9 mg/g, 6.0 mg/g, 6.1 mg/g,
6.2 mg/g, 6.3 mg/g, 6.4 mg/g, 6.5 mg/g, 6.6 mg/g, 6.7 mg/g, 6.8
mg/g, 6.9 mg/g, 7.0 mg/g, 7.1 mg/g, 7.2 mg/g, 7.3 mg/g, 7.4 mg/g,
7.5 mg/g, 7.6 mg/g, 7.7 mg/g, 7.8 mg/g, 7.9 mg/g, 8.0 mg/g, 8.1
mg/g, 8.2 mg/g, 8.3 mg/g, 8.4 mg/g, 8.5 mg/g, 8.6 mg/g, 8.7 mg/g,
8.8 mg/g, 8.9 mg/g, 9.0 mg/g, 9.1 mg/g, 9.2 mg/g, 9.3 mg/g, 9.4
mg/g, 9.5 mg/g, 9.6 mg/g, 9.7 mg/g, 9.8 mg/g, 9.9 mg/g, 10.0 mg/g,
10.1 mg/g, 10.2 mg/g, 10.3 mg/g, 10.4 mg/g, 10.5 mg/g, 10.6 mg/g,
10.7 mg/g, 10.8 mg/g, 10.9 mg/g, 11.0 mg/g, 11.1 mg/g, 11.2 mg/g,
11.3 mg/g, 11.4 mg/g, 11.5 mg/g, 11.6 mg/g, 11.7 mg/g, 11.8 mg/g,
11.9 mg/g, 12.0 mg/g, 12.1 mg/g, 12.2 mg/g, 12.3 mg/g, 12.4 mg/g,
12.5 mg/g, 12.6 mg/g, 12.7 mg/g, 12.8 mg/g, 12.9 mg/g, 13.0 mg/g,
13.1 mg/g, 13.2 mg/g, 13.3 mg/g, 13.4 mg/g, 13.5 mg/g, 13.6 mg/g,
13.7 mg/g, 13.8 mg/g, 13.9 mg/g, 14.0 mg/g, 14.1 mg/g, 14.2 mg/g,
14.3 mg/g, 14.4 mg/g, 14.5 mg/g, 14.6 mg/g, 14.7 mg/g, 14.8 mg/g,
14.9 mg/g, 15.0 mg/g, 15.1 mg/g, 15.2 mg/g, 15.3 mg/g, 15.4 mg/g,
15.5 mg/g, 15.6 mg/g, 15.7 mg/g, 15.8 mg/g, 15.9 mg/g, 16.0 mg/g,
16.1 mg/g, 16.2 mg/g, 16.3 mg/g, 16.4 mg/g, 16.5 mg/g, 16.6 mg/g,
16.7 mg/g, 16.8 mg/g, 16.9 mg/g, 17.0 mg/g, 17.1 mg/g, 17.2 mg/g,
17.3 mg/g, 17.4 mg/g, 17.5 mg/g, 17.6 mg/g, 17.7 mg/g, 17.8 mg/g,
17.9 mg/g. 18.0 mg/g. 18.1 mg/g. 18.2 mg/g. 18.3 mg/g. 18.4 mg/g
18.5 mg/g. 18.6 mg/g. 18.7 mg/g, 18.8 mg/g, 18.9 mg/g, 19.0 mg/g,
19.1 mg/g, 19.2 mg/g, 19.3 mg/g, 19.4 mg/g, 19.5 mg/g, 19.6 mg/g,
19.7 mg/g, 19.8 mg/g, 19.9 mg/g, and 20.0 mg/g (nicotine/gram
tobacco)). In some of the aforementioned embodiments, said tobacco
is genetically modified and comprises one or more of the isolated
nucleic acids, isolated nucleic acid cassettes, or isolated nucleic
acid constructs, described herein.
[0269] Nicotine-containing fractions, nicotine, or nicotine salts
of organic acids are added to the reduced-nicotine transgenic
tobacco by contacting said tobacco (e.g., spraying or additive
application), with or without propylene glycol, solvent, flavoring,
or water at any stage of the harvesting, curing, fermenting, aging,
reconstituting, expanding, or otherwise processing of the tobacco,
preferably at a stage that is post-cure, when flavorings and
additives are provided. By "exogenous nicotine" is meant nicotine,
nicotine derivatives, nicotine analogs, nicotine-containing
fractions (e.g., extracts of Nicotiana), and nicotine salts of
organic acids obtained from a source outside of the transgenic
tobacco to which the exogenous nicotine is applied. In this manner,
a transgenic tobacco with virtually any amount of nicotine can be
obtained.
[0270] In some embodiments, the exogenous nicotine (e.g.,
commercially available nicotine salts, liquid, or a
nicotine-containing extract prepared from a Nicotiana plant or
portion thereof) is contacted with a reduced-alkaloid transgenic
tobacco (e.g., a transgenic tobacco comprising a reduced amount of
nicotine and/or TSNA as prepared as described herein) after the
transgenic tobacco has been made substantially free of microbes
(e.g., bacteria, yeast, mold, or fungi). The reduced alkaloid
transgenic tobacco can be made substantially-free of microbes
(e.g., an aseptic preparation) by employing sterilization, heat
treatment, pasteurization, steam treatment, gas treatment, and
radiation (e.g., gamma, microwave, and ultraviolet). The term
"substantially-free of microbes" in some contexts can mean an
amount of bacteria, mold, fungi, or yeast that is reduced to the
point that the conversion of nicotine or total alkaloid to TSNA is
negligible, for example, a tobacco product comprises (e.g., on the
leaf or tobacco rod) or delivers (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods) a collective content of TSNAs
(e.g., NNN, NAT, NAB, or NNK) of less than or equal to 5.0 .mu.g/g,
4.0 .mu.g/g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0 .mu.g/g, or 0.5 .mu.g/g)
after prolonged storage (e.g., at least 1-30 days, 30-90 days
90-180 days. 180-270 days. 270 days-365 days. 1 year-1.5 years,
1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0
years-4 years, and 4.0 years-5.0 years)). The term
"substantially-free of microbes" also includes the term
"substantially-free of bacteria," which means in some contexts that
the tobacco or tobacco product is substantially-free of
Arthrobacter, Proteus, nicotine oxidizing bacteria, such as P-34,
Psuedomonas, Xantomonas, or Zoogloea strains of bacteria. For
example, a tobacco or tobacco product is substantially-free of
bacteria or a particular strain of bacteria when said tobacco or
tobacco product has less than or equal to 20% of the bacteria or a
specific strain of bacteria normally present on the tobacco or
tobacco product in the absence of application of a technique to rid
the tobacco or tobacco product of bacteria (e.g., less than or
equal to 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, or 20%). With respect to transgenic
tobacco described herein, the term "substantially-free of bacteria"
can refer to tobacco or a tobacco product containing said
transgenic tobacco that has less than or equal to 20% of the
bacteria normally present on the strain of tobacco prior to genetic
modification and/or application of a technique to rid the tobacco
or tobacco product of bacteria (e.g., less than or equal to 1%, 2%,
3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, or 20%).
[0271] Once the exogenous nicotine has been contacted with the
microbe-free tobacco, it is preferably processed and packaged
aseptically and the tobacco product is maintained in an airtight
container so as to not re-introduce microbes that convert the
exogenous nicotine to TSNAs. By using the aseptic processing,
manufacturing, and packaging procedures, described herein, one can
maintain an amount of total TSNA (e.g., the collective content of
NNN, NAT, NAB, and NNK) in a commercially available tobacco
product, which comprises exogenous nicotine, such that said product
comprises (e.g., on the leaf or tobacco rod) or delivers (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods) an
mount that is less than or equal to 5.0 .mu.g/g (e.g., less than or
equal to 5.0 .mu.g/g, 4.0 .mu.g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, or 0.05 .mu.g/g) for at least 1 week, 1 month, or 1-5
years after packaging (e.g., at least 1-30 days, 30-90 days, 90-180
days, 180-270 days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0
years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years,
and 4.0 years-5.0 years). In some embodiments, the exogenous
nicotine is contacted with a tobacco comprising one of the nucleic
acid constructs described herein and comprising (e.g., on the leaf
or tobacco rod) or delivering (e.g., side-stream or main-stream
smoke by the FTC and/or ISO methods) a collective content of NNN,
NAT, NAB, and NNN that is less than or equal to 2.0 .mu.g/g (e.g.,
less than or equal to 2.0 .mu.g/g, 1.5 .mu.g/g, 1.0 .mu.g/g, 0.5
.mu.g/g, 0.3 .mu.g/g, or 0.1 .mu.g/g). In some embodiments, an
amount on the leaf or tobacco rod or the delivery (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods) is
maintained to a level such that a collective content of NNN, NAT,
NAB, and NNN of less than or equal to 5.0 .mu.g/g (e.g., less than
or equal to 5.0 .mu.g/g, 4.0 .mu.g, 3.0 .mu.g/g, 2.0 .mu.g/g, 1.0
.mu.g/g, or 0.05 .mu.g/g) in a tobacco product containing said
tobacco can be maintained for at least at least 1 week, 1 month, or
1-5 years after packaging (e.g., at least 1-30 days, 30-90 days,
90-180 days, 180-270 days, 270 days-365 days, 1 year-1.5 years,
1.5-2.0 years, 2.0 years-2.5 years, 2.5 years-3.0 years, 3.0
years-4 years, and 4.0 years-5.0 years). Accordingly, several
embodiments address the problem of gradually increasing TSNA levels
in alkaloid-containing tobacco products by employing processing,
storage, and packaging methods that reduce the amount of microbial
flora on the tobacco, limit the re-introduction of microbes during
processing and maintain a reduced amount of microbes (e.g.,
bacteria) once the product is packaged, stored, and sold. Tobacco
and tobacco products comprising tobacco having a reduced amount of
endogenous nicotine and an amount of exogenous nicotine can be
analyzed by various methods to confirm that said tobacco and said
tobacco products are "reduced risk" or have less of a potential to
contribute to a tobacco-related disease, as compared to the parent
strain of tobacco having conventional amounts of endogenous
nicotine or a reference tobacco.
[0272] It addition, it may be desirable to blend tobacco of varying
nicotine levels to create the cessation product having the desired
level of nicotine. This blending process is typically performed
after the curing process, and may be performed by conventional
methods. Preferred tobacco blending approaches are provided in
Examples 19 and 20. In some embodiments, blending of the transgenic
tobacco is conducted to prepare the tobacco so that it will contain
specific amounts of nicotine and/or TSNA in specific products.
Preferably, the blending is conducted so that tobacco products of
varying amounts of nicotine and/or TSNAs are made in specific
products.
[0273] A mixture that contains different types of tobacco is
desirably substantially homogeneous throughout in order to avoid
undesirable fluctuations in taste or nicotine levels. Typically,
tobacco to be blended may have a moisture content between 30 and
75%. As an example, the tobacco is first cut or shredded to a
suitable size, then mixed in a mixing device, such as a rotating
drum or a blending box. One such known mixing device is a tumbling
apparatus that typically comprises a rotating housing enclosing
mixing paddles which are attached to and, therefore, rotate with
the housing to stir the tobacco components together in a tumbling
action as the drum turns.
[0274] After the desired tobaccos are thoroughly mixed, the
resulting tobacco blend is removed from the mixing apparatus and
bulked to provide a continuous, generally uniform quantity of the
tobacco blend. The tobacco is then allowed to remain relatively
undisturbed (termed the "bulking step") for the required period of
time before subsequent operations are performed. The bulking step
typically takes 30 minutes or less, and may be carried out on a
conveyor belt. The conveyor belt allows the blended tobacco to
remain in bulk form in an undisturbed condition while it is
continuously moving the tobacco blend through the process from the
mixing stage to the expansion stage.
[0275] The tobacco blend is typically expanded by the application
of steam. The tobacco mixture is typically subjected to at least
0.25 pounds of saturated steam at atmospheric conditions per pound
of blended tobacco for at least 10 seconds to provide an increase
in moisture of at least 2 weight percent to the tobacco blend.
After the tobacco blend has been expanded, it is dried. A typical
drying apparatus uses heated air or superheated steam to dry the
tobacco as the tobacco is conveyed by the heated air or steam
stream through a drying chamber or series of drying chambers.
Generally, the wet bulb temperature of the drying air may be from
about 150 degrees F. to about 211 degrees F. The tobacco blend is
typically dried to a moisture content of from about 60 percent to
about 5 percent. The dried, expanded tobacco blend is then in a
suitable mode to be processed into the tobacco-use cessation
product as described below.
[0276] Some blending approaches begin with tobacco prepared from
varieties that have extremely low amounts of nicotine, nornicotine,
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. Other approaches blend only low
nicotine/TSNA tobaccos (e.g., genetically modified Burley,
genetically modified Virginia flue, and genetically modified
Oriental tobaccos that contain reduced amounts of nicotine and/or
TSNAs). 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.
[0277] By one approach, a step 1 tobacco product is 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 or nicotine and/or TSNA reduction 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 per 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.
[0278] 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
Burley 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
Burley 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
Burley 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.
[0279] By a preferred method, conventional Virginia flue tobacco
was blended with genetically modified Burley (i.e., Burley
containing a significantly reduced amount of nicotine and
nitrosamine) to yield a blended tobacco that was incorporated into
three levels of reduced nicotine cigarettes (delivery in
main-stream smoke by the FTC method): a step 1 cigarette containing
0.6 mg nicotine, a step 2 cigarette containing 0.3 mg nicotine, and
a step 3 cigarette containing less than 0.05 mg nicotine. The
amount of total TSNA was found to range between approximately 0.17
.mu.g/g-0.6 .mu.g/g (delivery in main-stream smoke by the FTC
method).
[0280] In some cigarettes, approximately, 28% of the blend was
Virginia flue tobacco, approximately 29% of the blend was
genetically modified (i.e., reduced nicotine Burley), approximately
14% of the blend was Oriental, approximately 17% of the blend was
expanded flue-cured stem, and approximately 12% was standard
commercial reconstituted tobacco. The amount of total TSNAs in
cigarettes containing this blend was approximately 1.5 .mu.g/g
(delivery in main-stream smoke by the FTC method).
[0281] 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. It should also be appreciated that
reconstituted, expanded, chemically treated, or microbial treated
tobacco can be blended with the transgenic tobacco described
herein. 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, flavor, and
smokability. In this manner, a variety of types of tobacco products
having varying level of nicotine and/or nitrosamine, as well as,
appearance, flavor and smokability can be created.
[0282] Furthermore, it should also be appreciated that the blending
steps described above are not necessary for all embodiments. That
is, a tobacco product within the scope of the present invention can
be made by using the reduced nicotine tobacco described herein
without blending with another type of tobacco.
[0283] Nicotine Reduction and/or Tobacco-Use Cessation Programs
Methods
[0284] The methods described herein facilitate nicotine reduction,
TSNA reduction, and/or tobacco-use cessation by allowing the
individual to retain the secondary factors of addiction such as
smoke intake, oral fixation, and taste, while reducing the
addictive nicotine levels consumed. Eventually, complete cessation
is made possible because the presence of addiction for nicotine is
decreased while the individual is allowed to maintain dependence on
the secondary factors addiction as described above.
[0285] As mentioned above, 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. Additionally, as mentioned above, exogenous
nicotine can be added to the tobacco or tobacco product. Further,
two or more varieties of tobacco (e.g., transgenic reduced alkaloid
Burley, transgenic reduced alkaloid Flue-cured, and/or transgenic
reduced alkaloid Oriental) can be blended so as to achieve a
desired taste while maintaining nicotine levels at less than 7,000
ppm, 5,000 ppm, 3000 ppm, 2000 ppm, 1000 ppm, or 500 ppm and TSNA
levels at 0.5 ug/g or less (delivery in main-stream smoke by the
FTC method). In this manner, differences in amounts of nicotine
and/or TSNAs can be incrementally adjusted.
[0286] In some embodiments, a stepwise nicotine and/or TSNA
reduction and/or tobacco-use cessation program can be established
using the reduced nicotine and/or TSNA products described above. As
an example, the program participant can be identified as an
individual that desires a reduction in the consumption of nicotine
and/or TSNAs or as an individual that desires cessation of tobacco
use. The program participant may, optionally, determine his or her
current level of nicotine intake. The program participant then
begins the program at step 1, with a tobacco product having a
reduced amount of nicotine and/or TSNAs, as compared to the tobacco
product that was used prior to beginning the program. After a
period of time, the program participant proceeds to step 2, using a
tobacco product with less nicotine and/or TSNAs than the products
used in step 1. The program participant, after another period of
time, reaches step 3, wherein the program participant begins using
a tobacco product with less nicotine than the products in step 2,
and so on. Ultimately, the program participant uses a tobacco
product having an amount of nicotine and/or TSNAs that is less than
that which is sufficient to become addictive or to maintain an
addiction. Thus, the nicotine reduction and/or tobacco-use
cessation program limits the exposure of a program participant to
nicotine and/or TSNAs and, concomitantly, the harmful effect of
nicotine, yet retains the secondary factors of addiction, including
but not limited to, smoke intake, oral fixation, and taste.
[0287] For example, (with reference to the delivery of nicotine in
main-stream smoke by the FTC method) a smoker can begin the program
smoking cigarettes having 5 mg of nicotine, move to smoking
cigarettes with 3 mg of nicotine, followed by cigarettes having 1
mg nicotine, followed by cigarettes having 0.5 mg nicotine,
followed by cigarettes having less than 0.1 mg nicotine until the
consumer decides to smoke only the cigarettes having virtually no
nicotine and nitrosamines or quitting smoking altogether.
Preferably, a three-step program is followed whereby at step 1,
cigarettes containing 0.6 mg nicotine are used; at step 2,
cigarettes containing 0.3 mg nicotine are used; and at step 3,
cigarettes containing less than 0.1 mg nicotine are used. More
preferably, a three-step program is followed whereby at step 1,
cigarettes containing 0.6 mg nicotine are used; at step 2,
cigarettes containing 0.3 mg nicotine are used; and at step 3,
cigarettes containing less than 0.05 mg nicotine used. Accordingly,
the reduced nicotine and/or TSNA products described herein provide
the basis for an approach to reduce the carcinogenic potential in a
human in a step-wise fashion.
[0288] In another example, (with reference to the delivery of
nicotine in main-stream smoke by the FTC method) a smoker can begin
the program smoking cigarettes having 5 mg of nicotine and 1.5
.mu.g of nitrosamine, move to smoking cigarettes with 3 mg of
nicotine and 1 .mu.g of nitrosamine, followed by cigarettes having
1 mg nicotine and 0.5 .mu.g nitrosamine, followed by cigarettes
having 0.5 mg nicotine and 0.25 .mu.g nitrosamine, followed by
cigarettes having less than 0.1 mg nicotine and less than 0.1 .mu.g
TSNA until the consumer decides to smoke only the cigarettes having
virtually no nicotine and nitrosamines or quitting smoking
altogether. Preferably, a three-step program is followed whereby at
step 1, cigarettes containing 0.6 mg nicotine and less than 2
.mu.g/g TSNA are used; at step 2, cigarettes containing 0.3 mg
nicotine and less than 1 .mu.g/g TSNA are used; and at step 3,
cigarettes containing less than 0.1 mg nicotine and less than 0.7
.mu.g/g TSNA are used. More preferably, a three-step program is
followed whereby at step 1, cigarettes containing 0.6 mg nicotine
and less than 2 .mu.g/g TSNA are used; at step 2, cigarettes
containing 0.3 mg nicotine and less than 1 .mu.g/g TSNA are used;
and at step 3, cigarettes containing less than 0.5 mg nicotine and
less than 0.7 .mu.g/g TSNA are used. Accordingly, the reduced
nicotine and/or TSNA products described herein provide the basis
for an approach to reduce the carcinogenic potential in a human in
a step-wise fashion.
[0289] In a program to reduce a smoker's intake of TSNAs, (with
reference to the delivery of nicotine in main-stream smoke by the
FTC method) a smoker can begin the program, for example, by smoking
cigarettes having 1.5 .mu.g of nitrosamine, move to smoking
cigarettes with 1 .mu.g of nitrosamine, followed by cigarettes
having 0.5 .mu.g nitrosamine, followed by cigarettes having 0.25
.mu.g nitrosamine, followed by cigarettes having less than 0.1
.mu.g TSNA until the consumer decides to smoke only the cigarettes
having virtually no nitrosamines or quitting smoking altogether.
Preferably, a three-step program is followed whereby at step 1,
cigarettes containing less than 2 .mu.g/g TSNA are used; at step 2,
cigarettes containing less than 1 g/g TSNA are used; and at step 3,
cigarettes containing less than 0.7 .mu.g/g TSNA are used. More
preferably, a three-step program is followed whereby at step 1,
cigarettes containing less than 2 .mu.g/g TSNA are used; at step 2,
cigarettes containing less than 1 .mu.g/g TSNA are used; and at
step 3, cigarettes containing less than 0.7 .mu.g/g TSNA are used.
Accordingly, the reduced TSNA products described herein provide the
basis for an approach to reduce the carcinogenic potential in a
human in a step-wise fashion.
[0290] In some embodiments, the amount of tar in the cigarettes
provided remains approximately the same from step-to-step. For
example, the cigarettes provided at each step could comprise (e.g.,
on the leaf or tobacco rod) or deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods) at least, less
than, or equal to about 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg,
3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8
mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg,
12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg,
16.5 m, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5
mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg,
25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29
mg, 29.5 mg, or 30 mg of tar. Preferably, each cigarette comprises
or delivers about 8.0 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9.0 mg, 9.25
mg, or 9.5 mg of tar. It should be understood that variances
between the amounts of tar in each cigarette can be acceptable in
some embodiments so long as the cigarettes retain approximately the
same taste characteristics as the cigarettes provided at the
previous level. Preferably, cigarettes provided at each level can
have variances of up to about 5 mg of tar. More preferably,
cigarettes provided at each level can have variances of up to about
2.5 mg of tar. Most preferably, cigarettes provided at each level
have can have variances of up to about 1.5 mg of tar.
[0291] Embodiments also include tobacco products, which are
prepared with a variety of amounts of nicotine. This can be done,
by blending various types of tobacco, by addition of exogenous
nicotine, and/or by utilizing genetically engineered tobacco having
a specific amount of nicotine. These stepwise tobacco products are
made to have reduced levels of TSNAs and varying amounts of
nicotine. As an example, cigarettes may comprise (e.g., on the leaf
or tobacco rod) or deliver (e.g., side-stream or main-stream smoke
by the FTC and/or ISO methods), for example, at least, less than or
equal to 0.01 mg, 0.05 mg, 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, or 5.0 mg of nicotine per cigarette.
More preferably, blended cigarettes comprise (e.g., on the leaf or
tobacco rod) or deliver (e.g., side-stream or main-stream smoke by
the FTC and/or ISO methods) less than or equal to 0.01 mg, 0.05 mg,
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, 11.0 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 of nicotine. Most
preferably, the cigarettes contain less than 0.01 mg, 0.05 mg, 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 of nicotine.
[0292] Another aspect of the invention is a stepwise nicotine
reduction and/or tobacco-use cessation program using the reduced
nicotine and/or TSNA products described above in combination with
conventional NRT products. As the tobacco user progresses through
the program, the amount of nicotine and/or TSNAs present in the
tobacco product and/or the conventional NRT product is reduced.
[0293] As an example of a combination nicotine and/or TSNA
reduction and/or tobacco-use cessation program, the program
participant initially determines his or her current level of
nicotine intake. The program participant then begins the program
with a tobacco product having a reduced amount of nicotine, as
compared to the tobacco product that was used prior to beginning
the program. During the use of the low nicotine tobacco product,
the user also uses an NRT (e.g., a patch, inhaler, nasal spray,
lozenge, or a gum). In this respect, the tobacco cessation program
provides nicotine in addition to that present in the reduced
nicotine tobacco product, yet lowers the amount of inhaled
nicotine--which is thought to reduce indices of nicotine
dependence. Thus, the nicotine reduction and/or tobacco-use
cessation program limits the exposure of a program participant to
inhaled nicotine and, concomitantly, reduces the indices of
nicotine dependence and retains the secondary factors of addiction,
including smoke intake, oral fixation, and taste.
[0294] Such a program can utilize, for example, cigarettes that
comprise (e.g., on the leaf or tobacco rod) or deliver (e.g.,
side-stream or main-stream smoke by the FTC and/or ISO methods),
for example, at least, less than or equal to 0.01 mg, 0.05 mg, 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, or
5.0 mg of nicotine. Such a program can utilize conventional NRT
products having, for example, about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6
mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16
mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, or 25
mg of nicotine. The methods described herein facilitate tobacco-use
cessation by allowing the individual to retain the secondary
factors of addiction such as smoke intake, oral fixation, and
taste, while gradually reducing the addictive nicotine levels
consumed. Eventually, complete cessation of tobacco use is made
possible because the presence of addiction for nicotine is
gradually decreased while the individual is allowed to maintain
dependence on the secondary factors, above. Preferred examples of a
nicotine reduction and/or tobacco-use cessation program are
provided in Examples 21-25.
[0295] In another aspect of the invention, the cigarettes of
varying levels of nicotine are packaged to clearly indicate the
level of nicotine present, and marketed as a smoking cessation
program. A preferred approach to produce a product for nicotine
reduction and/or tobacco-use cessation program is provided in
Example 26. Individuals may wish to step up the program by skipping
gradation levels of nicotine per cigarette or staying at certain
steps until ready to proceed to the next level. Significantly,
aspects of the invention allow a consumer to individually select
the amount of nicotine that is ingested by selection of a
particular tobacco product described herein. Furthermore, because
the secondary factors of addiction are maintained, dependence on
nicotine can be reduced rapidly.
[0296] The nicotine reduction and/or tobacco-use cessation program
limits the exposure of a program participant to nicotine while
retaining the secondary factors of addiction. These secondary
factors include but are not limited to, smoke intake, oral
fixation, and taste. Because the secondary factors are still
present, the program participant may be more likely to be
successful in the nicotine reduction and/or tobacco-use cessation
program than in programs that rely on supplying the program
participant with nicotine but remove the above-mentioned secondary
factors. Ultimately, the program participant uses a tobacco product
having an amount of nicotine that is less than that which is
sufficient to become addictive.
[0297] In another aspect of the invention, individuals would choose
to obtain only cigarettes that deliver (e.g., side-stream or
main-stream smoke by the FTC and/or ISO methods) less than 0.05 mg
nicotine per cigarette. Some individuals, such as individuals
needing to stop nicotine intake immediately (for example,
individuals with medical conditions or individuals using drugs that
interact with nicotine) may find this method useful. For some
individuals, the mere presence of a cigarette in the mouth can be
enough to ease withdrawal from nicotine addiction. Gradually, the
addictive properties of smoking can decrease since there is no
nicotine in the cigarettes. These individuals are then able to quit
smoking entirely.
[0298] In another aspect of the invention, packs of cigarettes
containing the gradations of nicotine levels are provided as a
"smoking cessation kit." An individual who wishes to quit smoking
can buy the entire kit of cigarettes at the beginning of the
program. Thus any temptation that may occur while buying cigarettes
at the cigarette counter is avoided. Thus, the success of this
method may be more likely for some individuals. A preferred example
of such a kit is provided in Example 26.
[0299] Various nicotine reduction and/or smoking cessation kits are
prepared, geared to heavy, medium, or light smokers. The kits
provide all of the materials needed to quit smoking in either a
two-week period (fast), a one-month period (medium) or in a
two-month period (slow), depending on the kit. Each kit contains a
set number of packs of cigarettes modified according the present
invention, containing (with reference to delivery of nicotine in
the mainstream smoke by the FTC method) either step 1 cigarettes
containing 0.6 mg nicotine, step 2 cigarettes containing 0.3 mg
nicotine, and step 3 cigarettes containing less than 0.05 mg
nicotine. For example, 1 pack a day smokers would receive 7 packs
of cigarettes, each pack containing the above amounts of nicotine
per each cigarette. Several weeks worth of additional cigarettes
containing less than 0.05 mg nicotine/cigarette would also be
provided in the kit, to familiarize the consumer with smoking no
nicotine cigarettes. The kit may also contain a diary for keeping
track of daily nicotine intake, motivational literature to keep the
individual interested in continuing the cessation program, health
information on the benefits of smoking cessation, and web site
addresses to find additional anti-smoking information, such as chat
groups, meetings, newsletters, recent publications, and other
pertinent links.
[0300] The examples which follow are set forth to illustrate the
present invention, and are not to be construed as limiting
thereof.
EXAMPLE 1
Isolation and Sequencing
[0301] TobRD2 cDNA (Conkling et. al., Plant Phys. 93, 1203 (1990))
encodes QPTase, which is predicted to be a cytosolic protein.
Comparisons of the NtQPT1 amino acid sequence with the GenBank
database 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
Transformation of Tobacco Plants
[0302] DNA of the QPTase gene, 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 QPTase-encoding gene and TobRD2
promoter/antisense QPTase-encoding gene.
[0303] 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 Nic1-/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)).
[0304] Transformed cells are selected and regenerated into
transgenic tobacco plants called R.sub.o. The R.sub.o 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.
[0305] R.sub.o 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 3
Tobacco Having Reduced Nicotine and/or TSNA Levels
[0306] 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, and the expression 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)).
[0307] 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.
[0308] Nicotine levels of T.sub.1 progeny segregating 3:1 were
measured qualitatively using a micro-assay technique. Approximately
.about.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.
[0309] 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."
[0310] 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.
[0311] 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 nornicotine). 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 nornicotine
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.
[0312] 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.
[0313] Vector 21-41 was also grown on approximately 5000 acres by
greater than 600 farmers in five states (Pennsylvania, Mississippi,
Louisiana, Iowa, and Illinois). The US Department of Agriculture,
Agriculture Marketing Service (USDA-AMS) quantified nicotine levels
(expressed as percent nicotine per dry weight) using the FTC method
of 2,701 samples taken from these farms. Nicotine levels ranged
from 0.01% to 0.57%. The average percent nicotine level for all
these samples was 0.09%, with the median of 0.07%. Burley tobacco
cultivars typically have nicotine levels between 2% and 4% dry
weight (Tso, T. C., 1972, Physiology and Biochemistry of Tobacco
Plants. Dowden, Hutchinson, and Ross, Inc. Stroudsbury).
EXAMPLE 4
Regulation of NtQPT1 Gene Expression Using Molecular Decoys
[0314] Nucleotide sequence located between -1000 and -600 or -700
bp of the NtQPT1 promoter is inserted in tandem arrays into a
plant-Agrobacterium shuttle vector and subsequently transformed
into tobacco via methods known to one skilled in the art. Plants
stably transformed with said vector are assessed for the level of
expression of NtQPT1 and for nicotine and/or TSNA content. These
experiments demonstrate that tobacco transformed with molecular
decoys that interact with Nic gene products exhibit a reduced level
of expression of NtQPT1.
EXAMPLE 5
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
Molecular Decoys
[0315] Multiple copies of an approximately 300 or 400 nucleotide
long fragment of the NtQPT1 promoter (e.g., including nucleotide
sequence located between -1000 and -600 or -700 bp of the NtQPT1
promoter), are affixed to microparticles (e.g., by precipitation)
that are suitable for the ballistic transformation of a plant cell
(e.g., 1 to 5 .mu.m gold spheres). The microparticles are propelled
into tobacco plant cells (e.g., Burley 21 LA) using any suitable
ballistic cell transformation methodology, so as to produce
transformed plant cells. Plants are then regenerated from the
transformed plant cells. 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))
[0316] Transformed cells, tissues, and seedlings are grown on
Murashige-Skoog (MS) medium (with or without the selection
compound, e.g., antibiotic, depending on whether a selectable
marker was used. One-hundred independent transformants of Burley 21
LA (T.sub.0) are allowed to self. Progeny of the selfed plants
(T.sub.1) are germinated. Nicotine levels of T.sub.1 progeny are
measured qualitatively using a micro-assay technique. Approximately
200 mg fresh tobacco leaves are collected and ground in 1 ml
extraction solution. (Extraction solution: 1 ml Acetic acid in 100
ml H.sub.2O) Homogenate is centrifuged for 5 min at 14,000.times.g
and supernatant removed to a clean tube, to which the following
reagents are 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 is
prepared and diluted to create a standard series for calibration.
Absorbance at 460 nm is read and nicotine content of test samples
are determined using the standard calibration curve.
[0317] T.sub.1 progeny that have less than 10% of the nicotine
levels of the Burley 21 LA parent are allowed to self to produce
T.sub.2 progeny. Homozygous T.sub.2 progeny are then identified.
Nicotine levels in homozygous and heterozygous T.sub.2 progeny are
also qualitatively determined using the micro-assay. Leaf samples
of homozygous T.sub.2 progeny can also be 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 will have
nicotine levels that are substantially reduced as compared to the
untransformed tobacco (e.g., .about.70 ppm). Because the nicotine
levels in such plants are substantially reduced, the TSNA levels in
these plants are concomitantly reduced.
[0318] These experiments demonstrate that tobacco transformed with
molecular decoys that interact with Nic gene products exhibit a
reduced amount of nicotine and/or TSNA. Plants with multiple tandem
insertions of the molecular decoy that have reduced NtQPT1
expression and reduced nicotine/TSNA levels are used to generate
commercially valuable tobacco products.
EXAMPLE 6
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0319] A transgenic Flue-cured tobacco with a reduced amount of
nicotine and TSNAs was created using an RNAi approach. FIG. 1
illustrates an RNAi construct that was used to create a reduced
nicotine tobacco, wherein the root-specific promoter RD2 (Bp
1-2010) was used to drive expression of an RNAi cassette comprising
an antisense full-length QPTase cDNA (Bp 2011-3409) linked to a 382
bp fragment of the cucumber aquaporin gene (Bp 3410-3792), which is
linked to a sense full-length QPTase cDNA (Bp 3793-5191) and the
GapC terminator (Bp5192-5688) (see SEQ. ID. No. 21). This first
RNAi construct also comprises a GUS-selection cassette comprising
the GapC promoter (Bp 1-1291), which drives expression of the GUS
gene (Bp 1292-3103), linked to the GapC terminator (Bp 3104-3600)
(see SEQ. ID. No. 24). This first RNAi construct was ligated into a
binary vector, pBin19 which was then introduced into Agrobacterium
tumefaciens. Leaf disks from flue-cured variety K326 were then
transformed with Agrobacterium that contained the RNAi construct
comprising the RNAi cassette and the GUS selection cassette.
GUS-based selection was then employed to select positively
transformed plantlets (buds), which were then regenerated to
plants. Leaf samples were then harvested and the alkaloid content
was then determined. The alkaloid content of samples obtained from
some of the transgenic lines created with this first RNAi construct
was 6000 ppm. Since the total alkaloid content in tobacco is about
90% nicotine, it is understood by those skilled in the art that the
transgenic Flue-cured tobacco created using the construct shown in
FIG. 1 has reduced levels of nicotine, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
EXAMPLE 7
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0320] FIG. 2 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a QPTase inhibition cassette (SEQ. ID. No. 22)
and a norflurazone selection cassette (SEQ. ID. No. 25). Starting
from the right border (RB), the QPTase inhibition cassette
comprises an RD2 promoter (Bp 1-2010) operably linked to an
antisense fragment (360 bp) (Bp 2011-2370) of the QPTase gene,
joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense
fragment of the QPTase gene (360 bp) (Bp 3502-3861), which is
joined to the GAD2 terminator (Bp 3862-4134). The selection
cassette comprises the Actin 2 promoter (Bp 1-1161) operably linked
to a mutant phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined
to the GapC terminator (Bp 2891-3387) at the left border (LB).
[0321] Flue-cured tobacco was transformed with the construct shown
in FIG. 2 using Agrobacterium-mediated transformation and 1,140
independent lines were selected, regenerated, and transplanted in
the greenhouse. Of the 1,140 independent lines, 1097 plants were
harvested and tested for alkaloid content. A total of 608 lines
were identified as having less than 1,000 ppm total alkaloid and
139 lines were identified as having less than 500 ppm total
alkaloid. Accordingly, the transgenic Flue-cured tobacco created
using the construct shown in FIG. 2 has significantly reduced
levels of nicotine and TSNA, as compared to a conventional tobacco,
a reference tobacco, or the parental strain of tobacco prior to
genetic modification.
EXAMPLE 8
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0322] Burley tobacco was also transformed with the construct shown
in FIG. 2 using Agrobacterium-mediated transformation and 385
independent lines were selected, regenerated, and transplanted in
the greenhouse. Of the 385 independent lines, 350 lines of plants
were harvested and tested for alkaloid content. A total of 142
lines were identified as having less than 1,000 ppm total alkaloid
and 10 lines were identified as having less than 500 ppm total
alkaloid. Accordingly, it is understood by those skilled in the art
that the transgenic Burley tobacco created using the construct
shown in FIG. 2 also has significantly reduced levels of nicotine
and TSNA, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
EXAMPLE 9
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0323] Oriental tobacco was transformed with the construct shown in
FIG. 2 using Agrobacterium-mediated, Transbacter-mediated or
biolistic transformation and 61 independent lines were selected,
regenerated, and transplanted in the greenhouse. All 61 lines were
tested for alkaloid content and a total of 10 lines were identified
as having less than 1,500 ppm total alkaloids and a total of 3
lines were identified as having less than 1,000 ppm total alkaloid.
Accordingly, it is understood by those skilled in the art that the
transgenic Oriental tobacco created using the construct shown in
FIG. 2 also has significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
EXAMPLE 10
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0324] FIG. 3 illustrates another RNAi construct that can be used
to create a reduced nicotine and TSNA transgenic tobacco. This RNAi
construct has a PMTase inhibition cassette (SEQ. ID. No. 5) and a
norflurazone selection cassette (SEQ. ID. No. 25). Starting from
the right border (RB), the PMTase inhibition cassette comprises an
RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic
acid (241 bp) (Bp 2011-2251) of a PMTase gene, joined to a FAD2
intron (Bp 2252-3382), which is joined to a sense nucleic acid of
the PMTase gene (241 bp) (Bp 3383-3623), which is joined to the
GAD2 terminator (Bp 3624-3896). The selection cassette comprises
the Actin 2 promoter (Bp 1-1161) operably linked to a mutant
phytoene desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC
terminator (Bp 2891-3387) at the left border (LB).
[0325] Flue-cured tobacco will be transformed with the construct
shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Flue-cured tobacco that will be created using the construct shown
in FIG. 3 will have significantly reduced levels of nicotine and
TSNA, as compared to a conventional tobacco, a reference tobacco,
or the parental strain of tobacco prior to genetic
modification.
EXAMPLE 11
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0326] Burley tobacco will be transformed with the construct shown
in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated (see
e.g., Broothaerts et al., Nature 433:629 (2005), herein expressly
incorporated by reference in its entirety) or biolistic
transformation and independent lines will be selected, regenerated,
and transplanted in the greenhouse. Most of the independent lines
grown in the greenhouse will be harvested and tested for alkaloid
content. It is expected that approximately 50% of the lines tested
will have less than 1,000 ppm total alkaloid and approximately 10%
of the lines tested will have less than 500 ppm total alkaloid.
Accordingly, it is expected that the transgenic Burley tobacco that
will be created using the construct shown in FIG. 3 will have
significantly reduced levels of nicotine and TSNA, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
EXAMPLE 12
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0327] Oriental tobacco will also be transformed with the construct
shown in FIG. 3 using Agrobacterium-mediated, Transbacter-mediated
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Oriental tobacco that will be created using the construct shown in
FIG. 3 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
EXAMPLE 13
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0328] FIG. 4 illustrates another RNAi construct that was used to
create a reduced nicotine and TSNA transgenic tobacco. This RNAi
construct has a A622 inhibition cassette (SEQ. ID. No. 3) and a
norflurazone selection cassette (SEQ. ID. No. 25). Starting from
the right border (RB), the A622 inhibition cassette comprises an
RD2 promoter (Bp 1-2010) operably linked to an antisense nucleic
acid (628 bp) (Bp 2011-2638) of the A622 gene, joined to a FAD2
intron (Bp 2639-3769), which is joined to a sense nucleic acid of
the A622 gene (628 bp) (Bp 3770-4397), which is joined to the GAD2
terminator (Bp 4398-4670). The selection cassette comprises the
Actin 2 promoter (Bp 1-1161) operably linked to a mutant phytoene
desaturase gene (PDSM1) (Bp 1162-2890) joined to the GapC
terminator (Bp 2891-3387) at the left border (LB).
[0329] Flue-cured tobacco was transformed with the construct shown
in FIG. 4 using Agrobacterium-mediated transformation and 270
independent lines were selected, regenerated, and transplanted in
the greenhouse. Of the 270 independent lines, 259 plants were
harvested and tested for alkaloid content. A total of 131 lines
were identified as having less than 1,000 ppm total alkaloid and 45
lines were identified as having less than 500 ppm total alkaloid.
Accordingly, it is understood by those skilled in the art that the
transgenic Flue-cured tobacco created using the construct shown in
FIG. 4 also has significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco or the
parental strain of tobacco prior to genetic modification.
[0330] Several lines that were transformed with this construct were
unexpectedly found to have conventional levels of nicotine but a
significantly reduced amount of nornicotine. That is, 9 lines were
found to have nicotine levels ranging from 2.17 mg/g to 3.99 mg/g
and nornicotine levels less than or equal to 0.00 to 0.06 mg/g (see
Table 2). TABLE-US-00009 TABLE 2 Transgenic tobacco having reduced
nornicotine and conventional amounts of nicotine Alkaloid
Nornicotine Nicotine new I.D (ppm) (mg/g) (mg/g) VDG 0 20 2486.53
##STR9## 2.30 VDG 0 32 4683.01 ##STR10## 3.48 VDG 0 45 4490.79
##STR11## 3.94 VDG 0 52 2855.58 ##STR12## 2.61 VDG 0 54 2291.89
##STR13## 2.17 VDG 0 77 4857.86 ##STR14## 3.99 VDG 0 97 3072.40
##STR15## 2.58 VDG 107 4921.31 ##STR16## 3.59 VDG 116 4960.64
##STR17## 3.56 Control- 5005.22 0.28 4.02 8 Control- 5711.97 0.34
5.35 20 Control- 5196.25 0.24 4.52 28 *Highlighted entries show
transgenic tobacco lines having a reduced amount of nornicotine and
conventional amounts of nicotine.
EXAMPLE 14
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0331] Burley tobacco will be transformed with the construct shown
in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated or
biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Burley tobacco that will be created using the construct shown in
FIG. 4 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification. It is
also expected that some lines of tobacco created with the
afore-mentioned nucleic acid construct will retain conventional
amounts of nicotine but will comprise a reduced amount of
nornicotine, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
EXAMPLE 15
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
RNAi
[0332] Oriental tobacco will also be transformed with the construct
shown in FIG. 4 using Agrobacterium-mediated, Transbacter-mediated,
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Oriental tobacco that will be created using the construct shown in
FIG. 4 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification. It is
also expected that some lines of tobacco created with the
afore-mentioned nucleic acid construct will retain conventional
amounts of nicotine but will comprise a reduced amount of
nornicotine, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
EXAMPLE 16
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
a Double Knock-Out RNAi Construct
[0333] FIG. 5 illustrates a double-knock-out RNAi construct, which
has been created to develop a reduced nicotine and TSNA transgenic
tobacco. This double-knock-out RNAi construct has a QPTase/A622
inhibition cassette (SEQ. ID. No.23) and a norflurazone selection
cassette (SEQ. ID. No. 25). Starting from the right border (RB),
the QPTase/A622 inhibition cassette comprises an RD2 promoter (Bp
1-2010) operably linked to a QPTase antisense nucleic acid (360 bp)
(Bp 2011-2370) of a QPTase gene, which is joined to a A622
antisense nucleic acid (628 bp) (Bp 2371-2998) of a A622 gene,
which is joined to a FAD2 intron (Bp 2999-4129), which is joined to
a sense nucleic acid of the A622 gene (628 bp) (Bp 4130-4757),
which is joined to a sense nucleic acid of the QPTase gene (360 bp)
(Bp 4758-5117), which is joined to the GAD2 terminator (Bp
5118-5390). The selection cassette comprises the Actin 2 promoter
(Bp 1-1161) operably linked to a mutant phytoene desaturase gene
(PDSM1) (Bp 1162-2890) joined to the GapC terminator (Bp 2891-3387)
at the left border (LB).
[0334] Flue-cured tobacco was transformed with the construct shown
in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated, or
biolistic transformation and 444 independent lines were selected,
regenerated, and transplanted in the greenhouse. Each of these
lines were analyzed for alkaloid content and 240 lines were found
to have less than 1,000 ppm total alkaloid and 18 lines were found
to have less than 500 ppm total alkaloid. Accordingly, it is
understood by those skilled in the art that the transgenic
Flue-cured tobacco created Using the construct shown in FIG. 5 also
has significantly reduced levels of nicotine and TSNA, as compared
to a conventional tobacco, a reference tobacco, or the parental
strain of tobacco prior to genetic modification.
EXAMPLE 17
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
a Double Knock-Out RNAi Construct
[0335] Burley tobacco will be transformed with the construct shown
in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated or
biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Burley tobacco that will be created using the construct shown in
FIG. 5 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
EXAMPLE 18
Tobacco Having Reduced Nicotine and/or TSNA Levels Generated Using
a Double Knock-Out RNAi Construct
[0336] Oriental tobacco will also be transformed with the construct
shown in FIG. 5 using Agrobacterium-mediated, Transbacter-mediated,
or biolistic transformation and independent lines will be selected,
regenerated, and transplanted in the greenhouse. Most of the
independent lines grown in the greenhouse will be harvested and
tested for alkaloid content. It is expected that approximately 50%
of the lines tested will have less than 1,000 ppm total alkaloid
and approximately 10% of the lines tested will have less than 500
ppm total alkaloid. Accordingly, it is expected that the transgenic
Oriental tobacco that will be created using the construct shown in
FIG. 5 will have significantly reduced levels of nicotine and TSNA,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
EXAMPLE 19
Low Nicotine and Nitrosamine Blended Tobacco
[0337] 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. With reference to nicotine and/or TSNA delivery in
mainstream smoke by the FTC method, 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. Other approaches
blend only low nicotine/TSNA tobaccos (e.g., genetically modified
Burley, genetically modified Virginia flue, genetically modified
Oriental tobaccos that contain reduced amounts of nicotine and/or
TSNAs, and tobacco that has been treated to remove nicotine).
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.
[0338] By one approach, a step 1 tobacco product is 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 per 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.
[0339] 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
Burley 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
Burley 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
Burley 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.
[0340] 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, flavor, and smokability. In this manner, a variety of
types of tobacco products having varying level of nicotine and/or
nitrosamine, as well as, appearance, flavor and smokability can be
created.
EXAMPLE 20
Low Nicotine and TSNA Blended Tobacco
[0341] By a preferred method, conventional Virginia flue tobacco
was blended with genetically modified Burley (i.e., Burley
containing a significantly reduced amount of nicotine and
nitrosamine) to yield a blended tobacco that was incorporated into
three levels of reduced nicotine cigarettes (Quest 1.RTM.,
Quest2.RTM., and Quest3.RTM.): a step 1 cigarette containing 0.6 mg
nicotine, a step 2 cigarette containing 0.3 mg nicotine, and a step
3 cigarette containing less than 0.05 mg nicotine (nicotine
delivery in mainstream smoke by the FTC method). The amount of
total TSNA was found to range between approximately 0.17
.mu.g/g-0.6 .mu.g/g (TSNA delivery in mainstream smoke by the FTC
method).
[0342] In some cigarettes, approximately, 28% of the blend was
Virginia flue tobacco, approximately 29% of the blend was
genetically modified (i.e., reduced nicotine Burley), approximately
14% of the blend was Oriental, approximately 17% of the blend was
expanded flue-cured stem, and approximately 12% was standard
commercial reconstituted tobacco. The amount of total TSNAs in
cigarettes containing this blend was approximately 1.5 .mu.g/g
(TSNA delivery in mainstream smoke by the FTC method).
[0343] TABLES 3, 4, and 5 show the results of a study that analyzed
the amount of nicotine present in the blended tobacco used to make
the Quest 1.RTM. and Quest2.RTM. cigarettes, as compared to the
Vector 21-41 tobacco used to generate the Quest3.RTM. tobacco
product, which is not a blended cigarette. TABLE-US-00010 TABLE 3
QUEST 1 Average mg/g Lot#031805- BLEND Nicotine per dry 1H9R Sample
ID weight basis Range % CV Quest 1 (1) 14.291 0.230 0.48 Quest 1
(2) 14.958 0.230 1.54 Quest 1 (3) 15.207 0.021 0.14 Quest 3
"control" 0.533 0.007 1.28 Q1 AVG 14.818 STD 0.474 % CV 0.032
[0344] Analysis done according to 1999 National Registry
Method.
[0345] Nicotine reported in a dry weight basis TABLE-US-00011 TABLE
4 GC ANAL. DATE Lot#031 Nicotine 105- Content CLINIC 2F7P after
QUEST 2 TRIAL Nicotine corrected BLEND Weight Content for % mg/g
Avg. Sample of Tob. from GC % Moisture of mg/g ID (g) (mg) Moist.
(mg) Tob. Nic. Range % CV 1 A 1.002 8.136 8.230 8.213 B 1.000 8.173
1.14 8.267 8.267 8.240 0.038 0.47 2 A 1.001 7.679 7.810 7.802 B
1.003 7.662 1.67 7.792 7.768 7.785 0.017 0.22 3 A 1.001 6.666 6.809
6.802 B 1.000 7.020 2.1 7.171 7.171 6.986 0.261 3.73 Q3 A 1.002
0.488 0.497 0.496 control B 1.001 0.501 1.89 0.510 0.510 0.503
0.010 1.93
[0346] TABLE-US-00012 TABLE 5 Average mg/g QUEST 3 Nicotine per dry
Sample ID weight basis Range % CV 1 0.902 0.006 0.63 2 0.905 0.010
1.14 3 0.885 0.008 0.88 Q3 control 0.503 0.010 1.93 AVG 0.897 STD
0.011 % CV 1.20%
[0347] Analysis done according to 1999 National Registry
Method.
[0348] Nicotine reported in a dry weight basis
[0349] TABLE 6 shows the amounts of nicotine present in the
mainstream smoke generated by Quest 1.RTM., Quest2.RTM., and
Quest3.RTM. tobacco products (as determined by the FTC method), as
compared to two different commercially sold cigarettes (cigarette
A, a "100" type and cigarette B, a "light" type) and a reference
cigarette (2R4F). The data shows that the Quest 1.RTM.,
Quest2.RTM., and Quest3.RTM. products deliver significantly less
nicotine than the two commercially available cigarettes and the
reference cigarette. It should be noted that the Quest 3.RTM.
analyzed in this study is not a blended tobacco in that it only
contains Vector 21-41 tobacco. TABLE-US-00013 TABLE 6 Client Arista
MS TPM CO Water Nicotine Tar Run# Port# Code Code Puffs/Cigt
(mg/cigt) (mg/cigt) (mg/cigt) (mg/cigt) (mg/cigt) C00420 E Cig A
3557 11.3 24.74 16.66 3.96 1.48 19.30 C00420 J Cig A 3557 11.3
23.58 16.31 2.49 1.52 19.58 C00420 Q Cig A 3557 11.6 24.66 18.05
3.56 1.53 19.57 C00421 J Cig A 3557 11.2 24.14 17.36 2.79 1.49
19.86 C00421 O Cig A 3557 11.5 23.66 16.82 2.74 1.49 19.43 C00421 Q
Cig A 3557 11.0 23.96 18.04 2.32 1.47 20.17 Average 11.3 24.12
17.21 2.98 1.50 19.65 sd 0.2 0.49 0.73 0.64 0.02 0.32 % RSD 1.9 2.0
4.3 21.7 1.4 1.6 C00420 C Cig B 3558 8.6 12.72 11.60 1.09 0.83
10.79 C00420 I Cig B 3558 8.1 12.18 12.69 0.86 0.81 10.50 C00420 K
Cig B 3558 8.1 12.56 10.90 0.99 0.81 10.76 C00420 M Cig B 3558 8.1
12.46 10.93 1.07 0.80 10.59 C00420 O Cig B 3558 8.1 12.20 10.96
0.94 0.81 10.44 C00421 R Cig B 3558 8.6 13.50 11.70 0.95 0.86 11.70
Average 8.3 12.60 11.46 0.98 0.82 10.80 Sd 0.3 0.49 0.70 0.09 0.02
0.46 % RSD 3.4 3.9 6.1 8.8 2.5 4.3 C00421 A Quest 1 3559 7.1 11.12
11.46 1.09 0.48 9.54 C00421 D Quest 1 3559 6.6 11.22 11.36 0.93
0.49 9.80 C00421 F Quest 1 3559 6.6 10.98 10.68 1.08 0.47 9.44
C00421 H Quest 1 3559 6.3 10.78 10.44 1.12 0.47 9.20 C00421 K Quest
1 3559 6.8 11.20 11.28 1.18 0.45 9.57 C00421 M Quest 1 3559 6.7
11.04 11.19 0.65 0.48 9.91 Average 6.7 11.06 11.07 1.01 0.47 9.58
sd 0.3 0.16 0.41 0.19 0.01 0.26 % RSD 3.8 1.5 3.7 19.1 3.0 2.7
C00420 D Quest 2 3560 6.4 10.88 11.89 0.70 0.29 9.90 C00420 S Quest
2 3560 6.6 10.52 12.03 1.03 0.27 9.21 C00421 B Quest 2 3560 6.7
12.18 11.91 1.67 0.31 10.20 C00421 I Quest 2 3560 6.2 10.44 10.88
0.97 0.25 9.21 C00421 L Quest 2 3560 6.2 10.78 11.25 1.32 0.28 9.18
Average 6.4 10.96 11.59 1.14 0.28 9.54 sd 0.2 0.71 0.50 0.37 0.02
0.48 % RSD 3.9 6.4 4.3 32.7 7.0 5.0 C00420 B Quest 3 3561 6.1 8.72
9.79 0.79 0.049 7.89 C00420 P Quest 3 3561 6.1 7.94 9.76 0.44 0.047
7.46 C00420 R Quest 3 3561 6.3 8.58 10.39 0.58 0.052 7.95 C00421 C
Quest 3 3561 6.2 9.22 10.30 0.89 0.045 8.28 C00421 N Quest 3 3561
6.2 8.62 10.38 0.95 0.047 7.62 Average 6.2 8.62 10.12 0.73 0.048
7.84 sd 0.1 0.46 0.32 0.22 0.003 0.32 % RSD 1.5 5.3 3.2 29.6 5.7
4.1 C00420 N 2R4F 912 9.0 10.80 11.50 0.62 0.79 9.39 C00421 G 2R4F
912 9.1 12.24 13.32 0.92 0.83 10.49 C00421 P 2R4F 912 9.3 11.50
12.09 0.78 0.80 9.92 Average 9.1 11.51 12.30 0.77 0.81 9.93 sd 0.2
0.72 0.93 0.15 0.02 0.55 % RSD 1.8 6.3 7.5 19.4 2.9 5.5
[0350] TABLE 7 shows the amounts of TSNAs present in mainstream
smoke generated by Quest 1.RTM., Quest2.RTM., and Quest3.RTM. (as
determined by the FTC method), as compared to two different
commercially sold cigarettes (cigarette A, a "100" type and
cigarette B, a "light" type) and a reference cigarette (2R4F). The
data shows that the Quest 1.RTM., Quest2.RTM., and Quest3.RTM.
products deliver significantly less TSNAs than the two commercially
available cigarettes and the reference cigarette. It should be
noted that the Quest 3.RTM. analyzed in this study is not a blended
tobacco in that it only contains Vector 21-41 tobacco.
TABLE-US-00014 TABLE 7 ##STR18## ##STR19## ##STR20## ##STR21##
##STR22## ##STR23## Note: Shaded cells contain values between the
Limit of Detection (LOD) and Limit of Quantitation (LOQ) for the
method.
[0351] In one study, a Canadian intense smoking regimen was
performed, wherein it was demonstrated that Quest 3.RTM. delivers
0.05 mg/cig using extreme smoking parameters. (See TABLES 8 and 9).
Note, that Quest 1.RTM. delivers 1.07 mg/cig under these conditions
as opposed to 0.60 using FTC smoking conditions. Similar results
are observed for Quest 2.RTM.. TABLE-US-00015 TABLE 8 Puff Count
Nicotine Tar Matrix Condition Sample (/cig) (mg/cig) (mg/cig) Code
Code ID Average St Dev Average St Dev Average St Dev MS N 040501
8.6 0.2 1.07 0.06 22.1 1.4 MS N 040502 8.6 0.2 0.606 0.030 21.8 1.4
MS N 040503 8.0 0.3 0.050 0.003 19.8 1.2 Glossary of Abbreviations
Condition Code: N - puff volume, 55 mL; interval, 30 sec; duration,
2 sec; vent blocking, 100%. Brand Sample ID Description 040501
Quest .RTM. Lights 1 Low Nicotine 040502 Quest .RTM. Lights 2 Extra
Low Nicotine 040503 Quest .RTM. Lights 3 Nicotine Free
[0352] TABLE-US-00016 TABLE 9 Yields of "Tar" and Nicotine in
Mainstream Tobacco Smoke: `intense` Conditions* Sample Weight Puff
Count MS TPM CO Water Nicotine Tar ID (mg/cig) (per cig) (mg/cig)
(mg/cig) (mg/cig) (mg/cig) (mg/cig) 040501 1044 8.1 32.4 10.2 1.04
21.2 040501 1043 8.4 35.2 11.4 1.05 22.7 040501 1062 8.6 35.4 11.7
1.08 22.6 040501 1057 8.7 33.0 11.1 1.04 20.9 040501 1059 8.7 35.4
12.5 1.07 21.8 040501 1061 8.8 35.4 11.2 1.12 23.0 040501 1067 8.7
31.6 9.70 1.04 20.8 040501 1054 8.4 35.0 10.9 1.09 23.0 040501 1060
8.7 32.3 9.58 1.07 21.7 040501 1055 8.5 36.1 12.3 1.01 22.8 040501
1058 8.8 38.4 12.6 1.16 24.6 040501 1042 8.3 31.7 9.92 0.977 20.8
040501 1042 9.0 36.4 11.7 1.18 23.5 040501 1061 8.9 32.9 10.5 1.07
21.3 040501 1054 8.3 32.4 10.5 1.03 20.9 040501 1064 8.6 32.4 10.7
1.02 20.7 040501 1068 8.4 32.7 10.8 1.04 20.8 040501 1051 8.4 31.0
10.1 0.995 20.0 040501 1037 8.3 38.1 11.7 1.13 25.2 040501 1058 8.6
35.1 10.7 1.16 23.3 Average 1055 8.6 34.1 11.0 1.07 22.1 Std. Dev.
9 0.2 2.2 0.9 0.06 1.4 Coeff. Var. 0.9 2.7 6.4 8.2 5.4 6.5 040502
1066 8.6 32.7 10.9 0.642 21.2 040502 1067 8.7 36.5 12.2 0.659 23.7
040502 1062 8.6 29.1 9.22 0.576 19.3 040502 1039 8.7 32.5 10.5
0.599 21.3 040502 1058 8.5 34.8 11.5 0.572 22.7 040502 1049 8.9
36.0 12.0 0.608 23.5 040502 1051 8.8 31.4 10.3 0.608 20.5 040502
1049 8.2 34.2 11.4 0.585 22.2 040502 1053 8.5 35.6 12.0 0.639 23.0
040502 1073 8.8 34.8 11.0 0.633 23.2 040502 1054 8.2 29.6 9.43
0.529 19.6 040502 1062 8.6 32.9 10.3 0.577 22.0 040502 1068 8.6
35.2 11.4 0.608 23.1 040502 1061 8.9 35.9 11.8 0.633 23.5 040502
1089 8.8 35.4 12.1 0.618 22.6 040502 1084 8.6 34.2 11.6 0.593 22.0
040502 1028 8.6 33.3 11.8 0.586 20.9 040502 1097 9.0 32.5 11.5
0.617 20.4 040502 1077 8.7 30.5 9.65 0.625 20.3 040502 1060 8.4
31.3 9.46 0.616 21.3 Average 1062 8.6 33.4 11.0 0.606 21.8 Std.
Dev. 17 0.2 2.2 1.0 0.030 1.4 Coeff. Var. 1.6 2.5 6.6 8.9 5.0 6.2
040503 1005 8.0 29.9 9.95 0.046 19.9 040503 996 7.4 26.7 8.50 0.045
18.1 040503 1029 8.4 30.4 10.3 0.054 20.1 040503 1015 7.9 31.9 10.7
0.051 21.2 040503 1026 8.2 30.8 10.0 0.050 20.7 040503 1011 8.3
28.2 8.91 0.049 19.2 040503 1014 8.4 29.7 9.26 0.052 20.4 040503
1013 8.5 29.6 9.57 0.050 19.9 040503 1002 7.7 29.5 9.86 0.046 19.6
040503 1007 8.3 31.0 10.6 0.053 20.4 040503 1012 8.0 30.0 10.3
0.047 19.6 040503 1000 7.8 32.8 12.1 0.050 20.7 040503 1005 7.7
32.4 11.7 0.046 20.6 040503 1023 8.1 28.3 9.57 0.048 18.7 040503
1028 8.5 25.5 8.39 0.047 17.1 040503 1010 8.3 31.5 10.5 0.051 20.9
040503 1009 7.9 32.2 11.8 0.054 20.4 040503 1011 8.1 30.0 11.2
0.048 18.8 040503 1016 7.7 31.7 10.0 0.058 21.6 040503 994 7.3 25.4
7.47 0.053 17.9 Average 1011 8.0 29.9 10.0 0.050 19.8 Std. Dev. 10
0.3 2.1 1.2 0.003 1.2 Coeff. Var. 1.0 4.2 7.2 11.7 6.8 6.0 *puff
volume, 55 mL; interval, 30 sec; duration, 2 sec; vent blocking,
100%. See text for additional details.
EXAMPLE 21
Nicotine Reduction and/or Smoking Cessation Program Utilizing
Reduced Nicotine Tobacco Products
[0353] The following example describes a nicotine reduction and/or
smoking cessation program utilizing the low nicotine, low TSNA
tobacco products of the present invention. The modified tobacco
containing very low levels of TSNAs and essentially no nicotine was
mixed with tobacco having a known amount of nicotine to create
specific, stepwise levels of nicotine per cigarette. As an example,
Virginia flue tobacco was blended with genetically modified Burley
(i.e., Burley containing a significantly reduced amount of nicotine
and nitrosamine) to yield a blended tobacco that was incorporated
into three levels of reduced nicotine cigarettes (with reference to
nicotine and/or TSNA delivery in mainstream smoke by the FTC
method): a step 1 cigarette containing 0.6 mg nicotine, a step 2
cigarette containing 0.3 mg nicotine, and a step 3 cigarette
containing less than 0.05 mg nicotine. The stepwise packs of
cigarettes were clearly marked as to their nicotine content, and
the step in the stepwise nicotine reduction program was also
clearly marked on the package. Each week, the user purchases packs
containing cigarettes having the next lower level of nicotine, but
limits himself to no more cigarettes per day than consumed
previously. The user may define his/her own rate of nicotine
reduction and/or smoking cessation according to individual needs by
choosing a) the number of cigarettes smoked per day b) the starting
nicotine levels c) the change in nicotine level per cigarette each
week, and d) the final level of nicotine consumed per day. To keep
better track of the program, the individual keeps a daily record of
total nicotine intake, as well as the number of cigarettes consumed
per day. Eventually, the individual will be consuming tobacco
products with essentially no nicotine. Since the nicotine-free
tobacco products of the final step are non-addictive, it should
then be much easier to quit the use of the tobacco products
altogether.
EXAMPLE 22
Nicotine Reduction and/or Smoking Cessation Method Using Reduced
Nicotine Cigarettes
[0354] The effectiveness of Quest.RTM. cigarettes was evaluated in
a limited study. The chemical and physical characteristics of
Quest.RTM. cigarettes are found in TABLES 3-10 (Examples 20 and
23). The primary objective of this prospective randomized
controlled clinical trial was a continuous four-week period of
abstinence from smoking. The study consisted of three treatment
arms, comprised of 15 subjects each:
1) Quest 3.RTM.+nicotine patch
2) Quest 3.RTM.+placebo patch
3) Quest 1.RTM., Quest 2.RTM. and Quest 3'.
[0355] The study was 18 weeks in duration. During the first week of
the study, subjects smoked only their usual brand of cigarette.
From Weeks 2 through 5, Quest.RTM. cigarettes were introduced while
subjects gradually reduced the amount of usual brand smoked (Groups
1 and 2) or the amount of nicotine per cigarette (Group 3). By Week
6, subjects were expected to be smoking Quest 3.RTM. cigarettes
exclusively. From Weeks 6 through 11, subjects acclimated
themselves to nicotine-free smoking. They were gradually weaned off
Quest 3.RTM. (with phased discontinuation of nicotine patches for
Groups 1 and 2) from Week 12 through Week 14 in anticipation of the
four-week abstinence period during Weeks 15 through 18. Secondary
endpoints evaluated included compensatory smoking behavior and
withdrawal symptoms.
[0356] The overall success rate in achieving abstinence was 16
percent (7/45). Group 2 had the highest success, with approximately
33 percent of subjects (5/15) achieving four-week continuous
abstinence. Smokers in Group 3 attained a 13 percent (2/15)
abstinence rate. Group 1 experienced the lowest rates of success,
with no subjects reaching the primary abstinence outcome. In
addition, subjects who complied with the protocol regimen of use of
nicotine-free Quest 3.RTM. cigarettes exclusively in the weeks
leading up to their quit-smoking date had higher abstinence success
rates (54 percent, 7/13). The failure of Group 1 (0 percent quit
rate) was perceived as a methodology issue, and it was suggested
that future studies incorporating a similar combination therapy
have subjects wear the patch beyond the quit date.
[0357] Subjects rated Quest.RTM. cigarettes as less satisfying than
their usual brand and, therefore, did not crave them as much as
their regular brand of cigarette. This outcome was perceived by the
author as beneficial since the lack of a craving for Quest.RTM.
cigarettes may aid smoking cessation and also deter sustained use.
Even though Quest.RTM. was less satisfying, subjects did not
compensate for the lack of nicotine by smoking more cigarettes or
inhaling more smoke in each puff. Across the three groups, the
number of cigarettes smoked and the expired CO levels did not
increase during the six weeks that subjects had free access to
Quest.RTM. cigarettes.
[0358] The results of this pilot study indicate that Quest.RTM.
cigarettes, especially in the format of decreasing levels of
nicotine such as that which exists in Quest.RTM. 1,2,3, have the
potential to be efficacious as a smoking cessation aid.
[0359] Jed Rose and F M Behm also conducted a blinded, randomized
study designed to determine if compensatory smoking behaviors
differ depending on whether highly ventilated filtered or
reduced-nicotine filtered cigarettes are being consumed by the same
subjects. Rose J E, Behm F M. "Effects of low nicotine content
cigarettes on smoke intake" Nicotine and Tobacco Research
6:1-11(2004a) (see below).
[0360] A total of 16 smokers were enrolled in this study. The study
consisted of two separate 8-hour sessions where subjects randomly
smoked either low-nicotine filtered or highly ventilated filter
cigarettes ad libitum during each session, with a counterbalanced
order of smoking conditions presented across subjects. The
standardized smoke yield of nicotine was 0.02 mg/cigarette for the
Quest.RTM. brand low-nicotine cigarettes, and 0.2 mg/cigarette for
the highly ventilated filter cigarettes. Endpoints evaluated
included expired carbon monoxide, puff volume and number of puffs,
craving, arousal, reward, and airway sensations.
[0361] The number of cigarettes smoked during each 8-hour session
was significantly higher when subjects consumed the highly
ventilated filter cigarettes (mean=11.9) versus the low-nicotine
filtered cigarettes (mean=10.4). Cumulative puff volume taken from
the last cigarette of the session (for which puffing topography was
monitored) was also significantly higher for the highly ventilated
filtered cigarettes (800 cc) than for the low-nicotine filtered
cigarettes (500 cc). Subjects tended to take more puffs from the
highly ventilated filtered cigarettes, though the difference was
not statistically significant.
[0362] The investigators devised a scale to evaluate expired-air
carbon monoxide, which they called the "compensation index."
Expired-air CO values, as measured at the end of each 8-hour
session, were divided by the FTC standardized values for CO
delivery for each type of cigarette. Thus, a higher ratio would
indicate a compensatory increase in smoking behavior. Even though
expired CO levels were higher for the low-nicotine cigarettes (due
to a higher smoke yield), the compensation index was significantly
higher for the highly ventilated filter cigarettes (index=1.1) than
for the low-nicotine filtered cigarettes (index=2.4). (The
compensation index was graphed incorrectly in the paper and an
erratum reflecting the corrected values was submitted.)
[0363] The authors concluded that compensatory smoking behaviors
increased significantly during the highly ventilated filter
cigarette sessions, relative to the low-nicotine filtered
cigarettes. While the study provided some evidence that smokers
would be unlikely to compensate when switching from their usual
brand of cigarette to low-nicotine cigarettes, these suggestions
are indirect and uncontrolled since there was no session involving
smokers' usual brand of cigarette. However, baseline CO readings
obtained when subjects were smoking their usual brands of
cigarettes were similar to the readings obtained when smoking
Quest.RTM.. The data obtained from these studies and others to date
provide strong evidence that tobacco-use cessation programs that
employ Quest 1.RTM., Quest 2.RTM. and Quest 3.RTM. with or without
NRT (e.g., the nicotine patch) are effective approaches to achieve
cessation of tobacco use.
EXAMPLE 23
A Prospective, Double Blind, Randomized, Active-Controlled,
Parallel Group, Multicenter Test to Evaluate the Effectiveness of
Reduced Nicotine Cigarettes Alone and in Combination with Nicotine
Replacement Therapy as a Smoking Cessation Aid
[0364] This Phase II study will assess the magnitude of
effectiveness that can be obtained with Quest 1.RTM., Quest 2.RTM.
and Quest 3.RTM. alone and in combination with NRT compared to
conventional pharmacologic therapy, i.e., the NRT patch alone, as a
smoking cessation aid.
[0365] This will be a prospective, randomized, multicenter,
double-blind, parallel, active controlled Phase II trial in which a
total of 345 healthy smokers who are motivated to quit smoking will
be enrolled and followed over 8 months at approximately 5 sites.
Healthy smokers with a desire to quit smoking will be screened for
eligibility. This screening will include documentation of relevant
medical/smoking history including documentation of current usual
brand, physical examination, urine pregnancy test (in women of
child-bearing potential), drug screen, exhaled carbon monoxide
(CO), saliva cotinine level and the completion of four
questionnaires. Following screening, eligible subjects will be
randomized in a 1:1:1 ratio to one of the three treatment arms:
[0366] Group 1: Quest 1.RTM., Quest 2.RTM. and Quest 3.RTM. plus
NRT (patch)
[0367] Group 2: Quest 1.RTM., Quest 2.RTM. and Quest 3.RTM. plus
placebo patch
[0368] Group 3: Active Control cigarettes (conventional) plus NRT
(patch)
[0369] Quest Smoking Cessation Product is a tobacco-based
(botanical) medical product in cigarette form. As a `finished
good`, the product has been developed to meet specifications
similar to conventional cigarettes in both design and use, as
illustrated by the following product specifications. TABLE-US-00017
TABLE 10 QUEST .RTM. CHEMICAL AND PHYSICAL SPECIFICATIONS Quest 1
.RTM. Quest 2 .RTM. Quest 3 .RTM. Chemical: Tar (mg/cigt) 9.0 .+-.
1.0 8.0 .+-. 1.25 8.5 .+-. 1.0 Nicotine (mg/cigt) 0.59 .+-. 0.06
0.30 .+-. 0.05 <0.05 Moisture 12.4% .+-. 1.0% 12.4% .+-. 1.0%
12.4% .+-. 1.0% Carbon Monoxide 12.5 max 12.5 max 12.5 max
(mg/cigt) Physical: Total Weight (mg/cigt) 984 .+-. 70 984 .+-. 70
943 .+-. 70 Circumference (mm) 24.6 .+-. 0.1 24.6 .+-. 0.1 24.6
.+-. 0.1 Pressure Drop 115 .+-. 10 115 .+-. 10 123 .+-. 10 (mm
H.sub.2O) Hardness (%), 74 .+-. 3 74 .+-. 3 74 .+-. 3
unequlilibrated
[0370] The genetically modified plant used to produce the low
nicotine yielding tobacco product has been designated Vector 21-41.
This line is a transgenic tobacco that produces very low nicotine
levels by disrupting the normal expression of a quinolinic acid,
phosphoribosyltransferase, a key enzyme in the biosynthetic pathway
leading to the production of nicotine. Vector 21-41 tobacco is a
comparable plant to traditional wild-type tobacco and more
significantly, its non-transgenic parent, Burley 21 LA. Vector
21-41 does not express any novel agronomic trait such as insect
resistance or herbicide tolerance.
[0371] The nicotine patch is an over-the-counter (OTC) product;
brand names include Nicoderm CQ.RTM. and Nicotrol.RTM. and generic
products are available as well. The protocol specifies compliance
with current labeling for OTC patches. Careful consideration has
been made to limit active patch exposure to 10 weeks with no more
than 6 weeks of exposure to the highest dosage form (21 mg) and no
more than 2 weeks exposure at lower weaning dosages of 14 mg and 7
mg respectively. The patch will be replaced daily (every 24 hours)
as directed. In order to ensure blinding, a placebo patch will be
utilized in this study as well.
[0372] The Active Control cigarette to be used by Group 3 is a
conventional, American-blended cigarette with a tar content of
10.2.+-.0.5 mg/cigarette and a nicotine content of 0.80.+-.0.10
mg/cigarette, however, the cigarettes are "sham-faded" (i.e., the
nicotine content of the Active Control does not decrease, but it is
administered in accordance with that of the Quest.RTM. Smoking
Cessation Product to simulate the fading) every two weeks in
parallel with the fading provided by Quest 1.RTM., Quest 2.RTM. and
Quest 3.RTM. in Groups 1 and 2.
[0373] The objective of the Active Control is to deliver a constant
nicotine concentration with the same appearance and taste as the
investigational product. This matching reduces confounding and/or
bias in the trial outcome 1) that may be introduced by other
features of the device that differ from the Active Control; or 2)
that may lead to breaking of the blinding.
[0374] All participating subjects are exposed to 6 weeks of smoking
(either Quest 1.RTM., Quest 2.RTM., Quest 3.RTM. or conventional
"faded" cigarettes) and 12 weeks of application of the nicotine
and/or placebo patch. The last 2 weeks of smoking and first 2 weeks
of patch use overlap (Week 5 and 6).
[0375] Subjects in Group 1 and 2 will transition from their usual
brand (UB) cigarettes to Quest cigarettes. During the first 6 weeks
(Week 1-Week 6), subjects in Groups 1 and 2 will smoke ad libitum
for two weeks at each Quest.RTM. nicotine level beginning with
Quest 1.RTM., then proceeding to Quest 2.RTM. and then to Quest
3.RTM.. At Week 5, Quest.RTM. use will be supplemented in the first
treatment group with the addition of a transdermal nicotine patch
(21 mg), while subjects in Group 2 will add a placebo patch. Visit
5 is considered the `quit date` (quit all smoking).
[0376] Subjects in Group 1 will receive the 21 mg transdermal
nicotine patch for an additional 4 weeks (total period Week 5-Week
10), followed by the 14 mg transdermal nicotine patch for 2 weeks
(Week 11 and 12), the 7 mg transdermal nicotine patch for 2 weeks
(Week 13 and 14) and a placebo patch for 2 weeks (Week 15 and 16).
Subjects in Group 2 will receive a placebo patch only starting at
Week 5 through Week 16.
[0377] The third treatment group is a blinded control for the
exposure to Quest 1.RTM., Quest 2.RTM. and Quest 3.RTM. in Group 1,
whereby subjects are provided with an Active Control cigarette
(nicotine dose comparable to a conventional, American-blended
cigarette), but the cigarettes are "sham-faded" every 2 weeks in
parallel with the fading provided by Quest 1.RTM., Quest 2.RTM. and
Quest 3.RTM. in Group 1. The NRT treatment for Group 3 is the
current standard of care, indicating the addition of a placebo
patch during Week 5 and 6 preceding the "quit date" at Week 7
(visit 5). Subjects in Group 3 will use the 21 mg transdermal
nicotine patch for 6 weeks (Week 7-Week 12), followed by the 14 mg
transdermal nicotine patch for 2 weeks (Week 13 and 14), and the 7
mg transdermal nicotine patch for 2 weeks (Week 15 and 16).
[0378] All patches, placebo and NRT, are 24-hour applications, and
should be re-applied daily. Additionally, all subjects will receive
behavioral support through a 10 minute individual counseling
session at visit 1 by a certified smoking cessation counselor and
printed materials (You Can Quit Smoking).
[0379] As described, Quest.RTM. use will be supplemented in the
first treatment arm with the addition of a transdermal nicotine
patch (21 mg) at the beginning of Week 5. The application of the
patch 2 weeks prior to the quit date is intentional and has been
included because this regimen has been associated with improved
durability of long-term abstinence rates. Following the quit date,
subjects will remain on a traditional patch schedule until the end
of Week 14. The current design incorporates 2 weeks of placebo
patch treatment following standard NRT treatment in order to
promote consistency when assigning abstinence rates, maintain
blinding and avoid within-trial study bias.
[0380] Schuurmans and colleagues (Schuurmans et al. 2004) found
that nicotine patch pre-treatment before cessation increased
sustained abstinence rates at 6 months. Overall sustained
abstinence was observed in 17 percent of subjects at 6 months; 22
percent in an experimental group verses 12 percent in a
placebo-controlled group. Historically, one would begin nicotine
replacement therapy on his/her quit day confronting the subject not
only with a sudden behavioral change, but also with a new route of
receiving nicotine. Pre-treatment with NRT may weaken the
association between nicotine intake (rewarding effect) and the
behavior of smoking thereby aiding in a disassociation of the
conditioned behavior pattern. Secondly, pre-patch treatment may
reduce the need for inhaled nicotine. Finally, prior patch
treatment may ease the transition from inhaled nicotine to
transdermal nicotine replacement by familiarization with the mode
of delivery. Similar support for patch pre-treatment has been
suggested by Rose et al. (unpublished) who found that pre-treatment
with a nicotine patch two weeks before the quit date resulted in 50
percent abstinence rates at 6 weeks.
[0381] Patch pre-treatment has been incorporated into the current
study design as a means to further test its potential benefit.
Smoking Quest 3.RTM. cigarettes may hold a particular advantage
when paired with patch pre-treatment since Quest 3.RTM. contains no
nicotine. Thus, smoking nicotine-free cigarettes during
pre-treatment may allow for an even easier transition to the patch
with subjects acclimating to their new nicotine source while still
afforded the behavioral and sensory aspects provided by the
cigarette.
[0382] The control group, Group 3, will comply with current
labeling requirements for OTC patches indicating that treatment
with active NRT will start following the quit date. Furthermore,
careful consideration was made to limit active patch exposure to 10
weeks with no more than 6 weeks of exposure to the highest dosage
form (21 mg) and no more than 2 weeks exposure at lower weaning
dosages of 14 mg and 7 mg respectively.
[0383] The primary endpoint, i.e., 4 weeks of continuous abstinence
(Weeks 7-10), will be assessed after treatment termination, thus at
Week 11 (Visit 7). Abstinence rates will be verified by self
reports and exhaled CO<10 ppm for each subject.
[0384] Quit rates at 3 and 6 months (at Visits 11 and 12) following
treatment termination will be verified by self reports and exhaled
CO<10 ppm.
[0385] Preference and satisfaction of Quest.RTM. over usual brand
will be determined by subjective questionnaires.
[0386] The severity of withdrawal symptoms including negative mood,
urges to smoke and difficulty concentrating will be determined by
subjective questionnaires.
[0387] Compensatory smoking will be determined by the number of
cigarettes smoked and chemically verified by CO levels.
[0388] Three hundred forty-five (345) smokers who have provided
informed consent for participation in this trial, who are capable
of comprehending the nature of the study and who are likely to
comply with the visit schedule are to be entered into the study
provided they conform to the following criteria: Subjects must be
considered by the investigator to be in general good health between
21 and 65 years of age; Subjects must have a history of smoking an
average of 15 cigarettes or more per day for at least one year;
Subjects must be motivated to quit smoking; Subjects must have a
corrected CO measurement of greater than 15 ppm at baseline;
Subjects must be willing and able to return for scheduled follow-up
examinations for a total of 8 months; Subjects must read,
understand, complete and be given a copy of the signed Informed
Consent form; Subjects must be able to read, understand and
complete the questionnaires independently.
[0389] Subjects will be excluded if they meet any of the following
criteria: using any form of NRT or other tobacco-based product
(chew, snuff, etc.); usual brand consists of a menthol variety;
known serious pathophysiology or topical or systemic disorders of
any kind that would confound the results of the study; Subjects who
are using illegal drugs at baseline; pregnant or lactating, or who
plan to become pregnant or lactating during the course of the
study; participating in any other clinical trial of an
investigational drug or device during the time of this clinical
investigation or within 30 days prior to screening visit; taking
anti-depressants, anti-psychotics, Clonidine, Zyban, or any of the
classes of drugs listed in the Accutest 10-Multidrug screen; have a
positive Accutest 10-Multidrug screen; systolic blood pressure over
140 mmHg and/or diastolic blood pressure over 90 mmHg; consume an
average of 3 or more drinks of alcohol per day; consume greater
than an average of 3 packs of cigarettes per day.
[0390] All study products will bear the following: "Caution: New
Drug--Limited by Federal Law to investigational use by qualified
investigators only. Keep out of the reach of children. For clinical
trial use only." Additionally, cigarette packs will communicate the
following Surgeon General's warning:
"SURGEON GENERAL'S WARNING: Quitting Smoking Now Greatly Reduces
Serious Risks to Your Health."
[0391] Quest 1.RTM., Quest 2.RTM., Quest 3.RTM. and the Active
Control will be packaged 20 cigarettes per pack and 10 packs per
carton and labeled in accordance with their study visit period:
[0392] Week 1 & 2
[0393] Week 3 & 4
[0394] Week 5 & 6
[0395] Quest 1.RTM., Quest 2.RTM., Quest 3.RTM. and the Active
Control will be packaged in identical packages, such that there
will be no visible markings, and they will be indistinguishable
from conventional cigarettes. Each carton of cigarettes will have a
label on the box. The label will contain protocol number, study
period, the three-digit subject identification number (preprinted),
and the new drug caution.
[0396] A date-coding stamp will be embossed into the bottom of each
cigarette pack and carton which will distinguish each Quest.RTM.
product from one another and from the Active Control. The
date-coding system will consist of an arbitrary number to avoid
bias and will be traceable to the lot number for each production
run of investigational drug product.
[0397] Labeling of the NRT patch and placebo patch will be similar
to the cigarettes. NRT patches in 21 mg, 14 mg and 7 mg will be
repackaged. Placebo patches will be manufactured to match the size,
appearance and texture of an active NRT.
[0398] NRT patches in 21 mg, 14 mg and 7 mg and placebo patches
will be repackaged in an identical manner and in accordance with
the randomization scheme, as described above for the cigarette
packaging. A total of 20 patches will be packaged in one box. The
placebo patch box will be labeled with a subject identification
number and one of the following time periods: Week 5 & 6, Week
7 & 8, Week 9 & 10, Week 11 & 12, Week 13 & 14, or
Week 15 & 16. The 21 mg NRT patch box will be labeled with a
subject identification number and one of the following time
periods: Week 5 & 6, Week 7 & 8, Week 9 & 10, or Week
11 & 12. The 14 mg NRT patch box will be labeled with a subject
identification number and one of the following time periods: Week
11 & 12, or Week 13 & 14. The 7 mg NRT patch box will be
labeled with a subject identification number and one of the
following time periods: Week 13 & 14, or Week 15 & 16.
[0399] Each study kit will contain the appropriate number of
cigarette cartons and patches for one study subject. The kit will
be labeled with the protocol number, subject identification number
(preprinted), subject initials, and the new drug caution.
[0400] Healthy male and female smokers interested in quitting
smoking will be recruited from the community. The investigative
staff will explain the study purpose, procedures and subject
responsibilities to the potential participant. The subject's
willingness and ability to meet the follow-up requirements will be
determined. When it has been established that the subject is likely
to be eligible for participation, written informed consent will be
obtained. The subject will sign and date the informed consent form,
and the person explaining the consent will also sign and date the
consent form. One copy of the informed consent form will be
retained with the subject records and a signed copy will be
provided to the subject.
[0401] Once the written consent has been provided, the subject will
undergo complete screening procedures, consisting of relevant
medical history, physical examination, urine pregnancy test (in
women of child-bearing potential), drug screen, exhaled CO, saliva
cotinine and the completion of four questionnaires.
[0402] Prior to the study, Subject Identification (Subject ID)
numbers will be randomly assigned a treatment group (1, 2 or 3) in
a 1:1:1 ratio by an independent statistician. Balanced blocks of
equal size will be assigned to each study treatment. The study is
double-blind indicating that the investigator and subject will be
blinded to the treatment. Only the statistician responsible for
developing the randomization key and those in charge of
manufacturing and packaging the Test Articles will view the
randomization scheme. The kits will be pre-packaged, display a
Subject ID number and contain all cigarettes and patches to be
dispensed over the course of the trial. The maximum number of
subjects enrolled at any one site is 100.
[0403] Once subject eligibility has been confirmed, the subject
will be scheduled to return to the site in 7 days. At that time
they will be enrolled in the study, and the next sequential Subject
ID number will be assigned. This number will consist of 5 digits in
the following format: XX-XXX, the first 2 digits represent the
pre-assigned site number and the final three digits will be taken
from the next sequential kit available. For example, the first
patient enrolled at site "02", which had been allocated the next
available kit number 180, would be assigned the Subject ID number
02-180. A subject who is screened, but not entered into the study
(a screen fail) will be recorded on a screen log.
[0404] The Subject ID number and subject initials are to be
recorded on all study documents and will link the study treatment
and the study documents to the subject's name and (medical) record.
To maintain confidentiality, the subject's name or any other
personal identifiers should not be recorded on any study document
other than the informed consent form.
[0405] Subjects will be examined and evaluated according to the
following schedule of visits: Visit 1: Screening/Baseline (day -7
through day 0); Visit 2: Randomization (day 0).+-.2 days post
screening; Visit 3: 2 weeks.+-.3 days post randomization; Visit 4:
4 weeks.+-.3 days post randomization; Visit 5: 6 weeks.+-.3 days
post randomization (quit date); Visit 6: 8 weeks.+-.3 days post
randomization; Visit 7: 10 weeks.+-.3 days post randomization;
Visit 8: 12 weeks.+-.3 days post randomization; Visit 9: 14
weeks.+-.3 days post randomization; Visit 10: 16 weeks.+-.3 days
post randomization; and Visit 11: 18 weeks.+-.7 days post
randomization (3 month quit rate); Visit 12: 31 weeks.+-.7 days
post randomization (6 months quit rate).
[0406] The clinical parameters to be evaluated are: Self report
diaries (visits 1 through 5); saliva cotinine concentration (all
Visits); Exhaled, background, and corrected CO (all visits); blood
pressure and heart rate (all visits); Minnesota smoking withdrawal
questionnaire (all visits); Cigarette evaluation questionnaire
(visits 1 through 5); Sensory questionnaire (visits 1 through 5);
Fagerstrom Test of Nicotine Dependency (visits 1 through 5); and
DNA blood analysis.
[0407] A Case Report Form (CRF) booklet will be provided by Vector
Tobacco or its designee for each subject enrolled in the study. The
appropriate Case Report Form will be completed at each visit. The
investigator will sign each subject's completed CRF once he/she has
reviewed all data contained within. All CRFs will be completed in a
legible manner in black ink. Any corrections will be made by
drawing a single line through the incorrect entry, entering the
correct information and initialing and dating the change.
[0408] All clinical data generated in the study will be submitted
to Vector Tobacco or its designee for quality assurance review,
data entry, and statistical analysis. All forms will be reviewed
for completeness and evident recording errors will be rectified by
contact with the appropriate clinical site. Informed consent must
be obtained prior to any study-specific procedure and use of the
study product. A signed and dated informed consent form will be
obtained from each subject in accordance with ICH GCPs and with
local regulatory and legal requirements. The original, completed
informed consent form must be retained by the investigator as part
of the study records and a signed copy must be given to the
subject. All study visits should take place after 12 pm.
[0409] The following procedures and observations will be performed
at visit 1 (day -7 through day 0): signed written informed consent
will be obtained; screening for inclusion and exclusion criteria
will be completed; drug screen will be performed; demographic
information will be collected; relevant medical history will be
taken; smoking history (including number of cigarettes smoked per
day, and documentation of current usual brand) will be taken; You
Can Quit Smoking booklet will be dispensed; ten minute counseling
session will be provided; concomitant medication history will be
completed (for the first visit, this will need to state all
medications taken within the previous 7 days); physical exam will
be performed; urine pregnancy testing will be performed; blood
pressure and heart rate will be determined; height will be
measured; weight will be measured; diary card and instructions for
completing it will be given (subjects will be smoking their usual
brand of cigarettes during this week); saliva will be analyzed for
cotinine; exhaled CO, background CO, and corrected CO
concentrations will be measured; Smoking Withdrawal Questionnaire
will be completed; Cigarette Evaluation Questionnaire will be
completed (for usual brand); Sensory Questionnaire will be
completed (for usual brand); Fagerstrom Test of Nicotine Dependency
will be completed; and subjects will be scheduled to return to the
site in 7 days (.+-.2 Days) for visit 2.
[0410] The following procedures and observations will be performed
at visit 2 (day 0): blood for DNA analysis will be drawn (if
applicable); randomization assigned; confirmation of
inclusion/exclusion criteria; concomitant medication history will
be completed (for this and all subsequent visits, this will need to
state all medications taken since last visit); blood pressure and
heart rate will be determined; weight will be measured; diary
entries will be reviewed with subject, feedback provided and new
diary cards dispensed; saliva will be analyzed for cotinine;
Exhaled CO, background CO, and corrected CO concentrations will be
measured; first 2 weeks of randomized treatment will be dispensed;
Smoking Withdrawal Questionnaire will be completed; Cigarette
Evaluation Questionnaire will be completed (for usual brand);
Sensory Questionnaire will be completed (for usual brand);
Fagerstrom Test of Nicotine Dependency will be completed; subjects
will be instructed to bring their unused cigarette packs and diary
to the site; serious adverse events will be reviewed and recorded;
and subjects will be scheduled to return to the site in two weeks
(.+-.3 Days) for visit 3.
[0411] The following procedures and observations will be performed
at visit 3 (week 2): concomitant medication history will be
completed (for this and all subsequent visits, this will need to
state all medications taken since last visit); blood pressure and
heart rate will be determined; weight will be measured; diary card
will be supplied to document cigarette usage; saliva will be
analyzed for cotinine; exhaled CO, background CO, and corrected CO
concentrations will be measured; alcohol intake will be assessed;
week 3 & 4 of assigned randomized treatment will be dispensed;
Smoking Withdrawal Questionnaire will be completed; Cigarette
Evaluation Questionnaire will be completed; Sensory Questionnaire
will be completed; Fagerstrom Test of Nicotine Dependency will be
completed; use will be determined by the number of returned
cigarettes; adverse events will be reviewed and recorded; subjects
will be scheduled to return to the site in two weeks (.+-.3 Days)
for visit 4; and subjects will be reminded to bring their unused
cigarette packs and diary to the site.
[0412] The following procedures and observations will be performed
at visit 4 (week 4): concomitant medication history will be
completed (for this and all subsequent visits, this will need to
state all medications taken since last visit); blood pressure and
heart rate will be determined; weight will be measured; diary card
will be supplied to document cigarette usage; saliva will be
analyzed for cotinine; exhaled CO, background CO, and corrected CO
concentrations will be measured; alcohol intake will be assessed;
week 5 & 6 of assigned randomized treatment (cigarettes and
patches) will be dispensed; Smoking Withdrawal Questionnaire will
be completed; Cigarette Evaluation Questionnaire will be completed;
Sensory Questionnaire will be completed; Fagerstrom Test of
Nicotine Dependency will be completed; use will be determined by
the number of returned cigarettes; adverse events will be reviewed
and recorded; subjects will be scheduled to return to the site in
two weeks (.+-.3 Days) for visit 5; and subjects will be reminded
to bring their unused cigarette packs, unused patches and diary to
the site.
[0413] The following procedures and observations will be performed
at visit 5 (week 6--quit date): the quit date, as of this visit,
will be emphasized; complete concomitant medication history (for
this and all subsequent visits, this will need to state all
medications taken since last visit); blood pressure and heart rate
will be determined; weight will be measured; saliva analysis for
cotinine; exhaled CO, background CO, and corrected CO
concentrations will be measured; alcohol intake will be assessed;
week 7 & 8 of assigned randomized treatment (patches only) will
be dispensed; Smoking Withdrawal Questionnaire will be completed;
Cigarette Evaluation Questionnaire will be completed; Sensory
Questionnaire will be completed; Fagerstrom Test of Nicotine
Dependency will be completed; use will be determined by the number
of returned cigarettes; compliance will be determined by the number
of returned patches; review and record adverse events; subjects
will be scheduled to return to the site in two weeks (+3 Days) for
visit 6; and remind the subjects to bring their unused patches to
the site.
[0414] The following procedures and observations will be performed:
complete concomitant medication history (for this and all
subsequent visits, this will need to state all medications taken
since last visit); blood pressure and heart rate will be
determined; weight will be measured; saliva analysis for cotinine;
exhaled CO, background CO, and corrected CO concentrations will be
measured; alcohol intake will be assessed; week 9 & 10 of
assigned randomized treatment (patches only) will be dispensed;
smoking Withdrawal Questionnaire will be completed; compliance will
be determined by the number of returned patches; all subjects will
be asked if they have smoked since the last visit and if they have
smoked within the last 7 days; review and record adverse events;
subjects will be scheduled to return to the site in two weeks
(.+-.3 Days) for visit 7; and remind the subjects to bring their
unused patches to the site.
[0415] The following procedures and observations will be performed
at visit 7 (week 10): concomitant medication history will be
completed (for this and all subsequent visits, this will need to
state all medications taken since last visit); blood pressure and
heart rate will be determined; weight will be measured; saliva will
be analyzed for cotinine; exhaled CO, background CO, and corrected
CO concentrations will be measured; alcohol intake will be
assessed; week 11 & 12 of assigned randomized treatment
(patches only) will be dispensed; smoking Withdrawal Questionnaire
will be completed; compliance will be determined by the number of
returned patches; subjects will be asked if they have smoked since
the last visit and if they have smoked within the last 7 days;
adverse events will be reviewed and recorded; subjects will be
scheduled to return to the site in two weeks (.+-.3 Days) for visit
8; and subjects will be reminded to bring their unused patches to
the site.
[0416] The following procedures and observations will be performed
at visit 8 (week 12): concomitant medication history will be
completed (for this and all subsequent visits, this will need to
state all medications taken since last Visit); blood pressure and
heart rate will be determined; weight will be measured; saliva will
be analyzed for cotinine; exhaled CO, background CO, and corrected
CO concentrations will be measured; alcohol intake will be
assessed; week 13 & 14 of assigned randomized treatment
(patches only) will be dispensed; smoking Withdrawal Questionnaire
will be completed; compliance will be determined by the number of
returned patches; subjects will be asked if they have smoked since
the last visit and if they have smoked within the last 7 days;
adverse events will be reviewed and recorded; subjects will be
scheduled to return to the site in two weeks (.+-.3 Days) for visit
9; and subjects will be reminded to bring their unused patches to
the site.
[0417] The following procedures and observations will be performed
at visit 9: concomitant medication history will be completed (for
this and all subsequent visits, this will need to state all
medications taken since last visit); blood pressure and heart rate
will be determined; weight will be measured; saliva will be
analyzed for cotinine; exhaled CO, background CO, and corrected CO
concentrations will be measured; alcohol intake will be assessed;
week 15 & 16 of assigned randomized treatment (patches only)
will be dispensed; smoking Withdrawal Questionnaire will be
completed; compliance will be determined by the number of returned
patches; subjects will be asked if they have smoked since the last
visit and if they have smoked within the last 7 days; adverse
events will be reviewed and recorded; subjects will be scheduled to
return to the site in two weeks (.+-.3 Days) for visit 10; and
subjects will be reminded to bring their unused patches to the
site.
[0418] The following procedures and observations will be performed
at visit 10 (week 16): concomitant medication history will be
completed (for this and all subsequent visits, this will need to
state all medications taken since last visit); physical exam will
be performed; Urine pregnancy testing will be performed; blood
pressure and heart rate will be determined; weight will be
measured; saliva will be analyzed for cotinine; exhaled CO,
background CO, and corrected CO concentrations will be measured;
alcohol intake will be assessed; Smoking Withdrawal Questionnaire
will be completed; compliance will be determined by the number of
returned patches; subjects will be asked if they have smoked since
the last visit and if they have smoked within the last 7 days;
adverse events will be reviewed and recorded; and subjects will be
scheduled to return to the site in two weeks (.+-.3 Days) for visit
11.
[0419] The following procedures and observations will be performed
at visit 11 (week 18): concomitant medication history will be
completed (for this and all subsequent visits, this will need to
state all medications taken since last visit); blood pressure and
heart rate will be determined; weight will be measured; saliva will
be analyzed for cotinine; exhaled CO, background CO, and corrected
CO concentrations will be measured; alcohol intake will be
assessed; smoking Withdrawal Questionnaire will be completed;
subjects will be asked if they have smoked since the last visit and
if they have smoked within the last 7 days; adverse events will be
reviewed and recorded; and subjects will be notified that they will
receive a follow-up phone call in approximately 13 weeks to
determine their smoking status.
[0420] For visit 12 (week 31), the subject will be questioned
regarding their smoking status by telephone. Subjects who indicate
that they have relapsed within the past 7 days will be questioned
about adverse events and terminated from the study. Subjects who
indicate that they are still abstinent, or have smoked only since
the last visit and not within the past 7 days will be asked to come
to the site within one week of the phone call (.+-.3 days) for the
following procedures and observations: concomitant medication
history will be completed (for this and all subsequent visits, this
will need to state all medications taken since last visit); blood
pressure and heart rate will be determined; weight will be
measured; saliva will be analyzed for cotinine; exhaled CO,
background CO, and corrected CO concentrations will be measured;
alcohol intake will be assessed; Smoking Withdrawal Questionnaire
will be completed; adverse events will be reviewed and recorded;
and subjects will exit the study.
[0421] Subjects are considered to have completed the study if they
have completed all follow-up examinations through visit 12.
[0422] Subjects may be terminated from the study at the discretion
of the investigator only for reasons related to the study treatment
regimen that would jeopardize the subjects' health and/or welfare
if they were to continue in the study. Terminated subjects will be
considered to have completed the study and should not be replaced.
However, every effort will be made to follow terminated subjects
(due to an adverse event) for safety reasons using the appropriate
case report forms until the planned end of the study period.
Notification of a subject termination due to an adverse event will
be made immediately to Vector Tobacco or its designee.
[0423] Subjects may be discontinued from the study for
non-treatment-related reasons only when no other option is
possible. Reasons for discontinuation include, but are not
necessarily limited to, 1) voluntary withdrawal from the study by
the subject; 2) subject has moved from the area and is determined
to be lost to follow-up; 3) subject is unwilling or unable to
cooperate with study requirements (assigned treatment, follow-up
visits, etc.). The reason for discontinuation will be recorded on
the appropriate case report form. Discontinued subjects will not be
replaced in the study.
[0424] Subjects who do not quit smoking after visit 5 quit date or
start smoking again after the visit 5 quit date and prior to visit
10 will be discontinued from the study. All assessments for that
specific visit should be performed including a physical and
pregnancy test, if applicable.
[0425] Prior to discontinuing a subject, every effort should be
made to contact the subject to either encourage the subject to
maintain compliance with the protocol or to obtain as much
follow-up data as possible regarding the subject's current status.
Efforts to contact the subject will consist of documentation of at
least three attempts to contact the subject by phone followed by at
least two certified letters with return receipt. A study completion
form must be completed for all subjects who complete, discontinue,
or are terminated from the study.
EXAMPLE 24
A Nicotine Reduction and/or Smoking Cessation Method Using a
Reduced Nicotine Tobacco Product in Conjunction with a Nicotine
Patch
[0426] The efficacy of a nicotine reduction and/or smoking
cessation program using both a reduced nicotine cigarette and a
nicotine patch has been evaluated. The primary objective of this
prospective randomized controlled clinical trial was a continuous
four-week period of abstinence from smoking. The study consisted of
a group of 15 subjects (one of three treatment arms in the overall
study).
[0427] The study was 18 weeks in duration. During the first week of
the study, subjects smoked only their usual brand of cigarette.
From Weeks 2 through 5, Quest 3.RTM. cigarettes (having less than
0.05 mg nicotine) were introduced while subjects gradually reduced
the amount of usual brand smoked. By Week 6, subjects were expected
to be smoking Quest 3.RTM. cigarettes exclusively. From Weeks 6
through 11, subjects acclimated themselves to nicotine-free
smoking. They were gradually weaned off Quest 3.RTM. with phased
discontinuation of nicotine patches from Week 12 through Week 14 in
anticipation of the four-week abstinence period during Weeks 15
through 18. Secondary endpoints evaluated included compensatory
smoking behavior and withdrawal symptoms.
[0428] Subjects rated Quest.RTM. cigarettes as less satisfying than
their usual brand and, therefore, did not crave them as much as
their regular brand of cigarette. This outcome was perceived as
beneficial since the lack of a craving for Quest.RTM. cigarettes
may aid smoking cessation and also deter sustained use. Even though
Quest.RTM. was less satisfying, subjects did not compensate for the
lack of nicotine by smoking more cigarettes or inhaling more smoke
in each puff. The number of cigarettes smoked and the expired CO
levels did not increase during the six weeks that subjects had free
access to Quest.RTM. cigarettes. The results of this study showed
that Quest.RTM. cigarettes have the potential to be efficacious as
a smoking cessation aid.
EXAMPLE 25
A Nicotine Reduction and/or Smoking Cessation Method Using a
Reduced Nicotine Tobacco Product in Conjunction with a Nicotine
Patch
[0429] In a study by Rose et al. (presented at the Society for
Research in Nicotine and Tobacco 2005 Annual Meeting), fifteen
smokers (6 males, 9 females; mean FTND score 6.9) participated in a
study designed to investigate the neuroanatomical substrates of
nicotine dependence. Dependence was manipulated by having subjects
switch to smoking low nicotine content cigarettes while wearing
nicotine skin patches; this manipulation, which reduces exposure to
inhaled nicotine, has been shown to reduce indices of nicotine
dependence. Participants were assessed using positron emission
tomography (PET) to measure changes in regional cerebral metabolic
rate for glucose (rCMRglc) and regional cerebral blood flow (rCBF).
Subjects were scanned during three sessions conducted after
overnight abstinence from smoking: 1) at baseline; 2) after two
weeks of low nicotine content cigarettes (<0.1 mg nicotine
delivery)+nicotine patches (21 mg/24 h, removed the night before
test sessions); and 3) two weeks after returning to smoking their
usual brand of cigarettes (mean nicotine delivery 0.8 mg). Craving
for cigarettes decreased significantly at the second session (after
2 weeks exposure to low nicotine containing cigarettes+nicotine
patches) relative to the first and last sessions (p=0.03). FTND
score (assessed at each session) also decreased at the second
session (p=0.06). The right hemisphere anterior cingulated cortex
similarly showed a significant decrease in activation (based on
rCMRglc measures) at the second session (p=0.002). These results
confirmed previous findings that exposure to reduced nicotine
content cigarettes plus nicotine patches can lead to a reduction in
nicotine dependence, and offer additional support for the view that
activation of the anterior cingulated cortex is a neural correlate
of drug craving.
EXAMPLE 26
Nicotine Reduction and/or Smoking Cessation Kit Containing Packs of
Cigarettes with Low TSNA Levels and Stepwise Reductions in Nicotine
Levels
[0430] Various nicotine reduction and/or smoking cessation kits are
prepared, geared to heavy, medium, or light smokers. The kits
provide all of the materials needed to quit smoking in either a
two-week period (fast), a one-month period (medium) or in a
two-month period (slow), depending on the kit. Each kit contains a
set number of packs of cigarettes modified according the present
invention, containing either step 1 cigarettes containing -0.6 mg
nicotine, step 2 cigarettes containing 0.3 mg nicotine, and step 3
cigarettes containing less than 0.05 mg nicotine. For example, 1
pack a day smokers would receive 7 packs of cigarettes, each pack
containing the above amounts of nicotine per each cigarette.
Several weeks worth of additional cigarettes containing less than
0.05 mg nicotine/cigarette would also be provided in the kit, to
familiarize the consumer with smoking no nicotine cigarettes. The
kit may also contain a diary for keeping track of daily nicotine
intake, motivational literature to keep the individual interested
in continuing the cessation program, health information on the
benefits of smoking cessation, and web site addresses to find
additional anti-smoking information, such as chat groups, meetings,
newsletters, recent publications, and other pertinent links.
[0431] 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
26 1 1056 DNA Nicotiana tabacum 1 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 gcatga 1056 2 628
DNA Nicotiana tabacum 2 tgatcaagtg aacatcatca aagcaattaa agaagctgga
aatatcaaga gatttcttcc 60 ttcagaattt ggatttgatg tggatcatgc
tcgtgcaatt gaaccagctg catcactctt 120 cgctctaaag gtaagaatca
ggaggatgat agaggcagaa ggaattccat acacatatgt 180 aatctgcaat
tggtttgcag atttcttctt gcccaacttg gggcagttag aggccaaaac 240
ccctcctaga gacaaagttg tcatttttgg cgatggaaat cccaaagcaa tatatgtgaa
300 ggaagaagac atagcgacat acactatcga agcagtagat gatccacgga
cattgaataa 360 gactcttcac atgagaccac ctgccaatat tctatccttc
aacgagatag tgtccttgtg 420 ggaggacaaa attgggaaga ccctcgagaa
gttatatcta tcagaggaag atattctcca 480 gattgtacaa gagggacctc
tgccattaag gactaatttg gccatatgcc attcagtttt 540 tgttaatgga
gattctgcaa actttgaggt tcagcctcct acaggtgtcg aagccactga 600
gctatatcca aaagtgaaat acacaacc 628 3 4670 DNA Artificial Sequence
Artificially created chimeric nucleic acid sequence 3 ctcgaggatc
taaattgtga gttcaatctc ttccctattg gattgattat cctttctttt 60
cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat cctattttgt
120 ttctttactt atttatttgc ttctatgtct ttgtacaaag atttaaactc
tatggcacat 180 attttaaagt tgttagaaaa taaattcttt caagattgat
gaaagaactt tttaattgta 240 gatatttcgt agattttatt ctcttactac
caatataacg cttgaattga cgaaaatttg 300 tgtccaaata tctagcaaaa
aggtatccaa tgaaaatata tcatatgtga tcttcaaatc 360 ttgtgtctta
tgcaagattg atactttgtt caatggaaga gattgtgtgc atatttttaa 420
aatttttatt agtaataaag attctatata gctgttatag agggataatt ttacaaagaa
480 cactataaat atgattgttg ttgttagggt gtcaatggtt cggttcgact
ggttatttta 540 taaaatttgt accataccat ttttttcgat attctatttt
gtataaccaa aattagactt 600 ttcgaaatcg tcccaatcat gtcggtttca
cttcggtatc ggtaccgttc ggttaatttt 660 catttttttt taaatgtcat
taaaattcac tagtaaaaat agaatgcaat aacatacgtt 720 cttttatagg
acttagcaaa agctctctag acatttttac tgtttaaagg ataatgaatt 780
aaaaaacatg aaagatggct agagtataga tacacaacta ttcgacagca acgtaaaaga
840 aaccaagtaa aagcaaagaa aatataaatc acacgagtgg aaagatatta
accaagttgg 900 gattcaagaa taaagtctat attaaatatt caaaaagata
aatttaaata atatgaaagg 960 aaacatattc aatacattgt agtttgctac
tcataatcgc tagaatactt tgtgccttgc 1020 taataaagat acttgaaata
gcttagttta aatataaata gcataataga ttttaggaat 1080 tagtattttg
agtttaatta cttattgact tgtaacagtt tttataattc caaggcccat 1140
gaaaaattta atgctttatt agttttaaac ttactatata aatttttcat atgtaaaatt
1200 taatcggtat agttcgatat tttttcaatt tatttttata aaataaaaaa
cttaccctaa 1260 ttatcggtac agttatagat ttatataaaa atctacggtt
cttcagaaga aacctaaaaa 1320 tcggttcggt gcggacggtt cgatcggttt
agtcgatttt caaatattca ttgacactcc 1380 tagttgttgt tataggtaaa
aagcagttac agagaggtaa aatataactt aaaaaatcag 1440 ttctaaggaa
aaattgactt ttatagtaaa tgactgttat ataaggatgt tgttacagag 1500
aggtatgagt gtagttggta aattatgttc ttgacggtgt atgtcacata ttatttatta
1560 aaactagaaa aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa
atcggctgaa 1620 tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa
agaatcggtg actgttgatg 1680 atataaacaa agggcacatt ggtcaataac
cataaaaaat tatatgacag ctacagttgg 1740 tagcatgtgc tcagctattg
aacaaatcta aagaaggtac atctgtaacc ggaacaccac 1800 ttaaatgact
aaattaccct catcagaaag cagatggagt gctacaaata acacactatt 1860
caacaaccat aaataaaacg tgttcagcta ctaaaacaaa tataaataaa tctatgtttg
1920 taagcactcc agccatgtta atggagtgct attgcctgtt aactctcact
tataaaatag 1980 tagtagaaaa aatatgaacc aaaacacaac ggttgtgtat
ttcacttttg gatatagctc 2040 agtggcttcg acacctgtag gaggctgaac
ctcaaagttt gcagaatctc cattaacaaa 2100 aactgaatgg catatggcca
aattagtcct taatggcaga ggtccctctt gtacaatctg 2160 gagaatatct
tcctctgata gatataactt ctcgagggtc ttcccaattt tgtcctccca 2220
caaggacact atctcgttga aggatagaat attggcaggt ggtctcatgt gaagagtctt
2280 attcaatgtc cgtggatcat ctactgcttc gatagtgtat gtcgctatgt
cttcttcctt 2340 cacatatatt gctttgggat ttccatcgcc aaaaatgaca
actttgtctc taggaggggt 2400 tttggcctct aactgcccca agttgggcaa
gaagaaatct gcaaaccaat tgcagattac 2460 atatgtgtat ggaattcctt
ctgcctctat catcctcctg attcttacct ttagagcgaa 2520 gagtgatgca
gctggttcaa ttgcacgagc atgatccaca tcaaatccaa attctgaagg 2580
aagaaatctc ttgatatttc cagcttcttt aattgctttg atgatgttca cttgatcagt
2640 ccgtcgcttc tcttccattt cttctcattt tcgattttga ttcttatttc
tttccagtag 2700 ctcctgctct gtgaatttct ccgctcacga tagatctgct
tatactcctt acattcaacc 2760 ttagatctgg tctcgattct ctgtttctct
gtttttttct tttggtcgag aatctgatgt 2820 ttgtttatgt tctgtcacca
ttaataataa tgaactctct cattcataca atgattagtt 2880 tctctcgtct
acaaaacgat atgttgcatt ttcacttttc ttcttttttt ctaagatgat 2940
ttgctttgac caatttgttt agatctttat tttattttat tttctggtgg gttggtggaa
3000 attgaaaaaa aaaaaaacag cataaattgt tatttgttaa tgtattcatt
ttttggctat 3060 ttgttctggg taaaaatctg cttctactat tgaatctttc
ctggattttt tactcctatt 3120 gggtttttat agtaaaaata cataataaaa
ggaaaacaaa agttttatag attctcttaa 3180 accccttacg ataaaagttg
gaatcaaaat aattcaggat cagatgctct ttgattgatt 3240 cagatgcgat
tacagttgca tggcaaattt tctagatccg tcgtcacatt ttattttctg 3300
tttaaatatc taaatctgat atatgatgtc gacaaattct ggtggcttat acatcacttc
3360 aactgttttc ttttggcttt gtttgtcaac ttggttttca atacgatttg
tgatttcgat 3420 cgctgaattt ttaatacaag caaactgatg ttaaccacaa
gcaagagatg tgacctgcct 3480 tattaacatc gtattactta ctactagtcg
tattctcaac gcaatcgttt ttgtatttct 3540 cacattatgc cgcttctcta
ctctttattc cttttggtcc acgcattttc tatttgtggc 3600 aatccctttc
acaacctgat ttcccacttt ggatcatttg tctgaagact ctcttgaatc 3660
gttaccactt gtttcttgtg catgctctgt tttttagaat taatgataaa actattccat
3720 agtcttgagt tttcagcttg ttgattcttt tgcttttggt tttctgcagt
gatcaagtga 3780 acatcatcaa agcaattaaa gaagctggaa atatcaagag
atttcttcct tcagaatttg 3840 gatttgatgt ggatcatgct cgtgcaattg
aaccagctgc atcactcttc gctctaaagg 3900 taagaatcag gaggatgata
gaggcagaag gaattccata cacatatgta atctgcaatt 3960 ggtttgcaga
tttcttcttg cccaacttgg ggcagttaga ggccaaaacc cctcctagag 4020
acaaagttgt catttttggc gatggaaatc ccaaagcaat atatgtgaag gaagaagaca
4080 tagcgacata cactatcgaa gcagtagatg atccacggac attgaataag
actcttcaca 4140 tgagaccacc tgccaatatt ctatccttca acgagatagt
gtccttgtgg gaggacaaaa 4200 ttgggaagac cctcgagaag ttatatctat
cagaggaaga tattctccag attgtacaag 4260 agggacctct gccattaagg
actaatttgg ccatatgcca ttcagttttt gttaatggag 4320 attctgcaaa
ctttgaggtt cagcctccta caggtgtcga agccactgag ctatatccaa 4380
aagtgaaata cacaaccgca agtgtgttgc ctttgtgtgg aaatgaagag gtacttgcga
4440 ggactttgcg tttatcagtt tatgtgtttg tatatctatt tgatccagtt
attatggatt 4500 atatacgctt gaaactcatt ttaagccatt gttattgaac
gtttatcaaa tactttatta 4560 tgccaagcaa gtcaaacaca tgcttgttga
ttgaaatcaa gctatagaaa tctcttcttc 4620 acatacagca gtttagattc
acaatacaac aagcgaaacg ataaagtttc 4670 4 241 DNA Nicotiana tabacum 4
cattttacca tctttcgcca gaagtatgat cgagtcttaa tcaagtgaat aatgaacact
60 ggtagtacaa tcattggacc aagatcgagt cttaatcaag tgaataaata
agtgaaatgc 120 gacgtattgt aggagaattc tgcagtaatt atcataattt
ccaattcaca atcattgtaa 180 aattctttct ctgtggtgtt tcgtacttta
atataaattt tcctgctgaa gttttgaatc 240 g 241 5 3896 DNA Artificial
Sequence Artificially created chimeric nucleic acid sequence 5
ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat cctttctttt
60 cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat
cctattttgt 120 ttctttactt atttatttgc ttctatgtct ttgtacaaag
atttaaactc tatggcacat 180 attttaaagt tgttagaaaa taaattcttt
caagattgat gaaagaactt tttaattgta 240 gatatttcgt agattttatt
ctcttactac caatataacg cttgaattga cgaaaatttg 300 tgtccaaata
tctagcaaaa aggtatccaa tgaaaatata tcatatgtga tcttcaaatc 360
ttgtgtctta tgcaagattg atactttgtt caatggaaga gattgtgtgc atatttttaa
420 aatttttatt agtaataaag attctatata gctgttatag agggataatt
ttacaaagaa 480 cactataaat atgattgttg ttgttagggt gtcaatggtt
cggttcgact ggttatttta 540 taaaatttgt accataccat ttttttcgat
attctatttt gtataaccaa aattagactt 600 ttcgaaatcg tcccaatcat
gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660 catttttttt
taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt 720
cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg ataatgaatt
780 aaaaaacatg aaagatggct agagtataga tacacaacta ttcgacagca
acgtaaaaga 840 aaccaagtaa aagcaaagaa aatataaatc acacgagtgg
aaagatatta accaagttgg 900 gattcaagaa taaagtctat attaaatatt
caaaaagata aatttaaata atatgaaagg 960 aaacatattc aatacattgt
agtttgctac tcataatcgc tagaatactt tgtgccttgc 1020 taataaagat
acttgaaata gcttagttta aatataaata gcataataga ttttaggaat 1080
tagtattttg agtttaatta cttattgact tgtaacagtt tttataattc caaggcccat
1140 gaaaaattta atgctttatt agttttaaac ttactatata aatttttcat
atgtaaaatt 1200 taatcggtat agttcgatat tttttcaatt tatttttata
aaataaaaaa cttaccctaa 1260 ttatcggtac agttatagat ttatataaaa
atctacggtt cttcagaaga aacctaaaaa 1320 tcggttcggt gcggacggtt
cgatcggttt agtcgatttt caaatattca ttgacactcc 1380 tagttgttgt
tataggtaaa aagcagttac agagaggtaa aatataactt aaaaaatcag 1440
ttctaaggaa aaattgactt ttatagtaaa tgactgttat ataaggatgt tgttacagag
1500 aggtatgagt gtagttggta aattatgttc ttgacggtgt atgtcacata
ttatttatta 1560 aaactagaaa aaacagcgtc aaaactagca aaaatccaac
ggacaaaaaa atcggctgaa 1620 tttgatttgg ttccaacatt taaaaaagtt
tcagtgagaa agaatcggtg actgttgatg 1680 atataaacaa agggcacatt
ggtcaataac cataaaaaat tatatgacag ctacagttgg 1740 tagcatgtgc
tcagctattg aacaaatcta aagaaggtac atctgtaacc ggaacaccac 1800
ttaaatgact aaattaccct catcagaaag cagatggagt gctacaaata acacactatt
1860 caacaaccat aaataaaacg tgttcagcta ctaaaacaaa tataaataaa
tctatgtttg 1920 taagcactcc agccatgtta atggagtgct attgcctgtt
aactctcact tataaaatag 1980 tagtagaaaa aatatgaacc aaaacacaac
cgattcaaaa cttcagcagg aaaatttata 2040 ttaaagtacg aaacaccaca
gagaaagaat tttacaatga ttgtgaattg gaaattatga 2100 taattactgc
agaattctcc tacaatacgt cgcatttcac ttatttattc acttgattaa 2160
gactcgatct tggtccaatg attgtactac cagtgttcat tattcacttg attaagactc
2220 gatcatactt ctggcgaaag atggtaaaat ggtccgtcgc ttctcttcca
tttcttctca 2280 ttttcgattt tgattcttat ttctttccag tagctcctgc
tctgtgaatt tctccgctca 2340 cgatagatct gcttatactc cttacattca
accttagatc tggtctcgat tctctgtttc 2400 tctgtttttt tcttttggtc
gagaatctga tgtttgttta tgttctgtca ccattaataa 2460 taatgaactc
tctcattcat acaatgatta gtttctctcg tctacaaaac gatatgttgc 2520
attttcactt ttcttctttt tttctaagat gatttgcttt gaccaatttg tttagatctt
2580 tattttattt tattttctgg tgggttggtg gaaattgaaa aaaaaaaaaa
cagcataaat 2640 tgttatttgt taatgtattc attttttggc tatttgttct
gggtaaaaat ctgcttctac 2700 tattgaatct ttcctggatt ttttactcct
attgggtttt tatagtaaaa atacataata 2760 aaaggaaaac aaaagtttta
tagattctct taaacccctt acgataaaag ttggaatcaa 2820 aataattcag
gatcagatgc tctttgattg attcagatgc gattacagtt gcatggcaaa 2880
ttttctagat ccgtcgtcac attttatttt ctgtttaaat atctaaatct gatatatgat
2940 gtcgacaaat tctggtggct tatacatcac ttcaactgtt ttcttttggc
tttgtttgtc 3000 aacttggttt tcaatacgat ttgtgatttc gatcgctgaa
tttttaatac aagcaaactg 3060 atgttaacca caagcaagag atgtgacctg
ccttattaac atcgtattac ttactactag 3120 tcgtattctc aacgcaatcg
tttttgtatt tctcacatta tgccgcttct ctactcttta 3180 ttccttttgg
tccacgcatt ttctatttgt ggcaatccct ttcacaacct gatttcccac 3240
tttggatcat ttgtctgaag actctcttga atcgttacca cttgtttctt gtgcatgctc
3300 tgttttttag aattaatgat aaaactattc catagtcttg agttttcagc
ttgttgattc 3360 ttttgctttt ggttttctgc agcattttac catctttcgc
cagaagtatg atcgagtctt 3420 aatcaagtga ataatgaaca ctggtagtac
aatcattgga ccaagatcga gtcttaatca 3480 agtgaataaa taagtgaaat
gcgacgtatt gtaggagaat tctgcagtaa ttatcataat 3540 ttccaattca
caatcattgt aaaattcttt ctctgtggtg tttcgtactt taatataaat 3600
tttcctgctg aagttttgaa tcggcaagtg tgttgccttt gtgtggaaat gaagaggtac
3660 ttgcgaggac tttgcgttta tcagtttatg tgtttgtata tctatttgat
ccagttatta 3720 tggattatat acgcttgaaa ctcattttaa gccattgtta
ttgaacgttt atcaaatact 3780 ttattatgcc aagcaagtca aacacatgct
tgttgattga aatcaagcta tagaaatctc 3840 ttcttcacat acagcagttt
agattcacaa tacaacaagc gaaacgataa agtttc 3896 6 360 DNA Nicotiana
tabacum 6 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 7 1701 DNA Arabidopsis thaliana 7 atggttgtgt ttgggaatgt
ttctgcggcg aatttgcctt atcaaaacgg gtttttggag 60 gcactttcat
ctggaggttg tgaactaatg ggacatagct ttagggttcc cacttctcaa 120
gcgcttaaga caagaacaag gaggaggagt actgctggtc ctttgcaggt agtttgtgtg
180 gatattccaa ggccagagct agagaacact gtcaatttct tggaagctgc
tagtttatct 240 gcatccttcc gtagtgctcc tcgtcctgct aagcctttga
aagttgtaat tgctggtgct 300 ggattggctg gattgtcaac tgcaaagtac
ctggctgatg caggccacaa acctctgttg 360 cttgaagcaa gagatgttct
tggtggaaag atagctgcat ggaaggatga agatggggac 420 tggtatgaga
ctggtttaca tattttcttc ggtgcttatc cgaatgtgca gaatttattt 480
ggagaacttg ggatcaatga tcggttgcag tggaaggaac actccatgat ttttgctatg
540 ccaagtaaac ctggagaatt tagtagattt gacttcccag atgtcctacc
agcaccctta 600 aatggtattt gggctatttt gcggaacaac gagatgctga
catggccaga gaaaataaag 660 tttgctattg gacttttgcc agccatggtc
ggcggtcagg cttatgttga ggcccaagat 720 ggtttatcag tcaaagaatg
gatggaaaag cagggagtac ctgagcgcgt gaccgacgag 780 gtgtttattg
ccatgtcaaa ggcgctaaac tttataaacc ctgatgaact gtcaatgcaa 840
tgcattttga tagctttgaa ccggtttctt caggaaaaac atggttccaa gatggcattc
900 ttggatggta atcctccgga aaggctttgt atgccagtag tggatcatat
tcgatcacta 960 ggtggggaag tgcaacttaa ttctaggata aagaaaattg
agctcaatga cgatggcacg 1020 gttaagagtt tcttactcac taatggaagc
actgtcgaag gagacgctta tgtgtttgcc 1080 gctccagtcg atatcctgaa
gctcctttta ccagatccct ggaaagaaat accgtacttc 1140 aagaaattgg
ataaattagt tggagtacca gttattaatg ttcatatatg gtttgatcga 1200
aaactgaaga acacatatga tcacctactc tttagcagaa gtaaccttct gagcgtgtat
1260 gccgacatgt ccttaacttg taaggaatat tacgatccta accggtcaat
gctggagcta 1320 gtatttgcac cagcagagga atggatatca cggactgatt
ctgacatcat agatgcaaca 1380 atgaaagaac ttgagaaact cttccctgat
gaaatctcag ctgaccaaag caaagctaaa 1440 attctgaagt accatgtcgt
taagactcca agatctgggt acaagaccat cccaaactgt 1500 gaaccatgtc
gtcctctaca aagatcacct attgaaggat tctacttagc tggagattac 1560
acaaaacaga agtacttagc ttccatggaa ggcgctgtcc tctctggcaa attctgctct
1620 cagtctattg ttcaggatta cgagctactg gctgcgtctg gaccaagaaa
gttgtcggag 1680 gcaacagtat catcatcatg a 1701 8 1701 DNA Arabidopsis
thaliana 8 atggttgtgt ttgggaatgt ttctgcggcg aatttgcctt atcaaaacgg
gtttttggag 60 gcactttcat ctggaggttg tgaactaatg ggacatagct
ttagggttcc cacttctcaa 120 gcgcttaaga caagaacaag gaggaggagt
actgctggtc ctttgcaggt agtttgtgtg 180 gatattccaa ggccagagct
agagaacact gtcaatttct tggaagctgc tagtttatct 240 gcatccttcc
gtagtgctcc tcgtcctgct aagcctttga aagttgtaat tgctggtgct 300
ggattggctg gattgtcaac tgcaaagtac ctggctgatg caggccacaa acctctgttg
360 cttgaagcaa gagatgttct tggtggaaag atagctgcat ggaaggatga
agatggggac 420 tggtatgaga ctggtttaca tattttcttc ggtgcttatc
cgaatgtgca gaatttattt 480 ggagaacttg ggatcaatga tcggttgcag
tggaaggaac actccatgat ttttgctatg 540 ccaagtaaac ctggagaatt
tagtagattt gacttcccag atgtcctacc agcaccctta 600 aatggtattt
gggctatttt gcggaacaac gagatgctga catggccaga gaaaataaag 660
tttgctattg gacttttgcc agccatggtc ggcggtcagg cttatgttga ggcccaagat
720 ggtttatcag tcaaagaatg gatggaaaag cagggagtac ctgagcgcgt
gaccgacgag 780 gtgtttattg ccatgtcaaa ggcgctaaac tttataaacc
ctgatgaact gtcaatgcaa 840 tgcattttga tagctttgaa cccgtttctt
caggaaaaac atggttccaa gatggcattc 900 ttggatggta atcctccgga
aaggctttgt atgccagtag tggatcatat tcgatcacta 960 ggtggggaag
tgcaacttaa ttctaggata aagaaaattg agctcaatga cgatggcacg 1020
gttaagagtt tcttactcac taatggaagc actgtcgaag gagacgctta tgtgtttgcc
1080 gctccagtcg atatcctgaa gctcctttta ccagatccct ggaaagaaat
accgtacttc 1140 aagaaattgg ataaattagt tggagtacca gttattaatg
ttcatatatg gtttgatcga 1200 aaactgaaga acacatatga tcacctactc
tttagcagaa gtaaccttct gagcgtgtat 1260 gccgacatgt ccttaacttg
taaggaatat tacgatccta accggtcaat gctggagcta 1320 gtatttgcac
cagcagagga atggatatca cggactgatt ctgacatcat agatgcaaca 1380
atgaaagaac ttgagaaact cttccctgat gaaatctcag ctgaccaaag caaagctaaa
1440 attctgaagt accatgtcgt taagactcca agatctgtgt acaagaccat
cccaaactgt 1500 gaaccatgtc gtcctctaca aagatcacct attgaaggat
tctacttagc tggagattac 1560 acaaaacaga agtacttagc ttccatggaa
ggcgctgtcc tctctggcaa attctgctct 1620 cagtctattg ttcaggatta
cgagctactg gctgcgtctg gaccaagaaa gttgtcggag 1680 gcaacagtat
catcatcatg a 1701 9 1701 DNA Arabidopsis thaliana 9 atggttgtgt
ttgggaatgt ttctgcggcg aatttgcctt atcaaaacgg gtttttggag 60
gcactttcat ctggaggttg tgaactaatg ggacatagct ttagggttcc cacttctcaa
120 gcgcttaaga caagaacaag gaggaggagt actgctggtc ctttgcaggt
agtttgtgtg 180 gatattccaa ggccagagct agagaacact gtcaatttct
tggaagctgc tagtttatct 240
gcatccttcc gtagtgctcc tcgtcctgct aagcctttga aagttgtaat tgctggtgct
300 ggattggctg gattgtcaac tgcaaagtac ctggctgatg caggccacaa
acctctgttg 360 cttgaagcaa gagatgttct tggtggaaag atagctgcat
ggaaggatga agatggggac 420 tggtatgaga ctggtttaca tattttcttc
ggtgcttatc cgaatgtgca gaatttattt 480 ggagaacttg ggatcaatga
tcggttgcag tggaaggaac actccatgat ttttgctatg 540 ccaagtaaac
ctggagaatt tagtagattt gacttcccag atgtcctacc agcaccctta 600
aatggtattt gggctatttt gcggaacaac gagatgctga catggccaga gaaaataaag
660 tttgctattg gacttttgcc agccatggtc ggcggtcagg cttatgttga
ggcccaagat 720 ggtttatcag tcaaagaatg gatggaaaag cagggagtac
ctgagcgcgt gaccgacgag 780 gtgtttattg ccatgtcaaa ggcgctaaac
tttataaacc ctgatgaact gtcaatgcaa 840 tgcattttga tagctttgaa
ccggtttctt caggaaaaac atggttccaa gatggcattc 900 ttggatggta
atcctccgga aaggctttgt atgccagtag tggatcatat tcgatcacta 960
ggtggggaag tgcaacttaa ttctaggata aagaaaattg agctcaatga cgatggcacg
1020 gttaagagtt tcttactcac taatggaagc actgtcgaag gagacgctta
tgtgtttgcc 1080 gctccagtcg atatcctgaa gctcctttta ccagatccct
ggaaagaaat accgtacttc 1140 aagaaattgg ataaattagt tggagtacca
gttattaatg ttcatatatg gtttgatcga 1200 aaactgaaga acacatatga
tcacccactc tttagcagaa gtaaccttct gagcgtgtat 1260 gccgacatgt
ccttaacttg taaggaatat tacgatccta accggtcaat gctggagcta 1320
gtatttgcac cagcagagga atggatatca cggactgatt ctgacatcat agatgcaaca
1380 atgaaagaac ttgagaaact cttccctgat gaaatctcag ctgaccaaag
caaagctaaa 1440 attctgaagt accatgtcgt taagactcca agatctgtgt
acaagaccat cccaaactgt 1500 gaaccatgtc gtcctctaca aagatcacct
attgaaggat tctacttagc tggagattac 1560 acaaaacaga agtacttagc
ttccatggaa ggcgctgtcc tctctggcaa attctgctct 1620 cagtctattg
ttcaggatta cgagctactg gctgcgtctg gaccaagaaa gttgtcggag 1680
gcaacagtat catcatcatg a 1701 10 1061 DNA Nicotiana tabacum 10
aatatgaaag gaaacatatt caatacattg tagtttgcta ctcataatcg ctagaatact
60 ttgtgccttg ctaataaaga tacttgaaat agcttagttt aaatataaat
agcataatag 120 attttaggaa ttagtatttt gagtttaatt acttattgac
ttgtaacagt ttttataatt 180 ccaaggccca tgaaaaattt aatgctttat
tagttttaaa cttactatat aaatttttca 240 tatgtaaaat ttaatcggta
tagttcgata ttttttcaat ttatttttat aaaataaaaa 300 acttacccta
attatcggta cagttataga tttatataaa aatctacggt tcttcagaag 360
aaacctaaaa atcggttcgg tgcggacggt tcgatcggtt tagtcgattt tcaaatattc
420 attgacactc ctagttgttg ttataggtaa aaagcagtta cagagaggta
aaatataact 480 taaaaaatca gttctaagga aaaattgact tttatagtaa
atgactgtta tataaggatg 540 ttgttacaga gaggtatgag tgtagttggt
aaattatgtt cttgacggtg tatgtcacat 600 attatttatt aaaactagaa
aaaacagcgt caaaactagc aaaaatccaa cggacaaaaa 660 aatcggctga
atttgatttg gttccaacat ttaaaaaagt ttcagtgaga aagaatcggt 720
gactgttgat gatataaaca aagggcacat tggtcaataa ccataaaaaa ttatatgaca
780 gctacagttg gtagcatgtg ctcagctatt gaacaaatct aaagaaggta
catctgtaac 840 cggaacacca cttaaatgac taaattaccc tcatcagaaa
gcagatggag tgctacaaat 900 aacacactat tcaacaacca taaataaaac
gtgttcagct actaaaacaa atataaataa 960 atctatgttt gtaagcactc
cagccatgtt aatggagtgc tattgcctgt taactctcac 1020 ttataaaata
gtagtagaaa aaatatgaac caaaacacaa c 1061 11 711 DNA Nicotiana
tabacum 11 gaattcaatg gagaaggaaa atatttccag tgtaaacaca agtgaatgaa
gagaagccaa 60 aataatctct atcattcaag ccttaggtgg agattaaaaa
aattatttac tttcttatca 120 aagtaatagg tgatcaacag ctttcgtaaa
acgtcattag gagaatatta taatctcttt 180 tatgctgaag aacccacata
aggaagatca taaaatacat gactttcaga tgacttcttg 240 gagctttatt
tttaaagagt ggctagctgg tcagcaaaga ggtgctcgtc agatatcata 300
aaattttact attatttgtt ttaagaggga gatggggcac acatgcttgt gacaaaagta
360 agaggaagaa aggagacaga agaggaaata gatttggggg gggggggggg
ggtttcacaa 420 tcaaagaaaa tttttaaaat ggagagagaa atgagcacac
acatatacta acaaaatttt 480 actaataatt gcaccgagac aaacttatat
tttagttcca aaatgtcagt ctaaccctgc 540 acgttgtaat gaatttttaa
ctattatatt atatcgagtt gcgccctcca ctcctcggtg 600 tccaaattgt
atttaaatgc atagatgttt attgggagtg tacagcaagc tttcggaaaa 660
tacaaaccat aatactttct cttcttcaat ttgtttagtt taattttgaa a 711 12
1278 DNA Nicotiana tabacum 12 tgcgtcaaat ggataaacaa aaaaatagca
taagttagtt ttgttactcg agagttatgt 60 attataaggt atagggaaat
gactcaaaca taccactgaa cttaacgaaa cgacgcatat 120 atatactact
taacttaacg aaaaaggggt gagagtggat gggtgctggt aaataatgaa 180
gggtttatat aacgtcacgt gtcaaaattc gatagtagta gtttcgttag ttgtaatagc
240 atatatggcc caaagttata atatagataa tatgtttatg tccaactatt
aacgagtgac 300 atagacagtt cattttgtga agttcaatga catatttgag
ccctttccct tttattatct 360 ccttttattt gttctaataa aagaatggca
tttattatgt acatagacaa ataactattt 420 tctttggaat ataatttgtt
tatatatttt aaaatcatgt ctcaatttag tttgttttgt 480 gcatatttca
actattcaat tttgtccata tatttattac cttcccccat ttacaagcat 540
tgaaccgctt tgctcaccaa aacttatgca cattgcaaaa atatcatgta aaggttttat
600 gtatgctgta attaaggtct gaactcatcg tgattttatt tttaggcttc
attgaccact 660 accaaactct ttgatgctac attttctaat tatattggag
ttcgattata tccgaattcg 720 cgttgcgtag ggcccattcg agggaaaaca
ctccctatca aggatttttt catacccaga 780 gctcgaactc aagacatctg
gttaagggaa gaacagtctc atccactgca ccatatcctt 840 ttgtggtcaa
caagtaaatt ttatgtagaa ccaaaaacta tactcgaatt gataaaataa 900
atggtgtaaa atattgtttt ctttcttaca ttttggacag taaatatgta ggacaataat
960 aattagcgtg gggtcttaag aaaattagca tagatttcca gaaattccaa
atcaaccggc 1020 agttccaggt ttgaaaacta caactcattc cgacggttca
aacttcaaac catgcttgct 1080 gactcggctt cttctttctt tttcaccaag
acagagcagt agtcacgtga cacccctcac 1140 gtgcctcccc cctttatatt
tcagactgca accctacact ttcgctacat tcactaccat 1200 attcttttca
ctaagcaatt ttctctccta cttttcttta aacccctttt ttctccccta 1260
agccatggca tctagatc 1278 13 1079 DNA Nicotiana tabacum 13
atcttattgt ataaatatcc ataaacacat catgaaagac actttctttc acggtctgaa
60 ttaattatga tacaattcta atagaaaacg aattaaatta cgttgaattg
tatgaaatct 120 aattgaacaa gccaaccacg acgacgacta acgttgcctg
gattgactcg gtttaagtta 180 accactaaaa aaacggagct gtcatgtaac
acgcggatcg agcaggtcac agtcatgaag 240 ccatcaaagc aaaagaacta
atccaagggc tgagatgatt aattagttta aaaattagtt 300 aacacgaggg
aaaaggctgt ctgacagcca ggtcacgtta tctttacctg tggtcgaaat 360
gattcgtgtc tgtcgatttt aattattttt ttgaaaggcc gaaaataaag ttgtaagaga
420 taaacccgcc tatataaatt catatatttt ctctccgctt tgaattgtct
cgttgtcctc 480 ctcactttca tcggccgttt ttgaatctcc ggcgacttga
cagagaagaa caaggaagaa 540 gactaagaga gaaagtaaga gataatccag
gagattcatt ctccgttttg aatcttcctc 600 aatctcatct tcttccgctc
tttctttcca aggtaatagg aactttctgg atctacttta 660 tttgctggat
ctcgatcttg ttttctcaat ttccttgaga tctggaattc gtttaatttg 720
gatctgtgaa cctccactaa atcttttggt tttactagaa tcgatctaag ttgaccgatc
780 agttagctcg attatagcta ccagaatttg gcttgacctt gatggagaga
tccatgttca 840 tgttacctgg gaaatgattt gtatatgtga attgaaatct
gaactgttga agttagattg 900 aatctgaaca ctgtcaatgt tagattgaat
ctgaacactg tttaagttag atgaagtttg 960 tgtatagatt cttcgaaact
ttaggatttg tagtgtcgta cgttgaacag aaagctattt 1020 ctgattcaat
cagggtttat ttgactgtat tgaactcttt ttgtgtgttt gcagctcat 1079 14 1943
DNA Nicotiana tabacum 14 aagcttttta tttagctttt tcctccctat
ttcaatatat aatggctcaa tttttgtcag 60 atagcaataa aaccatacaa
gaaaataaaa caaatcacaa aatacaaaaa gaggttatat 120 ctccatgtat
gcaatttcat tatatgcata taagcatctt acgtataaaa aaaaagaggg 180
aatcatggac gtgtctttct aatccaagta gggtcaactt tatagggtcg gtgtatgtgt
240 agtttaatcg aaaaagaatt ccatcattag gtaatttaca attagatcct
taaattatac 300 aaatatataa gggtataaaa gttgatcaat atttcaggga
tattttagtc gttcaacatt 360 tagtataaat tattcgtact tttataataa
taaatagata gataaacata gatatagata 420 taaatataga tagataaatg
ggggatttgc atctataccc actttttggg tcacgtttta 480 atttgtgccc
gctttgcaaa aaaaattgca agcgtacaca ctttttcgcg taacttcagc 540
atacggggct aaagtagcaa agacagtcac gcaaaacttc agcatacttc agtctttgct
600 acttcagccc cgtatgctga agttatgcga aaagcgggta tgcttgtaat
ttttttgcaa 660 agcgagcata agttaaaacg tgacacaaaa agcgggtata
gatacaaatg gccctttttt 720 ttctagccaa attttattca tttttttgga
atactttttc actttatttt aaaattagtg 780 tttggttata aatttttaaa
tacaacttgg agttggactc caaagtcttt acatacttat 840 ttttagtttt
attaccctat tttttttaac atgagatatt tacttttaca gatctaaaaa 900
tgatattttc ttagttttaa cactataaat agccatgaag gcccatttcc tccctttgca
960 aaaagtatac ccaaacgcaa ctccgtcttc acctccaact ccaacttcat
aatttcaatt 1020 aaagtgaaaa ttattttaag agaccatttg gacatgataa
ttttttcact ttttccgaac 1080 ttttttttac tttttttcaa atcagtgttt
ggccataaaa ttttcatttt tcacttgaag 1140 ttgaattttt gaatttttcg
agaattcgaa aaaccccaga aagctgtttt tcaaaatttt 1200 cactcggatc
ctcacaaaac ttccaaaata acccaaaatt atattcatgt ccaacacaac 1260
tctaattttc aaataccatt ttcacttgaa aaagaaattc accttttttt tttttttgaa
1320 ctttacaatt cttatgtcca aacgccccct tcgaatctac ggccaacgtt
tattaagtaa 1380 ggaaagaaaa atggctataa taattatatc ccttttgaag
taaatataat tctaccaaat 1440 taattaatat gcttaaaaac aataaaaata
atcaaaattg ctagagagga caaccaatta 1500 gccgaagcat tgtcaagatt
gagcagggcg cagaatgaag aaagtagttt tttatctttt 1560 gatgccctac
gccttttgta ttaaaatact atatacaaga tttgaaaaag acgagttcca 1620
ttcaaaacag ttcccttgtc ccgaaatgtt cattgatgaa gtaatatgca cttttaaaat
1680 tatttttttc cagtttatcc taaaaaaaat attattttta taatcacata
gaaataatat 1740 atatcaaata acaaagggaa aaagaaagta gggaaagaaa
ataataattg aagtgggctg 1800 ggctttgaca tggaaaggaa tggcttagta
ataattgaag ttagcatcgg atctatttga 1860 agtgccactc atccctcaga
aaaacagtgt tagtattttc tctcacaaat tgattctgtg 1920 gtccgaattg
gagttcctaa atc 1943 15 690 DNA Arabidopsis thaliana 15 ttcgttgaaa
aatcatcgaa attttcgacg gattccaatg atcaaaaatt cgtcaataat 60
ttccaacgat attctgacta aactaaatct gatgaaatat ttttgacggc tttccaacca
120 aaatatttcg ttgtgacttg tcaaaaatcc gttagaatac taagcaactt
ttcgacagat 180 tttcagcaaa aatattcggt aatataacgt gttaaaaata
tgataaaaaa aaaaacttga 240 tgaatctact aaaactaaat tttcaatcat
atatatctat tattcatata tttcattcat 300 tttattattt ttctcttaac
aattatttag ttattctggt atcgtgtaat tatattcata 360 tgatttattc
tgatattgat tcggttagca tccggataaa tctgggttgg gctttttaac 420
ttggtttttc taagaaaaat tctaatatga tttggttagc atccggatta gtctagtttg
480 gtaggcctgc ctttgtgatt cttaactcgg tcttttgtat gggtttgaac
aattactaca 540 ccatttagat tcttctgacc catatcaaat aaagatccac
ttaggcccat tagggttaga 600 acaaacatga ggttgcagaa taaaaagggt
tcattttcct cactctcaag ttggatctca 660 aaaccctaat atctgaactt
cgccgtcgag 690 16 995 DNA Arabidopsis thaliana 16 ttctgttcgt
atatttgtaa ctattatgtg tatttttatt ttgttagtat tactaattca 60
agtggtttaa gttgttgaga ctctttaaaa tctaagcatt ttataaacaa taatatataa
120 ttattgttta ggctaaattt gtcactaatt aaggtttgga tacatagtgt
ctaaactaag 180 ctaataatat cacttaacgt ttacttgtaa cgctaggtga
tgatgtcgtc aagtcaattg 240 gtacaaggaa taaacgagtg gtcatatgac
attatgacca tatgaattca aactccagta 300 atccaatggt aattggattc
aatgatcaag acttgaacca cgtaatccac ccttatcctt 360 agaagctcat
aaatatcact aaagggacag gcaacactta accagtagtt gtccaataat 420
ttagttttcc aaaatgaaaa attattgttg tcatctattt taggtgtttt agttcaatgt
480 ggattcctcg tcctaacaaa tacttgacga atatatctag actataaaat
tggttatgag 540 ttctactttt ttttgtttgt gaaattatca aaatttgtta
tatttattta tttattctca 600 ttaatttgag tactaatttt taaattattt
atactaaaaa caattactaa gatacaaaaa 660 tggataagag catggtgtat
agatatttaa tgggatagaa tatttcccat aattgtatgt 720 gtgtgagagg
ttttgttttc gtaaggaaag aaacaaaaac catttgacca aagaaaagca 780
aaagaaggca aggaatcaaa caacaaatgt tgcaaggcag aaataatgga cgttatgtta
840 atgtagtgtc gtcacacgtg acttaaaaga gacgagtctg cgtgtcaaac
taaaaatgta 900 tgcaactata aaaatgggat ttgattatct ttttagtacc
gaagcctacc aaccacatgc 960 acactaattc tactcgccaa ataaagtgaa aagag
995 17 1017 DNA Arabidopsis thaliana 17 aagtaacttt tagaattgat
tcaatctttt tagaatagat tttttttttt tttttttttg 60 gatttcgctg
aggttttacc attttgttac tcagcatatt ttaacgatgt tgcatttgtg 120
tcccatatac gttattgtta gtgaaaaata taatgtaaga ataatttata taactatcct
180 actagcaaag ctaacgcaaa ttttgaactc gaactttagt taccgtgaat
gaaaataaca 240 gacttgaact ttataatact cgtagtatac gtaatttttg
ctttttgcag atatgcttgc 300 cactaataaa gtcataaatt ttatattttc
ataaactata gttatacact tttgactaaa 360 caaacaaaat cggtttagca
aaagaaaaag ttacttttct gatgaactag gataaggaat 420 tcggaactga
attttgctac gttctctctg gaccacacac actgaacacc cttttaagat 480
tttctccttc tctttttcaa cgtaatttat cttttgatca gaaacgacaa aaaagaagtc
540 taacaatatc aaacaatttt tttatagata tttttagata tttttcctgc
taattttatc 600 tagtgtagac aaacccaaat atacgattat tataaaaaca
cgaaatacca agtggacgac 660 tgaggttaat agatctagcc gtagaataaa
gatctgcatg aaaggcggtg agaatctaaa 720 cggtgataag accataacac
acggaacatc ggtacgctct cgaacgtaca agaatcgacg 780 acacacaaac
actccacaat tatttgaaca ctggacaatt attgaaccga cgtacgagaa 840
tcaatgcgct gagggtaaag acgtaaatga agaactagtt ttggagataa gagcggagaa
900 agattgcgac acatgtatgg tcaatattaa tctcatttag cttataaatt
tgggagcttc 960 ctctatcatt aattttcatt cataaatttt tcttcaattt
gaattttctc gagaaaa 1017 18 273 DNA Arabidopsis thaliana 18
gcaagtgtgt tgcctttgtg tggaaatgaa gaggtacttg cgaggacttt gcgtttatca
60 gtttatgtgt ttgtatatct atttgatcca gttattatgg attatatacg
cttgaaactc 120 attttaagcc attgttattg aacgtttatc aaatacttta
ttatgccaag caagtcaaac 180 acatgcttgt tgattgaaat caagctatag
aaatctcttc ttcacataca gcagtttaga 240 ttcacaatac aacaagcgaa
acgataaagt ttc 273 19 1131 DNA Arabidopsis thaliana 19 gtccgtcgct
tctcttccat ttcttctcat tttcgatttt gattcttatt tctttccagt 60
agctcctgct ctgtgaattt ctccgctcac gatagatctg cttatactcc ttacattcaa
120 ccttagatct ggtctcgatt ctctgtttct ctgttttttt cttttggtcg
agaatctgat 180 gtttgtttat gttctgtcac cattaataat aatgaactct
ctcattcata caatgattag 240 tttctctcgt ctacaaaacg atatgttgca
ttttcacttt tcttcttttt ttctaagatg 300 atttgctttg accaatttgt
ttagatcttt attttatttt attttctggt gggttggtgg 360 aaattgaaaa
aaaaaaaaac agcataaatt gttatttgtt aatgtattca ttttttggct 420
atttgttctg ggtaaaaatc tgcttctact attgaatctt tcctggattt tttactccta
480 ttgggttttt atagtaaaaa tacataataa aaggaaaaca aaagttttat
agattctctt 540 aaacccctta cgataaaagt tggaatcaaa ataattcagg
atcagatgct ctttgattga 600 ttcagatgcg attacagttg catggcaaat
tttctagatc cgtcgtcaca ttttattttc 660 tgtttaaata tctaaatctg
atatatgatg tcgacaaatt ctggtggctt atacatcact 720 tcaactgttt
tcttttggct ttgtttgtca acttggtttt caatacgatt tgtgatttcg 780
atcgctgaat ttttaataca agcaaactga tgttaaccac aagcaagaga tgtgacctgc
840 cttattaaca tcgtattact tactactagt cgtattctca acgcaatcgt
ttttgtattt 900 ctcacattat gccgcttctc tactctttat tccttttggt
ccacgcattt tctatttgtg 960 gcaatccctt tcacaacctg atttcccact
ttggatcatt tgtctgaaga ctctcttgaa 1020 tcgttaccac ttgtttcttg
tgcatgctct gttttttaga attaatgata aaactattcc 1080 atagtcttga
gttttcagct tgttgattct tttgcttttg gttttctgca g 1131 20 8956 DNA
Artificial Sequence Artificially created chimeric nucleic acid
sequence 20 ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat
cctttctttt 60 cttccaattt gtgtttcttt ttgcctaatt tattgtgtta
tcccctttat cctattttgt 120 ttctttactt atttatttgc ttctatgtct
ttgtacaaag atttaaactc tatggcacat 180 attttaaagt tgttagaaaa
taaattcttt caagattgat gaaagaactt tttaattgta 240 gatatttcgt
agattttatt ctcttactac caatataacg cttgaattga cgaaaatttg 300
tgtccaaata tctagcaaaa aggtatccaa tgaaaatata tcatatgtga tcttcaaatc
360 ttgtgtctta tgcaagattg atactttgtt caatggaaga gattgtgtgc
atatttttaa 420 aatttttatt agtaataaag attctatata gctgttatag
agggataatt ttacaaagaa 480 cactataaat atgattgttg ttgttagggt
gtcaatggtt cggttcgact ggttatttta 540 taaaatttgt accataccat
ttttttcgat attctatttt gtataaccaa aattagactt 600 ttcgaaatcg
tcccaatcat gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660
catttttttt taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt
720 cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg
ataatgaatt 780 aaaaaacatg aaagatggct agagtataga tacacaacta
ttcgacagca acgtaaaaga 840 aaccaagtaa aagcaaagaa aatataaatc
acacgagtgg aaagatatta accaagttgg 900 gattcaagaa taaagtctat
attaaatatt caaaaagata aatttaaata atatgaaagg 960 aaacatattc
aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc 1020
taataaagat acttgaaata gcttagttta aatataaata gcataataga ttttaggaat
1080 tagtattttg agtttaatta cttattgact tgtaacagtt tttataattc
caaggcccat 1140 gaaaaattta atgctttatt agttttaaac ttactatata
aatttttcat atgtaaaatt 1200 taatcggtat agttcgatat tttttcaatt
tatttttata aaataaaaaa cttaccctaa 1260 ttatcggtac agttatagat
ttatataaaa atctacggtt cttcagaaga aacctaaaaa 1320 tcggttcggt
gcggacggtt cgatcggttt agtcgatttt caaatattca ttgacactcc 1380
tagttgttgt tataggtaaa aagcagttac agagaggtaa aatataactt aaaaaatcag
1440 ttctaaggaa aaattgactt ttatagtaaa tgactgttat ataaggatgt
tgttacagag 1500 aggtatgagt gtagttggta aattatgttc ttgacggtgt
atgtcacata ttatttatta 1560 aaactagaaa aaacagcgtc aaaactagca
aaaatccaac ggacaaaaaa atcggctgaa 1620 tttgatttgg ttccaacatt
taaaaaagtt tcagtgagaa agaatcggtg actgttgatg 1680 atataaacaa
agggcacatt ggtcaataac cataaaaaat tatatgacag ctacagttgg 1740
tagcatgtgc tcagctattg aacaaatcta aagaaggtac atctgtaacc ggaacaccac
1800 ttaaatgact aaattaccct catcagaaag cagatggagt gctacaaata
acacactatt 1860 caacaaccat aaataaaacg tgttcagcta ctaaaacaaa
tataaataaa tctatgtttg 1920 taagcactcc agccatgtta atggagtgct
attgcctgtt aactctcact tataaaatag 1980 tagtagaaaa aatatgaacc
aaaacacaac ggttgtgtat ttcacttttg gatatagctc 2040 agtggcttcg
acacctgtag gaggctgaac ctcaaagttt gcagaatctc cattaacaaa 2100
aactgaatgg catatggcca aattagtcct taatggcaga ggtccctctt gtacaatctg
2160 gagaatatct tcctctgata gatataactt ctcgagggtc ttcccaattt
tgtcctccca 2220 caaggacact atctcgttga aggatagaat attggcaggt
ggtctcatgt gaagagtctt 2280 attcaatgtc cgtggatcat ctactgcttc
gatagtgtat gtcgctatgt cttcttcctt 2340 cacatatatt gctttgggat
ttccatcgcc aaaaatgaca actttgtctc taggaggggt 2400 tttggcctct
aactgcccca agttgggcaa gaagaaatct gcaaaccaat tgcagattac 2460
atatgtgtat ggaattcctt ctgcctctat catcctcctg attcttacct ttagagcgaa
2520 gagtgatgca gctggttcaa ttgcacgagc atgatccaca tcaaatccaa
attctgaagg 2580 aagaaatctc ttgatatttc cagcttcttt aattgctttg
atgatgttca cttgatcagt 2640 ccgtcgcttc tcttccattt cttctcattt
tcgattttga ttcttatttc tttccagtag 2700 ctcctgctct gtgaatttct
ccgctcacga tagatctgct tatactcctt acattcaacc 2760 ttagatctgg
tctcgattct ctgtttctct
gtttttttct tttggtcgag aatctgatgt 2820 ttgtttatgt tctgtcacca
ttaataataa tgaactctct cattcataca atgattagtt 2880 tctctcgtct
acaaaacgat atgttgcatt ttcacttttc ttcttttttt ctaagatgat 2940
ttgctttgac caatttgttt agatctttat tttattttat tttctggtgg gttggtggaa
3000 attgaaaaaa aaaaaaacag cataaattgt tatttgttaa tgtattcatt
ttttggctat 3060 ttgttctggg taaaaatctg cttctactat tgaatctttc
ctggattttt tactcctatt 3120 gggtttttat agtaaaaata cataataaaa
ggaaaacaaa agttttatag attctcttaa 3180 accccttacg ataaaagttg
gaatcaaaat aattcaggat cagatgctct ttgattgatt 3240 cagatgcgat
tacagttgca tggcaaattt tctagatccg tcgtcacatt ttattttctg 3300
tttaaatatc taaatctgat atatgatgtc gacaaattct ggtggcttat acatcacttc
3360 aactgttttc ttttggcttt gtttgtcaac ttggttttca atacgatttg
tgatttcgat 3420 cgctgaattt ttaatacaag caaactgatg ttaaccacaa
gcaagagatg tgacctgcct 3480 tattaacatc gtattactta ctactagtcg
tattctcaac gcaatcgttt ttgtatttct 3540 cacattatgc cgcttctcta
ctctttattc cttttggtcc acgcattttc tatttgtggc 3600 aatccctttc
acaacctgat ttcccacttt ggatcatttg tctgaagact ctcttgaatc 3660
gttaccactt gtttcttgtg catgctctgt tttttagaat taatgataaa actattccat
3720 agtcttgagt tttcagcttg ttgattcttt tgcttttggt tttctgcagt
gatcaagtga 3780 acatcatcaa agcaattaaa gaagctggaa atatcaagag
atttcttcct tcagaatttg 3840 gatttgatgt ggatcatgct cgtgcaattg
aaccagctgc atcactcttc gctctaaagg 3900 taagaatcag gaggatgata
gaggcagaag gaattccata cacatatgta atctgcaatt 3960 ggtttgcaga
tttcttcttg cccaacttgg ggcagttaga ggccaaaacc cctcctagag 4020
acaaagttgt catttttggc gatggaaatc ccaaagcaat atatgtgaag gaagaagaca
4080 tagcgacata cactatcgaa gcagtagatg atccacggac attgaataag
actcttcaca 4140 tgagaccacc tgccaatatt ctatccttca acgagatagt
gtccttgtgg gaggacaaaa 4200 ttgggaagac cctcgagaag ttatatctat
cagaggaaga tattctccag attgtacaag 4260 agggacctct gccattaagg
actaatttgg ccatatgcca ttcagttttt gttaatggag 4320 attctgcaaa
ctttgaggtt cagcctccta caggtgtcga agccactgag ctatatccaa 4380
aagtgaaata cacaaccgca agtgtgttgc ctttgtgtgg aaatgaagag gtacttgcga
4440 ggactttgcg tttatcagtt tatgtgtttg tatatctatt tgatccagtt
attatggatt 4500 atatacgctt gaaactcatt ttaagccatt gttattgaac
gtttatcaaa tactttatta 4560 tgccaagcaa gtcaaacaca tgcttgttga
ttgaaatcaa gctatagaaa tctcttcttc 4620 acatacagca gtttagattc
acaatacaac aagcgaaacg ataaagtttc ttctgttcgt 4680 atatttgtaa
ctattatgtg tatttttatt ttgttagtat tactaattca agtggtttaa 4740
gttgttgaga ctctttaaaa tctaagcatt ttataaacaa taatatataa ttattgttta
4800 ggctaaattt gtcactaatt aaggtttgga tacatagtgt ctaaactaag
ctaataatat 4860 cacttaacgt ttacttgtaa cgctaggtga tgatgtcgtc
aagtcaattg gtacaaggaa 4920 taaacgagtg gtcatatgac attatgacca
tatgaattca aactccagta atccaatggt 4980 aattggattc aatgatcaag
acttgaacca cgtaatccac ccttatcctt agaagctcat 5040 aaatatcact
aaagggacag gcaacactta accagtagtt gtccaataat ttagttttcc 5100
aaaatgaaaa attattgttg tcatctattt taggtgtttt agttcaatgt ggattcctcg
5160 tcctaacaaa tacttgacga atatatctag actataaaat tggttatgag
ttctactttt 5220 ttttgtttgt gaaattatca aaatttgtta tatttattta
tttattctca ttaatttgag 5280 tactaatttt taaattattt atactaaaaa
caattactaa gatacaaaaa tggataagag 5340 catggtgtat agatatttaa
tgggatagaa tatttcccat aattgtatgt gtgtgagagg 5400 ttttgttttc
gtaaggaaag aaacaaaaac catttgacca aagaaaagca aaagaaggca 5460
aggaatcaaa caacaaatgt tgcaaggcag aaataatgga cgttatgtta atgtagtgtc
5520 gtcacacgtg acttaaaaga gacgagtctg cgtgtcaaac taaaaatgta
tgcaactata 5580 aaaatgggat ttgattatct ttttagtacc gaagcctacc
aaccacatgc acactaattc 5640 tactcgccaa ataaagtgaa aagagccata
tattagcaaa tgcccacctt tatgagcttg 5700 tcaataggta tatatcttag
aacaaggaca tcaatggcaa aaatagcaag acatgaaatc 5760 aaattgtgcc
agacaagcaa cacagaaaaa gaaaccctcc acccacaacg ccctccaaaa 5820
actgtagtca ccttaattag ggcggtcata ttcaatgtgt aaagttctgt gcgaagaatc
5880 ttacagattt gctagctaaa gcaaaaagct aagtgactaa actccatatt
actgagagtc 5940 tgaaatgggc ttgcgaacca cgaagaagta cattggtgtg
aaaatccctt tcttggcacc 6000 accgacaaga ccttctgcag ctttctctaa
gaaagcttga accctttgac tacctttagg 6060 agcaagtccc acgtattcaa
gcgccgaaac cagatttctg gtgaaaagtc tgccaactgc 6120 tgttaggcgg
aagctactga gcgagaagtg actcgtatcc aaaggcaagt accatggaac 6180
aggtgagtca tcagccagat ccttgtccca tacaacttca aaaccagctt gtttggctgc
6240 ttcgaggcac tgtgttgtca atctaacctc agggaggcca tttccgagct
caatttcggc 6300 cttgatcctg ttgtgctctt cgttattggg gttgtaagaa
tcggtcatgc accactcata 6360 cacagcgaaa cattgaccag gcttcagcac
ccggtaaatc tctttatagc atcccaatgg 6420 atctggtgca tggcaggtag
cttctgtaag ttcctgtttt cacctgcacc atgaaaaata 6480 tactattact
attatttttc atttatttgt gtggtccata ttgctatgtg tgaaatgaaa 6540
aaatattttt tttctcaaac tacaatattg tcagaaagaa aggaattaat attccgaatt
6600 tataccaaaa aattaatttc ttttttctct ttggtaagct ggattctgtt
attctttggt 6660 aaaacggaga ataattttgt ttatcaactt ctgttgattt
tatgaacaat tctcaattaa 6720 ttgaaggggt agtttaaggc tgatgaatct
tttggatgag ttacttgagc agtatggatt 6780 gactcacatg actaactgct
tcactagctt ccaatatttt ttagttatta catgttgtgt 6840 atgttgatta
ttgtgctcta agcaatcgga ttctcttgtt aaataaaaac tatcatagtt 6900
tatttattca ataatcgagt ttgagctaac actcctgtct atctggaata caaaaggaaa
6960 gataataaaa gtttttggta ccttgaaaac tagaagtatc aggaagggga
gccttgaaca 7020 aaggtcaagt tgtctccgtt tgacctacat gtcatgttcg
agccattgat gcttgcatca 7080 ggatagactg cctacatcac cccctcttgc
ggtacggccc ttccccggac ctgcgtgaac 7140 gcgggatact ttgtgcaccg
gaaaactaca agtatcccta acacatatca ggattttagt 7200 gatatccctt
cactgccgtg ttcgataaag gttacataaa gttttaaatt tatgggtgct 7260
aaatatcaca gctaaatata cacattaaag atattactgc atccatatat gttgccatga
7320 ccatacatca agtatacatc cacccctaat ttttgagtgt ttttgagatg
cagcaaagtt 7380 gaaggagatt ataatagttt gatgtggaga gactaatttt
ttttttaaca tcactttcta 7440 agggtgctat cttttcacca ccatcactgg
tggcttgttg atttgtagct aatcattatc 7500 ttttgatgaa aacaaggaca
ttctttagtg cactaagatt gttaaacgtt cgtgcttcat 7560 tgtaaatgta
atatactcgc gcttgttggc atgaacactt ggaattgttt actggaacac 7620
tgcagagaag ctacctgcca tgcaccagat ccattgggat gctataaaga gatttaccgg
7680 gtgctgaagc ctggtcaatg tttcgctgtg tatgagtggt gcatgaccga
ttcttacaac 7740 cccaataacg aagagcacaa caggatcaag gccgaaattg
agctcggaaa tggcctccct 7800 gaggttagat tgacaacaca gtgcctcgaa
gcagccaaac aagctggttt tgaagttgta 7860 tgggacaagg atctggctga
tgactcacct gttccatggt acttgccttt ggatacgagt 7920 cacttctcgc
tcagtagctt ccgcctaaca gcagttggca gacttttcac cagaaatctg 7980
gtttcggcgc ttgaatacgt gggacttgct cctaaaggta gtcaaagggt tcaagctttc
8040 ttagagaaag ctgcagaagg tcttgtcggt ggtgccaaga aagggatttt
cacaccaatg 8100 tacttcttcg tggttcgcaa gcccatttca gactctcagt
aatatggagt ttagtcactt 8160 agctttttgc tttagctagc aaatctgtaa
gattcttcgc acagaacttt acacattgaa 8220 tatgaccgcc ctaattaagg
tgactacagt ttttggaggg cgttgtgggt ggagggtttc 8280 tttttctgtg
ttgcttgtct ggcacaattt gatttcatgt cttgctattt ttgccattga 8340
tgtccttgtt ctaagatata tacctattga caagctcata aaggtgggca tttgctaata
8400 tatggtttcc ctttgctttt gtgtaaacct caaaacttta tcccccatct
ttgattttat 8460 cccttgtttt tctgcttttt tcttctttct tgggttttaa
tttccggact taacgtttgt 8520 tttccggttt gcgagacata ttctatcgga
ttctcaactg tctgatgaaa taaatatgta 8580 atgttctata agtctttcaa
tttgatatgc atatcaacaa aaagaaaata ggacaatgcg 8640 gctacaaata
tgaaatttac aagtttaaga accatgagtc gctaaagaaa tcattaagaa 8700
aattagtttc acattcaatt cttgtcacat gattaacgag cttgagaggt ttagagtaac
8760 aatatcttga agcaaaagat gacccacttg aaatctagtg atggatacat
aagtggacgt 8820 gccttgttta ggataggatt ctggataaga gtctcgaata
ttcattttta ccaagtatat 8880 tcaaggatct tgtggatcat atatttcctc
aatcaaaggg acttgaccca aattcacata 8940 aagatatttt ggagtc 8956 21
5688 DNA Artificial Sequence Artificially created chimeric nucleic
acid sequence 21 ctcgaggatc taaattgtga gttcaatctc ttccctattg
gattgattat cctttctttt 60 cttccaattt gtgtttcttt ttgcctaatt
tattgtgtta tcccctttat cctattttgt 120 ttctttactt atttatttgc
ttctatgtct ttgtacaaag atttaaactc tatggcacat 180 attttaaagt
tgttagaaaa taaattcttt caagattgat gaaagaactt tttaattgta 240
gatatttcgt agattttatt ctcttactac caatataacg cttgaattga cgaaaatttg
300 tgtccaaata tctagcaaaa aggtatccaa tgaaaatata tcatatgtga
tcttcaaatc 360 ttgtgtctta tgcaagattg atactttgtt caatggaaga
gattgtgtgc atatttttaa 420 aatttttatt agtaataaag attctatata
gctgttatag agggataatt ttacaaagaa 480 cactataaat atgattgttg
ttgttagggt gtcaatggtt cggttcgact ggttatttta 540 taaaatttgt
accataccat ttttttcgat attctatttt gtataaccaa aattagactt 600
ttcgaaatcg tcccaatcat gtcggtttca cttcggtatc ggtaccgttc ggttaatttt
660 catttttttt taaatgtcat taaaattcac tagtaaaaat agaatgcaat
aacatacgtt 720 cttttatagg acttagcaaa agctctctag acatttttac
tgtttaaagg ataatgaatt 780 aaaaaacatg aaagatggct agagtataga
tacacaacta ttcgacagca acgtaaaaga 840 aaccaagtaa aagcaaagaa
aatataaatc acacgagtgg aaagatatta accaagttgg 900 gattcaagaa
taaagtctat attaaatatt caaaaagata aatttaaata atatgaaagg 960
aaacatattc aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc
1020 taataaagat acttgaaata gcttagttta aatataaata gcataataga
ttttaggaat 1080 tagtattttg agtttaatta cttattgact tgtaacagtt
tttataattc caaggcccat 1140 gaaaaattta atgctttatt agttttaaac
ttactatata aatttttcat atgtaaaatt 1200 taatcggtat agttcgatat
tttttcaatt tatttttata aaataaaaaa cttaccctaa 1260 ttatcggtac
agttatagat ttatataaaa atctacggtt cttcagaaga aacctaaaaa 1320
tcggttcggt gcggacggtt cgatcggttt agtcgatttt caaatattca ttgacactcc
1380 tagttgttgt tataggtaaa aagcagttac agagaggtaa aatataactt
aaaaaatcag 1440 ttctaaggaa aaattgactt ttatagtaaa tgactgttat
ataaggatgt tgttacagag 1500 aggtatgagt gtagttggta aattatgttc
ttgacggtgt atgtcacata ttatttatta 1560 aaactagaaa aaacagcgtc
aaaactagca aaaatccaac ggacaaaaaa atcggctgaa 1620 tttgatttgg
ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg actgttgatg 1680
atataaacaa agggcacatt ggtcaataac cataaaaaat tatatgacag ctacagttgg
1740 tagcatgtgc tcagctattg aacaaatcta aagaaggtac atctgtaacc
ggaacaccac 1800 ttaaatgact aaattaccct catcagaaag cagatggagt
gctacaaata acacactatt 1860 caacaaccat aaataaaacg tgttcagcta
ctaaaacaaa tataaataaa tctatgtttg 1920 taagcactcc agccatgtta
atggagtgct attgcctgtt aactctcact tataaaatag 1980 tagtagaaaa
aatatgaacc aaaacacaac aacatctcaa aatatttgaa gtaacacaga 2040
attttacata caccaaactt ataaatcaag tattttcatt gtaacaaatt ccatgaaaca
2100 tgaaaacaaa gctataatga aattaccaac tcaagcaata aggttggaaa
agagccatct 2160 gagatattcc agcaatttac atctttttgt ttgattacac
agtgaaggat cttttgtttg 2220 acaactagta aaatgattct tatttgcacc
tttcagctat tcagctgctt ttactccaac 2280 cctatagcag aagtaatggc
gctcatgctc gttttgtacg ccttccaact tcaagggcga 2340 gctctgtatc
gatcttcagg gaaatgtcaa gtgctttcac ggaatgcgtc agggcaccac 2400
tagaaatgta ggtaacacca gtttgtccaa tcttgtgtac tgtttcaagg gtaacatttc
2460 ctgaagcctc cgtatcaaac ctcccattga tcaattctac agcctcctta
agcatggata 2520 catcaatatc tccgttagat aatggaacaa ccatattgtc
cagcattatc ctagtcaacg 2580 aagtctttgt ttgagatgca tagtctagaa
cctcacgtac ttcttcaatt gtcctggttt 2640 caacctcaac ccctatttga
agtttatttt gctccaaata ctgatccaca gattttagag 2700 ctttgccgac
acctccagca gcagatatgt gattgtcttt tatcattacc atatcaaata 2760
agcccattct gtgattcttc cccccaccga tcaataccgc ccatttatcc accaaacgta
2820 atccaggagc agttttccta gtctccaaga tgtaagcagg gtgtgcagca
tctgccattt 2880 ccttagttag tgtagctatt ccactcattc tttgcataaa
attgagaaca accctctcag 2940 ctataacaat gttgtaagcg tttccttgta
ctttgccaaa tttcaagcct ttatgaactt 3000 tatcgccatc atttacatac
cactccacct ttaatgaagg atcaacttcc gcgaatatca 3060 tctcagcaag
tgcaattcct gctatgatcc cgtcttcctt tgctagaaaa tgagcatcgg 3120
attccatatc aagaggaatt gtcgccttac aagtcacatc tcctaaattc ccagcatctt
3180 cagagagtgc aagtttcata acttccttta aatcataagt tgggtgtgct
ggtggtttca 3240 cctctaatga ctccactctt gtattcttgg tggctattgc
tgacattttc accaccaacc 3300 ttggagctgt aattgcataa ggatgcactg
tagcagtgaa aggaatagct ctaaacatgg 3360 tttttttttg ggggggttgt
gaaatgaatt ttgtggaaaa tagtttttgg ggcacatcaa 3420 tcctgcggtg
acattcggaa tgtttctaac aagaaagata tcgttggtcc gagccttgct 3480
ctacatcata gctcagtgca taggggccct gtgcgggtgc gccttagtca agacattgca
3540 gcgagatcat tacaaccact atggcggtgg cgctaaccag ctcgttgatg
gttatagccg 3600 aggcactggc cttgctgttg agattatggg cacctttatt
cttctgtata ctgtcttctc 3660 cgccactgat cccaaacgca atgctagaga
ttcccatgtt cctgtcttgg ctccactccc 3720 cattggcttt gctgtcttca
ttgttcacct cgccaccatt cccgtcaccg gcactggcat 3780 caacccagcg
agcaaaaact attttccaca aaattcattt cacaaccccc ccaaaaaaaa 3840
accatgttta gagctattcc tttcactgct acagtgcatc cttatgcaat tacagctcca
3900 aggttggtgg tgaaaatgtc agcaatagcc accaagaata caagagtgga
gtcattagag 3960 gtgaaaccac cagcacaccc aacttatgat ttaaaggaag
ttatgaaact tgcactctct 4020 gaagatgctg ggaatttagg agatgtgact
tgtaaggcga caattcctct tgatatggaa 4080 tccgatgctc attttctagc
aaaggaagac gggatcatag caggaattgc acttgctgag 4140 atgatattcg
cggaagttga tccttcatta aaggtggagt ggtatgtaaa tgatggcgat 4200
aaagttcata aaggcttgaa atttggcaaa gtacaaggaa acgcttacaa cattgttata
4260 gctgagaggg ttgttctcaa ttttatgcaa agaatgagtg gaatagctac
actaactaag 4320 gaaatggcag atgctgcaca ccctgcttac atcttggaga
ctaggaaaac tgctcctgga 4380 ttacgtttgg tggataaatg ggcggtattg
atcggtgggg ggaagaatca cagaatgggc 4440 ttatttgata tggtaatgat
aaaagacaat cacatatctg ctgctggagg tgtcggcaaa 4500 gctctaaaat
ctgtggatca gtatttggag caaaataaac ttcaaatagg ggttgaggtt 4560
gaaaccagga caattgaaga agtacgtgag gttctagact atgcatctca aacaaagact
4620 tcgttgacta ggataatgct ggacaatatg gttgttccat tatctaacgg
agatattgat 4680 gtatccatgc ttaaggaggc tgtagaattg atcaatggga
ggtttgatac ggaggcttca 4740 ggaaatgtta cccttgaaac agtacacaag
attggacaaa ctggtgttac ctacatttct 4800 agtggtgccc tgacgcattc
cgtgaaagca cttgacattt ccctgaagat cgatacagag 4860 ctcgcccttg
aagttggaag gcgtacaaaa cgagcatgag cgccattact tctgctatag 4920
ggttggagta aaagcagctg aatagctgaa aggtgcaaat aagaatcatt ttactagttg
4980 tcaaacaaaa gatccttcac tgtgtaatca aacaaaaaga tgtaaattgc
tggaatatct 5040 cagatggctc ttttccaacc ttattgcttg agttggtaat
ttcattatag ctttgttttc 5100 atgtttcatg gaatttgtta caatgaaaat
acttgattta taagtttggt gtatgtaaaa 5160 ttctgtgtta cttcaaatat
tttgagatgt tgagctcgtg aaatggcctc tttagttttt 5220 gattgaatca
taggggtatt agttttctat ggccgggagt ggtcttcttg cttaattgta 5280
atggaataac cagagaggaa ctactgtgtt atctttgagg aatgttgggc ttttttcgtt
5340 tgaattatca tgaatgaaat tttacttttt cccaatacaa gtttgttttc
gtttcttggt 5400 ttttgttatc ccttggttta tgtcttggtt tggcttaaat
gattgaagat tacactacct 5460 atgtttctgc tattcctgtt gaagatcaca
tttgataata atgcatcgaa tgcattaaag 5520 tttcttattg gctctgtcaa
aagtattgaa ggtggatttt tctaattggc aagagaaagt 5580 attaaagagg
tgatttatta gtacttatat ttttctcagc atctctcttt cagtgttgga 5640
gcttcataaa attagcactt cagagtttca gtcgggagct gaattcga 5688 22 4134
DNA Artificial Sequence Artificially created chimeric nucleic acid
sequence 22 ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat
cctttctttt 60 cttccaattt gtgtttcttt ttgcctaatt tattgtgtta
tcccctttat cctattttgt 120 ttctttactt atttatttgc ttctatgtct
ttgtacaaag atttaaactc tatggcacat 180 attttaaagt tgttagaaaa
taaattcttt caagattgat gaaagaactt tttaattgta 240 gatatttcgt
agattttatt ctcttactac caatataacg cttgaattga cgaaaatttg 300
tgtccaaata tctagcaaaa aggtatccaa tgaaaatata tcatatgtga tcttcaaatc
360 ttgtgtctta tgcaagattg atactttgtt caatggaaga gattgtgtgc
atatttttaa 420 aatttttatt agtaataaag attctatata gctgttatag
agggataatt ttacaaagaa 480 cactataaat atgattgttg ttgttagggt
gtcaatggtt cggttcgact ggttatttta 540 taaaatttgt accataccat
ttttttcgat attctatttt gtataaccaa aattagactt 600 ttcgaaatcg
tcccaatcat gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660
catttttttt taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt
720 cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg
ataatgaatt 780 aaaaaacatg aaagatggct agagtataga tacacaacta
ttcgacagca acgtaaaaga 840 aaccaagtaa aagcaaagaa aatataaatc
acacgagtgg aaagatatta accaagttgg 900 gattcaagaa taaagtctat
attaaatatt caaaaagata aatttaaata atatgaaagg 960 aaacatattc
aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc 1020
taataaagat acttgaaata gcttagttta aatataaata gcataataga ttttaggaat
1080 tagtattttg agtttaatta cttattgact tgtaacagtt tttataattc
caaggcccat 1140 gaaaaattta atgctttatt agttttaaac ttactatata
aatttttcat atgtaaaatt 1200 taatcggtat agttcgatat tttttcaatt
tatttttata aaataaaaaa cttaccctaa 1260 ttatcggtac agttatagat
ttatataaaa atctacggtt cttcagaaga aacctaaaaa 1320 tcggttcggt
gcggacggtt cgatcggttt agtcgatttt caaatattca ttgacactcc 1380
tagttgttgt tataggtaaa aagcagttac agagaggtaa aatataactt aaaaaatcag
1440 ttctaaggaa aaattgactt ttatagtaaa tgactgttat ataaggatgt
tgttacagag 1500 aggtatgagt gtagttggta aattatgttc ttgacggtgt
atgtcacata ttatttatta 1560 aaactagaaa aaacagcgtc aaaactagca
aaaatccaac ggacaaaaaa atcggctgaa 1620 tttgatttgg ttccaacatt
taaaaaagtt tcagtgagaa agaatcggtg actgttgatg 1680 atataaacaa
agggcacatt ggtcaataac cataaaaaat tatatgacag ctacagttgg 1740
tagcatgtgc tcagctattg aacaaatcta aagaaggtac atctgtaacc ggaacaccac
1800 ttaaatgact aaattaccct catcagaaag cagatggagt gctacaaata
acacactatt 1860 caacaaccat aaataaaacg tgttcagcta ctaaaacaaa
tataaataaa tctatgtttg 1920 taagcactcc agccatgtta atggagtgct
attgcctgtt aactctcact tataaaatag 1980 tagtagaaaa aatatgaacc
aaaacacaac tttatcgcca tcatttacat accactccac 2040 ctttaatgaa
ggatcaactt ccgcgaatat catctcagca agtgcaattc ctgctatgat 2100
cccgtcttcc tttgctagaa aatgagcatc ggattccata tcaagaggaa ttgtcgcctt
2160 acaagtcaca tctcctaaat tcccagcatc ttcagagagt gcaagtttca
taacttcctt 2220 taaatcataa gttgggtgtg ctggtggttt cacctctaat
gactccactc ttgtattctt 2280 ggtggctatt gctgacattt tcaccaccaa
ccttggagct gtaattgcat aaggatgcac 2340 tgtagcagtg aaaggaatag
ctctaaacat gtccgtcgct tctcttccat ttcttctcat 2400 tttcgatttt
gattcttatt tctttccagt agctcctgct ctgtgaattt ctccgctcac 2460
gatagatctg cttatactcc ttacattcaa ccttagatct ggtctcgatt ctctgtttct
2520 ctgttttttt cttttggtcg agaatctgat gtttgtttat gttctgtcac
cattaataat 2580 aatgaactct ctcattcata caatgattag tttctctcgt
ctacaaaacg atatgttgca 2640 ttttcacttt tcttcttttt ttctaagatg
atttgctttg accaatttgt ttagatcttt 2700 attttatttt attttctggt
gggttggtgg aaattgaaaa aaaaaaaaac agcataaatt 2760 gttatttgtt
aatgtattca ttttttggct atttgttctg ggtaaaaatc tgcttctact 2820
attgaatctt tcctggattt tttactccta ttgggttttt atagtaaaaa tacataataa
2880 aaggaaaaca aaagttttat agattctctt aaacccctta cgataaaagt
tggaatcaaa 2940 ataattcagg atcagatgct ctttgattga ttcagatgcg
attacagttg
catggcaaat 3000 tttctagatc cgtcgtcaca ttttattttc tgtttaaata
tctaaatctg atatatgatg 3060 tcgacaaatt ctggtggctt atacatcact
tcaactgttt tcttttggct ttgtttgtca 3120 acttggtttt caatacgatt
tgtgatttcg atcgctgaat ttttaataca agcaaactga 3180 tgttaaccac
aagcaagaga tgtgacctgc cttattaaca tcgtattact tactactagt 3240
cgtattctca acgcaatcgt ttttgtattt ctcacattat gccgcttctc tactctttat
3300 tccttttggt ccacgcattt tctatttgtg gcaatccctt tcacaacctg
atttcccact 3360 ttggatcatt tgtctgaaga ctctcttgaa tcgttaccac
ttgtttcttg tgcatgctct 3420 gttttttaga attaatgata aaactattcc
atagtcttga gttttcagct tgttgattct 3480 tttgcttttg gttttctgca
gatgtttaga gctattcctt tcactgctac agtgcatcct 3540 tatgcaatta
cagctccaag gttggtggtg aaaatgtcag caatagccac caagaataca 3600
agagtggagt cattagaggt gaaaccacca gcacacccaa cttatgattt aaaggaagtt
3660 atgaaacttg cactctctga agatgctggg aatttaggag atgtgacttg
taaggcgaca 3720 attcctcttg atatggaatc cgatgctcat tttctagcaa
aggaagacgg gatcatagca 3780 ggaattgcac ttgctgagat gatattcgcg
gaagttgatc cttcattaaa ggtggagtgg 3840 tatgtaaatg atggcgataa
agcaagtgtg ttgcctttgt gtggaaatga agaggtactt 3900 gcgaggactt
tgcgtttatc agtttatgtg tttgtatatc tatttgatcc agttattatg 3960
gattatatac gcttgaaact cattttaagc cattgttatt gaacgtttat caaatacttt
4020 attatgccaa gcaagtcaaa cacatgcttg ttgattgaaa tcaagctata
gaaatctctt 4080 cttcacatac agcagtttag attcacaata caacaagcga
aacgataaag tttc 4134 23 5390 DNA Artificial Sequence Artificially
created chimeric nucleic acid sequence 23 ctcgaggatc taaattgtga
gttcaatctc ttccctattg gattgattat cctttctttt 60 cttccaattt
gtgtttcttt ttgcctaatt tattgtgtta tcccctttat cctattttgt 120
ttctttactt atttatttgc ttctatgtct ttgtacaaag atttaaactc tatggcacat
180 attttaaagt tgttagaaaa taaattcttt caagattgat gaaagaactt
tttaattgta 240 gatatttcgt agattttatt ctcttactac caatataacg
cttgaattga cgaaaatttg 300 tgtccaaata tctagcaaaa aggtatccaa
tgaaaatata tcatatgtga tcttcaaatc 360 ttgtgtctta tgcaagattg
atactttgtt caatggaaga gattgtgtgc atatttttaa 420 aatttttatt
agtaataaag attctatata gctgttatag agggataatt ttacaaagaa 480
cactataaat atgattgttg ttgttagggt gtcaatggtt cggttcgact ggttatttta
540 taaaatttgt accataccat ttttttcgat attctatttt gtataaccaa
aattagactt 600 ttcgaaatcg tcccaatcat gtcggtttca cttcggtatc
ggtaccgttc ggttaatttt 660 catttttttt taaatgtcat taaaattcac
tagtaaaaat agaatgcaat aacatacgtt 720 cttttatagg acttagcaaa
agctctctag acatttttac tgtttaaagg ataatgaatt 780 aaaaaacatg
aaagatggct agagtataga tacacaacta ttcgacagca acgtaaaaga 840
aaccaagtaa aagcaaagaa aatataaatc acacgagtgg aaagatatta accaagttgg
900 gattcaagaa taaagtctat attaaatatt caaaaagata aatttaaata
atatgaaagg 960 aaacatattc aatacattgt agtttgctac tcataatcgc
tagaatactt tgtgccttgc 1020 taataaagat acttgaaata gcttagttta
aatataaata gcataataga ttttaggaat 1080 tagtattttg agtttaatta
cttattgact tgtaacagtt tttataattc caaggcccat 1140 gaaaaattta
atgctttatt agttttaaac ttactatata aatttttcat atgtaaaatt 1200
taatcggtat agttcgatat tttttcaatt tatttttata aaataaaaaa cttaccctaa
1260 ttatcggtac agttatagat ttatataaaa atctacggtt cttcagaaga
aacctaaaaa 1320 tcggttcggt gcggacggtt cgatcggttt agtcgatttt
caaatattca ttgacactcc 1380 tagttgttgt tataggtaaa aagcagttac
agagaggtaa aatataactt aaaaaatcag 1440 ttctaaggaa aaattgactt
ttatagtaaa tgactgttat ataaggatgt tgttacagag 1500 aggtatgagt
gtagttggta aattatgttc ttgacggtgt atgtcacata ttatttatta 1560
aaactagaaa aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa atcggctgaa
1620 tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg
actgttgatg 1680 atataaacaa agggcacatt ggtcaataac cataaaaaat
tatatgacag ctacagttgg 1740 tagcatgtgc tcagctattg aacaaatcta
aagaaggtac atctgtaacc ggaacaccac 1800 ttaaatgact aaattaccct
catcagaaag cagatggagt gctacaaata acacactatt 1860 caacaaccat
aaataaaacg tgttcagcta ctaaaacaaa tataaataaa tctatgtttg 1920
taagcactcc agccatgtta atggagtgct attgcctgtt aactctcact tataaaatag
1980 tagtagaaaa aatatgaacc aaaacacaac tttatcgcca tcatttacat
accactccac 2040 ctttaatgaa ggatcaactt ccgcgaatat catctcagca
agtgcaattc ctgctatgat 2100 cccgtcttcc tttgctagaa aatgagcatc
ggattccata tcaagaggaa ttgtcgcctt 2160 acaagtcaca tctcctaaat
tcccagcatc ttcagagagt gcaagtttca taacttcctt 2220 taaatcataa
gttgggtgtg ctggtggttt cacctctaat gactccactc ttgtattctt 2280
ggtggctatt gctgacattt tcaccaccaa ccttggagct gtaattgcat aaggatgcac
2340 tgtagcagtg aaaggaatag ctctaaacat ggttgtgtat ttcacttttg
gatatagctc 2400 agtggcttcg acacctgtag gaggctgaac ctcaaagttt
gcagaatctc cattaacaaa 2460 aactgaatgg catatggcca aattagtcct
taatggcaga ggtccctctt gtacaatctg 2520 gagaatatct tcctctgata
gatataactt ctcgagggtc ttcccaattt tgtcctccca 2580 caaggacact
atctcgttga aggatagaat attggcaggt ggtctcatgt gaagagtctt 2640
attcaatgtc cgtggatcat ctactgcttc gatagtgtat gtcgctatgt cttcttcctt
2700 cacatatatt gctttgggat ttccatcgcc aaaaatgaca actttgtctc
taggaggggt 2760 tttggcctct aactgcccca agttgggcaa gaagaaatct
gcaaaccaat tgcagattac 2820 atatgtgtat ggaattcctt ctgcctctat
catcctcctg attcttacct ttagagcgaa 2880 gagtgatgca gctggttcaa
ttgcacgagc atgatccaca tcaaatccaa attctgaagg 2940 aagaaatctc
ttgatatttc cagcttcttt aattgctttg atgatgttca cttgatcagt 3000
ccgtcgcttc tcttccattt cttctcattt tcgattttga ttcttatttc tttccagtag
3060 ctcctgctct gtgaatttct ccgctcacga tagatctgct tatactcctt
acattcaacc 3120 ttagatctgg tctcgattct ctgtttctct gtttttttct
tttggtcgag aatctgatgt 3180 ttgtttatgt tctgtcacca ttaataataa
tgaactctct cattcataca atgattagtt 3240 tctctcgtct acaaaacgat
atgttgcatt ttcacttttc ttcttttttt ctaagatgat 3300 ttgctttgac
caatttgttt agatctttat tttattttat tttctggtgg gttggtggaa 3360
attgaaaaaa aaaaaaacag cataaattgt tatttgttaa tgtattcatt ttttggctat
3420 ttgttctggg taaaaatctg cttctactat tgaatctttc ctggattttt
tactcctatt 3480 gggtttttat agtaaaaata cataataaaa ggaaaacaaa
agttttatag attctcttaa 3540 accccttacg ataaaagttg gaatcaaaat
aattcaggat cagatgctct ttgattgatt 3600 cagatgcgat tacagttgca
tggcaaattt tctagatccg tcgtcacatt ttattttctg 3660 tttaaatatc
taaatctgat atatgatgtc gacaaattct ggtggcttat acatcacttc 3720
aactgttttc ttttggcttt gtttgtcaac ttggttttca atacgatttg tgatttcgat
3780 cgctgaattt ttaatacaag caaactgatg ttaaccacaa gcaagagatg
tgacctgcct 3840 tattaacatc gtattactta ctactagtcg tattctcaac
gcaatcgttt ttgtatttct 3900 cacattatgc cgcttctcta ctctttattc
cttttggtcc acgcattttc tatttgtggc 3960 aatccctttc acaacctgat
ttcccacttt ggatcatttg tctgaagact ctcttgaatc 4020 gttaccactt
gtttcttgtg catgctctgt tttttagaat taatgataaa actattccat 4080
agtcttgagt tttcagcttg ttgattcttt tgcttttggt tttctgcagt gatcaagtga
4140 acatcatcaa agcaattaaa gaagctggaa atatcaagag atttcttcct
tcagaatttg 4200 gatttgatgt ggatcatgct cgtgcaattg aaccagctgc
atcactcttc gctctaaagg 4260 taagaatcag gaggatgata gaggcagaag
gaattccata cacatatgta atctgcaatt 4320 ggtttgcaga tttcttcttg
cccaacttgg ggcagttaga ggccaaaacc cctcctagag 4380 acaaagttgt
catttttggc gatggaaatc ccaaagcaat atatgtgaag gaagaagaca 4440
tagcgacata cactatcgaa gcagtagatg atccacggac attgaataag actcttcaca
4500 tgagaccacc tgccaatatt ctatccttca acgagatagt gtccttgtgg
gaggacaaaa 4560 ttgggaagac cctcgagaag ttatatctat cagaggaaga
tattctccag attgtacaag 4620 agggacctct gccattaagg actaatttgg
ccatatgcca ttcagttttt gttaatggag 4680 attctgcaaa ctttgaggtt
cagcctccta caggtgtcga agccactgag ctatatccaa 4740 aagtgaaata
cacaaccatg tttagagcta ttcctttcac tgctacagtg catccttatg 4800
caattacagc tccaaggttg gtggtgaaaa tgtcagcaat agccaccaag aatacaagag
4860 tggagtcatt agaggtgaaa ccaccagcac acccaactta tgatttaaag
gaagttatga 4920 aacttgcact ctctgaagat gctgggaatt taggagatgt
gacttgtaag gcgacaattc 4980 ctcttgatat ggaatccgat gctcattttc
tagcaaagga agacgggatc atagcaggaa 5040 ttgcacttgc tgagatgata
ttcgcggaag ttgatccttc attaaaggtg gagtggtatg 5100 taaatgatgg
cgataaagca agtgtgttgc ctttgtgtgg aaatgaagag gtacttgcga 5160
ggactttgcg tttatcagtt tatgtgtttg tatatctatt tgatccagtt attatggatt
5220 atatacgctt gaaactcatt ttaagccatt gttattgaac gtttatcaaa
tactttatta 5280 tgccaagcaa gtcaaacaca tgcttgttga ttgaaatcaa
gctatagaaa tctcttcttc 5340 acatacagca gtttagattc acaatacaac
aagcgaaacg ataaagtttc 5390 24 3600 DNA Artificial Sequence
Artificially created chimeric nucleic acid sequence 24 tctagaatgt
tcgtgcgtca aatggataaa caaaaaaata gcataagtta gttttgttac 60
tcgagagtta tgtattataa ggtataggga aatgactcaa acataccact gaacttaacg
120 aaacgacgca tatatatact acttaactta acgaaaaagg ggtgagagtg
gatgggtgct 180 ggtaaataat gaagggttta tataacgtca cgtgtcaaaa
ttcgatagta gtagtttcgt 240 tagttgtaat agcatatatg gcccaaagtt
ataatataga taatatgttt atgtccaact 300 attaacgagt gacatagaca
gttcattttg tgaagttcaa tgacatattt gagccctttc 360 ccttttatta
tctcctttta tttgttctaa taaaagaatg gcatttatta tgtacataga 420
caaataacta ttttctttgg aatataattt gtttatatat tttaaaatca tgtctcaatt
480 tagtttgttt tgtgcatatt tcaactattc aattttgtcc atatatttat
taccttcccc 540 catttacaag cattgaaccg ctttgctcac caaaacttat
gcacattgca aaaatatcat 600 gtaaaggttt tatgtatgct gtaattaagg
tctgaactca tcgtgatttt atttttaggc 660 ttcattgacc actaccaaac
tctttgatgc tacattttct aattatattg gagttcgatt 720 atatccgaat
tcgcgttgcg tagggcccat tcgagggaaa acactcccta tcaaggattt 780
tttcataccc agagctcgaa ctcaagacat ctggttaagg gaagaacagt ctcatccact
840 gcaccatatc cttttgtggt caacaagtaa attttatgta gaaccaaaaa
ctatactcga 900 attgataaaa taaatggtgt aaaatattgt tttctttctt
acattttgga cagtaaatat 960 gtaggacaat aataattagc gtggggtctt
aagaaaatta gcatagattt ccagaaattc 1020 caaatcaacc ggcagttcca
ggtttgaaaa ctacaactca ttccgacggt tcaaacttca 1080 aaccatgctt
gctgactcgg cttcttcttt ctttttcacc aagacagagc agtagtcacg 1140
tgacacccct cacgtgcctc ccccctttat atttcagact gcaaccctac actttcgcta
1200 cattcactac catattcttt tcactaagca attttctctc ctacttttct
ttaaacccct 1260 tttttctccc ctaagccatg gcatctagat catgttacgt
cctgtagaaa ccccaacccg 1320 tgaaatcaaa aaactcgacg gcctgtgggc
attcagtctg gatcgcgaaa actgtggaat 1380 tgatcagcgt tggtgggaaa
gcgcgttaca agaaagccgg gcaattgctg tgccaggcag 1440 ttttaacgat
cagttcgccg atgcagatat tcgtaattat gcgggcaacg tctggtatca 1500
gcgcgaagtc tttataccga aaggttgggc aggccagcgt atcgtgctgc gtttcgatgc
1560 ggtcactcat tacggcaaag tgtgggtcaa taatcaggaa gtgatggagc
atcagggcgg 1620 ctatacgcca tttgaagccg atgtcacgcc gtatgttatt
gccgggaaaa gtgtacgtat 1680 caccgtttgt gtgaacaacg aactgaactg
gcagactatc ccgccgggaa tggtgattac 1740 cgacgaaaac ggcaagaaaa
agcagtctta cttccatgat ttctttaact atgccggaat 1800 ccatcgcagc
gtaatgctct acaccacgcc gaacacctgg gtggacgata tcaccgtggt 1860
gacgcatgtc gcgcaagact gtaaccacgc gtctgttgac tggcaggtgg tggccaatgg
1920 tgatgtcagc gttgaactgc gtgatgcgga tcaacaggtg gttgcaactg
gacaaggcac 1980 tagcgggact ttgcaagtgg tgaatccgca cctctggcaa
ccgggtgaag gttatctcta 2040 tgaactgtgc gtcacagcca aaagccagac
agagtgtgat atctacccgc ttcgcgtcgg 2100 catccggtca gtggcagtga
agggcgaaca gttcctgatt aaccacaaac cgttctactt 2160 tactggcttt
ggtcgtcatg aagatgcgga cttgcgtggc aaaggattcg ataacgtgct 2220
gatggtgcac gaccacgcat taatggactg gattggggcc aactcctacc gtacctcgca
2280 ttacccttac gctgaagaga tgctcgactg ggcagatgaa catggcatcg
tggtgattga 2340 tgaaactgct gctgtcggct ttaacctctc tttaggcatt
ggtttcgaag cgggcaacaa 2400 gccgaaagaa ctgtacagcg aagaggcagt
caacggggaa actcagcaag cgcacttaca 2460 ggcgattaaa gagctgatag
cgcgtgacaa aaaccaccca agcgtggtga tgtggagtat 2520 tgccaacgaa
ccggataccc gtccgcaagg tgcacgggaa tatttcgcgc cactggcgga 2580
agcaacgcgt aaactcgacc cgacgcgtcc gatcacctgc gtcaatgtaa tgttctgcga
2640 cgctcacacc gataccatca gcgatctctt tgatgtgctg tgcctgaacc
gttattacgg 2700 atggtatgtc caaagcggcg atttggaaac ggcagagaag
gtactggaaa aagaacttct 2760 ggcctggcag gagaaactgc atcagccgat
tatcatcacc gaatacggcg tggatacgtt 2820 agccgggctg cactcaatgt
acaccgacat gtggagtgaa gagtatcagt gtgcatggct 2880 ggatatgtat
caccgcgtct ttgatcgcgt cagcgccgtc gtcggtgaac aggtatggaa 2940
tttcgccgat tttgcgacct cgcaaggcat attgcgcgtt ggcggtaaca agaaagggat
3000 cttcactcgc gaccgcaaac cgaagtcggc ggcttttctg ctgcaaaaac
gctggactgg 3060 catgaacttc ggtgaaaaac cgcagcaggg aggcaaacaa
tgagagctcg tgaaatggcc 3120 tctttagttt ttgattgaat cataggggta
ttagttttct atggccggga gtggtcttct 3180 tgcttaattg taatggaata
accagagagg aactactgtg ttatctttga ggaatgttgg 3240 gcttttttcg
tttgaattat catgaatgaa attttacttt ttcccaatac aagtttgttt 3300
tcgtttcttg gtttttgtta tcccttggtt tatgtcttgg tttggcttaa atgattgaag
3360 attacactac ctatgtttct gctattcctg ttgaagatca catttgataa
taatgcatcg 3420 aatgcattaa agtttcttat tggctctgtc aaaagtattg
aaggtggatt tttctaattg 3480 gcaagagaaa gtattaaaga ggtgatttat
tagtacttat atttttctca gcatctctct 3540 ttcagtgttg gagcttcata
aaattagcac ttcagagttt cagtcgggag ctgaattcga 3600 25 3387 DNA
Artificial Sequence Artificially created chimeric nucleic acid
sequence 25 cgttttgacg agttcggatg tagtagtagc cattatttaa tgtacatact
aatcgtgaat 60 agtgaatatg atgaaacatt gtatcttatt gtataaatat
ccataaacac atcatgaaag 120 acactttctt tcacggtctg aattaattat
gatacaattc taatagaaaa cgaattaaat 180 tacgttgaat tgtatgaaat
ctaattgaac aagccaacca cgacgacgac taacgttgcc 240 tggattgact
cggtttaagt taaccactaa aaaaacggag ctgtcatgta acacgcggat 300
cgagcaggtc acagtcatga agccatcaaa gcaaaagaac taatccaagg gctgagatga
360 ttaattagtt taaaaattag ttaacacgag ggaaaaggct gtctgacagc
caggtcacgt 420 tatctttacc tgtggtcgaa atgattcgtg tctgtcgatt
ttaattattt ttttgaaagg 480 ccgaaaataa agttgtaaga gataaacccg
cctatataaa ttcatatatt ttctctccgc 540 tttgaattgt ctcgttgtcc
tcctcacttt catcggccgt ttttgaatct ccggcgactt 600 gacagagaag
aacaaggaag aagactaaga gagaaagtaa gagataatcc aggagattca 660
ttctccgttt tgaatcttcc tcaatctcat cttcttccgc tctttctttc caaggtaata
720 ggaactttct ggatctactt tatttgctgg atctcgatct tgttttctca
atttccttga 780 gatctggaat tcgtttaatt tggatctgtg aacctccact
aaatcttttg gttttactag 840 aatcgatcta agttgaccga tcagttagct
cgattatagc taccagaatt tggcttgacc 900 ttgatggaga gatccatgtt
catgttacct gggaaatgat ttgtatatgt gaattgaaat 960 ctgaactgtt
gaagttagat tgaatctgaa cactgtcaat gttagattga atctgaacac 1020
tgtttaagtt agatgaagtt tgtgtataga ttcttcgaaa ctttaggatt tgtagtgtcg
1080 tacgttgaac agaaagctat ttctgattca atcagggttt atttgactgt
attgaactct 1140 ttttgtgtgt ttgcagctca tatggttgtg tttgggaatg
tttctgcggc gaatttgcct 1200 tatcaaaacg ggtttttgga ggcactttca
tctggaggtt gtgaactaat gggacatagc 1260 tttagggttc ccacttctca
agcgcttaag acaagaacaa ggaggaggag tactgctggt 1320 cctttgcagg
tagtttgtgt ggatattcca aggccagagc tagagaacac tgtcaatttc 1380
ttggaagctg ctagtttatc tgcatccttc cgtagtgctc ctcgtcctgc taagcctttg
1440 aaagttgtaa ttgctggtgc tggattggct ggattgtcaa ctgcaaagta
cctggctgat 1500 gcaggccaca aacctctgtt gcttgaagca agagatgttc
ttggtggaaa gatagctgca 1560 tggaaggatg aagatgggga ctggtatgag
actggtttac atattttctt cggtgcttat 1620 ccgaatgtgc agaatttatt
tggagaactt gggatcaatg atcggttgca gtggaaggaa 1680 cactccatga
tttttgctat gccaagtaaa cctggagaat ttagtagatt tgacttccca 1740
gatgtcctac cagcaccctt aaatggtatt tgggctattt tgcggaacaa cgagatgctg
1800 acatggccag agaaaataaa gtttgctatt ggacttttgc cagccatggt
cggcggtcag 1860 gcttatgttg aggcccaaga tggtttatca gtcaaagaat
ggatggaaaa gcagggagta 1920 cctgagcgcg tgaccgacga ggtgtttatt
gccatgtcaa aggcgctaaa ctttataaac 1980 cctgatgaac tgtcaatgca
atgcattttg atagctttga accggtttct tcaggaaaaa 2040 catggttcca
agatggcatt cttggatggt aatcctccgg aaaggctttg tatgccagta 2100
gtggatcata ttcgatcact aggtggggaa gtgcaactta attctaggat aaagaaaatt
2160 gagctcaatg acgatggcac ggttaagagt ttcttactca ctaatggaag
cactgtcgaa 2220 ggagacgctt atgtgtttgc cgctccagtc gatatcctga
agctcctttt accagatccc 2280 tggaaagaaa taccgtactt caagaaattg
gataaattag ttggagtacc agttattaat 2340 gttcatatat ggtttgatcg
aaaactgaag aacacatatg atcacctact ctttagcaga 2400 agtaaccttc
tgagcgtgta tgccgacatg tccttaactt gtaaggaata ttacgatcct 2460
aaccggtcaa tgctggagct agtatttgca ccagcagagg aatggatatc acggactgat
2520 tctgacatca tagatgcaac aatgaaagaa cttgagaaac tcttccctga
tgaaatctca 2580 gctgaccaaa gcaaagctaa aattctgaag taccatgtcg
ttaagactcc aagatctggg 2640 tacaagacca tcccaaactg tgaaccatgt
cgtcctctac aaagatcacc tattgaagga 2700 ttctacttag ctggagatta
cacaaaacag aagtacttag cttccatgga aggcgctgtc 2760 ctctctggca
aattctgctc tcagtctatt gttcaggatt acgagctact ggctgcgtct 2820
ggaccaagaa agttgtcgga ggcaacagta tcatcatcat gagaaaaggg cgaattcgtt
2880 aaccgcagac gagctcgtga aatggcctct ttagtttttg attgaatcat
aggggtatta 2940 gttttctatg gccgggagtg gtcttcttgc ttaattgtaa
tggaataacc agagaggaac 3000 tactgtgtta tctttgagga atgttgggct
tttttcgttt gaattatcat gaatgaaatt 3060 ttactttttc ccaatacaag
tttgttttcg tttcttggtt tttgttatcc cttggtttat 3120 gtcttggttt
ggcttaaatg attgaagatt acactaccta tgtttctgct attcctgttg 3180
aagatcacat ttgataataa tgcatcgaat gcattaaagt ttcttattgg ctctgtcaaa
3240 agtattgaag gtggattttt ctaattggca agagaaagta ttaaagaggt
gatttattag 3300 tacttatatt tttctcagca tctctctttc agtgttggag
cttcataaaa ttagcacttc 3360 agagtttcag tcgggagctg aattcga 3387 26
1701 DNA Arabidopsis thaliana 26 atggttgtgt ttgggaatgt ttctgcggcg
aatttgcctt atcaaaacgg gtttttggag 60 gcactttcat ctggaggttg
tgaactaatg ggacatagct ttagggttcc cacttctcaa 120 gcgcttaaga
caagaacaag gaggaggagt actgctggtc ctttgcaggt agtttgtgtg 180
gatattccaa ggccagagct agagaacact gtcaatttct tggaagctgc tagtttatct
240 gcatccttcc gtagtgctcc tcgtcctgct aagcctttga aagttgtaat
tgctggtgct 300 ggattggctg gattgtcaac tgcaaagtac ctggctgatg
caggccacaa acctctgttg 360 cttgaagcaa gagatgttct tggtggaaag
atagctgcat ggaaggatga agatggggac 420 tggtatgaga ctggtttaca
tattttcttc ggtgcttatc cgaatgtgca gaatttattt 480 ggagaacttg
ggatcaatga tcggttgcag tggaaggaac actccatgat ttttgctatg 540
ccaagtaaac ctggagaatt tagtagattt gacttcccag atgtcctacc agcaccctta
600 aatggtattt gggctatttt gcggaacaac gagatgctga catggccaga
gaaaataaag 660 tttgctattg gacttttgcc agccatggtc ggcggtcagg
cttatgttga ggcccaagat 720 ggtttatcag tcaaagaatg gatggaaaag
cagggagtac ctgagcgcgt gaccgacgag 780 gtgtttattg ccatgtcaaa
ggcgctaaac tttataaacc ctgatgaact gtcaatgcaa 840 tgcattttga
tagctttgaa ccggtttctt caggaaaaac atggttccaa gatggcattc 900
ttggatggta atcctccgga aaggctttgt atgccagtag tggatcatat tcgatcacta
960 ggtggggaag tgcaacttaa ttctaggata aagaaaattg agctcaatga
cgatggcacg 1020 gttaagagtt tcttactcac taatggaagc actgtcgaag
gagacgctta tgtgtttgcc 1080 gctccagtcg atatcctgaa gctcctttta
ccagatccct ggaaagaaat accgtacttc 1140 aagaaattgg ataaattagt
tggagtacca gttattaatg ttcatatatg gtttgatcga 1200 aaactgaaga
acacatatga tcacctactc tttagcagaa gtaaccttct gagcgtgtat 1260
gccgacatgt ccttaacttg taaggaatat tacgatccta accggtcaat gctggagcta
1320 gtatttgcac cagcagagga atggatatca cggactgatt ctgacatcat
agatgcaaca 1380 atgaaagaac ttgagaaact cttccctgat gaaatctcag
ctgaccaaag caaagctaaa 1440 attctgaagt accatgtcgt taagactcca
agatctgtgt acaagaccat cccaaactgt 1500 gaaccatgtc gtcctctaca
aagatcacct attgaaggat tctacttagc tggagattac 1560 acaaaacaga
agtacttagc ttccatggaa ggcgctgtcc tctctggcaa attctgctct 1620
cagtctattg ttcaggatta cgagctactg gctgcgtctg gaccaagaaa gttgtcggag
1680 gcaacagtat catcatcatg a 1701
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